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Impaired action of anxiolytic drugs in mice deficient in cannabinoid CB1 receptors
Neuropharmacology 46 (2004) 966–973 www.elsevier.com/locate/neuropharm
Impaired action of anxiolytic drugs in mice deficient in cannabinoid CB1 receptors Leyre Urigu¨en a, Sandra Pe´rez-Rial a, Catherine Ledent b, Toma´s Palomo a, Jorge Manzanares a, a
Servicio de Psiquiatrı´a y Unidad de Investigacio´n, Pabello´n de Medicina Comunitaria, Hospital Universitario 12 de Octubre, Avda Co´rdoba s/n, 28041 Madrid, Spain b IRIBHN, Universite´ Libre de Bruxelles, Brussels, Belgium Received 20 June 2003; received in revised form 11 November 2003; accepted 7 January 2004
Abstract The role of cannabinoid CB1 receptors in the action of anxiolytics was examined. Deletion of CB1 receptors resulted in increased anxiety-like behaviours in light/dark box, elevated plus maze and social interaction tests. Mutant mice presented basal low corticosterone concentrations and low proopiomelanocortin gene expression in the anterior lobe of the pituitary gland compared to wildtype mice. Ten minutes of restraint stress resulted in a twofold increase in corticosterone concentrations in the plasma of mutant mice, compared to wild-type mice. Bromazepam (50 or 100 lg/kg) markedly increased the time spent in light area in wild-type animals, though both doses were without effect in mutant mice. Administration of buspirone (1 or 2 mg/kg) produced anxiolytic effects in wild-type mice. In contrast, only the highest dose of buspirone had anxiolytic results in mutant mice. Our findings reveal that CB1 receptors are involved in the regulation of emotional responses, and play a pivotal role in the action mechanism of anxiolytics. They suggest that alterations in the functional activity of the CB1 receptor may be related to the emergence of anxiety disorders, and may affect treatment with anxiolytics. # 2004 Elsevier Ltd. All rights reserved. Keywords: Cannabinoid CB1 receptor; Anxiety; Knockout mice; Anxiety-like behaviour; Benzodiazepine; Buspirone
1. Introduction The control of emotional states in the brain is subject to multiheterogenous regulation by several neurotransmitters and hormones. Increases or decreases in the functional activity of many of these neuromodulators represent the rationale underlying the molecular mechanisms of anxiolytic drugs. Thus, the discovery of neurochemical elements that alter the state of anxiety may help to identify new therapeutic targets and contribute to an understanding of differences in the response of anxiolytics in certain clinical situations. The role played by cannabinoid CB1 receptors in the action mechanism or the efficacy of anxiolytic drugs is largely unknown. However, a large body of evidence Corresponding author. Tel.: +34-91-390-8252; fax: +34-91-3908538. E-mail address: [email protected] (J. Manzanares).
0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.01.003
indicates the participation of cannabinoid receptors in the regulation of key elements affecting anxiety-like behaviours. It has been reported that acute administration of cannabinoids may cause anxiogenic responses in humans (Zuardi et al., 1982). Animal studies reveal that cannabinoids can induce both anxiolytic and anxiogenic-like responses depending on the doses and the familiarity of the environment (Rodriguez de Fonseca et al., 1996). In contrast, blockade of CB1 cannabinoid receptors with SR 141716A induced anxiety-like responses in the elevated plus maze and in the defensive withdrawal test in rats (Navarro et al., 1997). Furthermore, recent studies suggest that blockade of anandamide hydrolysis by inhibition of FAAH may result in anxiolytic action in two different validated animal models of anxiety (Kathuria et al., 2003). THC or cannabinoid receptor agonists produced pronounced alterations in the endogenous opioid system
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(Manzanares et al., 1999a; Valverde et al., 2000; Navarro et al., 2001), which has recently been proposed as being involved in the anxiolytic-like effects induced by THC (Berrendero and Maldonado, 2002). In addition, the endogenous cannabinoid system plays an important physiological role in the control of the hypothalamic-pituitary-adrenal (HPA) axis function: (1) THC or anandamide increases plasma corticotropin and corticosterone concentrations (Manzanares et al., 1999b; Weidenfeld et al., 1994), (2) THC or cannabinoid receptor agonists increase corticotropin releasing factor gene expression in the paraventricular nucleus of the hypothalamus (Corchero et al., 1999), (3) an increase in the release of CRF or in the secretion of corticosterone in the plasma occurred after spontaneous (Oliva et al., 2003) or antagonist-precipitated cannabinoid withdrawal syndrome (Rodrı´guez de Fonseca et al., 1997), and (4) administration of SR-141716A (at doses of 12.5 and 50 lg/rat, icv) produced a marked increase in the secretion of both ACTH and corticosterone (Manzanares et al., 1999b), suggesting that under basal conditions endogenous cannabinoid ligands might be tonically inhibiting the release of both hormones. Cannabinoid CB1 knockout mice represent an excellent tool for exploring the role of cannabinoid receptors in the neurobiology of anxiety and their involvement in the action mechanism of anxiolytic drugs. Recent findings supporting the role of cannabinoid CB1 receptors in the regulation of anxiety include studies showing that mutant mice exhibited an anxiogenic-like response in the light/dark box, increased anhedony in chronic unpredictable mild stress procedure (Martin et al., 2002), and reduced exploration in the elevated plus maze (Haller et al., 2002). In contrast, Marsicano et al. (2002) found no alterations in the time on open arms in the elevated maze between mutant and wild-type animals. In this study, we examined behavioural and neuroendocrinological alterations induced by deletion of cannabinoid CB1 receptors, its effect on restraint stressmediated regulation of plasma corticosterone secretion, and the differential response of anxiolytic drugs (bromazepam, buspirone) in the light/dark box test between mutant and wild-type mice. Our results led us to speculate that deletion of cannabinoid CB1 receptors produces a high state of anxiety-like behaviours, basal alterations in the function of the pituitary adrenal axis, hypersensitivity to stress and impaired action of anxiolytic drugs.
2. Materials and methods 2.1. Animals Male cannabinoid receptor (CB1) double mutant CB1 (/) mice and wild-type CB1 (+/+) littermates
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were used in all the experiments. All mice were born at the Complutense University vivarium from breeding pairs CB1 (+/). The generation of mice lacking the CB1 receptor was previously described by Ledent et al. (1999). Heterozygote mice from the sixth generation on a CD1 (Charles River, France) background were bred at our animal facilities and homozygotes from the same generation were selected for our experiments. Therefore, wild-type and CB1 mutant mice used in a given experiment originated from the same breeding series and were matched for age and weight (age ¼ 2 3 months; weight ¼ 25 35 g). Mice were maintained v at a constant temperature of 23 2 C and in a 12-h dark/light cycle (light from 7:00 to 19:00 h), with free access to food (commercial diet for rodents A04 Panlab, Barcelona, Spain) and water. Animals were housed in standard laboratory cages, each one containing groups of 8–10 individuals. All experimental procedures were carried out between 09:00 and 13:00 h. Behavioural tests were carried out under the same conditions. All the experiments in this study were performed in compliance with the Royal Decree 223/1988 of 14 March (BOE.8 18) and the Ministerial Order of 13 October 1989 (BOE 18) regarding protection of experimental animals, as well as with the European Council Directive of 24 November 1986 (86/609/EEC).
2.2. Drugs N-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro [4,5]decane-7,9-dione hydrobromide-hemicarbonate salt (buspirone hydrochloride) and 7-bromo-5-(2-pyridyl)3H-1,4-benzodiazepin-2(1H)-one (bromazepam) were obtained from Sigma–Aldrich (Madrid, Spain) and dissolved in NaCl. Drugs were administered as indicated in the figure legends.
2.3. Motor activity-open field test The open field consists of a transparent square cage 25 25 25 cm with a white plexiglas floor. The base of the cage is divided by lines into peripheral squares (12) and central squares (4). A lamp directed at the centre of the field provides illumination for the floor. Testing was conducted in a silent room with constant light. Mice were individually placed in the centre of the apparatus to initiate a 30-min test session. Each session was recorded with a video camera and analysed directly with the SMART (Spontaneous Motor Activity Recording & Tracking) v. 2.0 software system (Panlab, Barcelona, Spain) on a computer. Total, central and peripheral distance covered (m), divided into three 10-min intervals, was analysed.
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2.4. Assessment of anxiety-like behaviours
2.6. Corticosterone radioimmunoassay
2.4.1. Light/dark box The light/dark box test makes use of rodents’ natural aversion to bright areas compared with darker ones. In a two-compartment box, rodents will prefer dark areas, whereas anxiolytics should increase the time spent in the light compartment. The apparatus consisted of two metacrilate boxes (20 20 15 cm) one transparent and one black and opaque, separated by an opaque tunnel (4 cm). Light from a 60 W desk lamp placed 25 cm above the light box provided room illumination. Mice were individually tested in 5-min sessions. The floor of each box was cleaned between sessions. At the beginning of the session, mice were placed in the tunnel facing the dark box. The time spent by mice in the light area was recorded over 5-min periods. A mouse whose four paws were in the new box was considered as having changed boxes.
Blood samples were centrifuged (3000 rpm for 15 min) v and plasma was stored at 80 C. Plasma corticosterone concentrations were measured using a radioimmunoassay kit (Coat-A-Count rat/mice corticosterone, Diagnostic Product Corporation, Los Angeles, CA, USA). Lower sensitivity limit of the assay was 5.7 ng/ml. Intra-assay variability was 5.8%.
2.4.2. Elevated plus maze The elevated plus maze consisted of two open arms and two enclosed horizontal perpendicular arms 50 cm above the floor. The junction of four arms formed a central squared platform (5 5 cm). The test began with the animal being placed in the centre of the apparatus facing one of the enclosed arms and allowed to explore freely for 5 min. We considered arm entries as entry of four paws into the arm. The time spent in open arms and the number of open-arm entries were recorded. Number of entries and time spent in open arms were expressed as percentages of total entries and total test time, respectively.
2.8. In situ hybridisation histochemistry (ISHH)
2.4.3. Social interaction When two mice from separate cages are placed together in a small chamber in which neither has established territory, they engage in social interaction which includes a variety of behavioural patterns: sniffing, following, grooming, kicking, crawling under or over the partner, and touching or nearly touching their faces. On the day of the experiment, pairs of mice from different home cages were placed together in a small plastic cage (20 40 10 cm) with a cardboard lid and fresh wood litter on the floor (no change in the light level). The time that mice socially interacted was measured for 5 min. 2.5. Restraint stress procedure Unanaesthetised mice were confined in an acrylic cylindrical tube (inner size 10 3 3 cm) for 10 min. After this period of restraint stress, the animals were removed from the tubes and rapidly decapitated. Control animals remained in their home cages until decapitation.
2.7. Histology of adrenal glands Adrenal gland tissues were fixed in neutral-buffered formalin for 48 h, dehydrated in 70% ethanol, and embedded in paraffin. Tissues were sectioned at 8 lm thickness, deparaffinised, and stained with haematoxylin and eosine (H&E).
Pituitary gland sections were mounted onto gelatinv coated slides and stored at 80 C until the day of the assay. ISHH was performed as described previously (Young et al., 1986) using synthetic oligonucleotide probes complementary to proopiomelanocortin (POMC), nucleotides 96–134 (CTT CTT GCC CAC CGG CTT GCC CCA GCG CCA GCG GAA GTG CTC CAT GGA GTA GGA) of the rat POMC gene mRNA. Oligonucleotide probe was labelled using terminal deoxytransferase (Amersham, Madrid, Spain) to add 35Slabelled deoxyATP (1000 Ci mmol1; Amersham, Madrid, Spain) tail to the 30 end. The probe (in 50 ll of hybridisation buffer) was applied to each section and v left overnight at 37 C for hybridisation. Following hybridisation, sections were washed four times for 15 min each in 0.15 M NaCl, 0.015 M sodium citrate, pH v 7.2 (1X saline sodium citrate, SSC) at 55 C, followed by two 30-min washes in 1X SSC at room temperature, one brief water dip and blow-dry with air. The dried slides were apposed to Kodak BioMax MR-1 film (Amersham, Madrid, Spain) for 4 days. Autoradiograms were analysed with a PC computer using the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). Optical densities were calculated from the uncalibrated mode by subtracting from each measurement its corresponding background, and expressed in grey scale values. The background measurement was taken from an area of the slice with the lowest non-specific hybridisation signal and subtracted from the hybridisation signal measurement in the same slice. Results were presented considering mean control values as 100%.
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2.9. Statistical analyses Statistical analyses were performed using the Student t-test when comparing two groups, one-way analysis of variance followed by the Student Newman Keul test when comparing three or four groups, and repeatedmeasures ANOVA followed by the Student Newman Keul test when comparing groups at different time points. Differences were considered significant if the probability of error was less than 5%. 3. Results 3.1. Motor activity-open field
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wild-type and mutant mice were found at 10–20 and 20–30-min periods. Speed in central areas significantly increased at 1–10 and 10–20-min periods in CB1 (/) (data not shown). 3.2. Assessment of anxiety-like behaviours 3.2.1. Light/dark box test In the light/dark box test, CB1 (/) spent significantly (p < 0:001) less time in the light area (Fig. 2A) and displayed a pronounced (p < 0:001) reduction in the number of transitions between compartments (Fig. 2B) (Student t-test; t¼ 7:522, p < 0:001, 18 df, and t¼ 7:672, p < 0:001, 10 df).
To determine whether deletion of the cannabinoid CB1 receptor alters the spontaneous motor activity, we compared CB1 (+/+) and CB1 (/) mice in the open field for a period of 30 min. No differences were found between mutant and wild-type animals when considering the whole period of activity (distance) in central and peripheral areas of the open field (data not shown). However, subtle changes were found when considering this time period divided into three phases of 10 min each and separating central and peripheral areas. The results revealed that distance in peripheral areas of the open field significantly increased at 0–10min period (Fig. 1A) (RM ANOVA followed by Student Newman Keul test: F ð55; 71Þ ¼ 19:752, p¼ 0:015); no differences were found at 10–20-min or 20–30-min periods, whereas distance in central areas significantly decreased (Fig. 1B) in the first 10-min period in CB1 (/) mice compared to CB1 (+/+) mice (RM ANOVA followed by Student Newman Keul test: F ð55; 71Þ ¼ 6:297, p< 0:001); no differences between
3.2.2. Elevated plus maze In the elevated plus maze, the percentage (%) of time spent in the open arms, as well as percentage (%) of entries into the open arms, significantly decreased in CB1 (/) (Fig. 2C,D (Student t-test; t¼ 6:698, p< 0:001, 12 df, and t¼ 7:929, p< 0:001, 12 df).
Fig. 1. Assessment of spontaneous locomotor activity in CB1 (+/+) and CB1 (/) mice in the open field. Motor activity (peripheral and central activities) was measured in a 30-min session, divided into three periods of 10 min (n¼ 12). Columns represent the means and vertical lines +SEM of distance (m) in 12 mice; , values from CB1 (/) mice that are significantly different (p< 0:001, RM ANOVA in each 10-min period) from CB1 (+/+) mice; (A) peripheral and (B) central distance in the open field.
Fig. 2. Assessment of anxiety-like behaviours in CB1 (+/+) and CB1 (/) mice in the light/dark box, elevated plus maze and social interaction tests. Behaviour in each test was evaluated for a period of 5 min. Columns represent the means and vertical lines +SEM of A (time (s)), B (number of transitions), C (% time in open arms), D (% entries), E (time (s)) in 6–8 mice; , values from CB1 (/) mice (black columns) that are significantly different (p< 0:05, Student ttest) from CB1 (+/+) mice (white columns).
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3.2.3. Social interaction test In the social interaction test, the time in which mice from separate cages (unfamiliar situation) engaged in social interaction was significantly lower (p< 0:001) in CB1 (/) (Fig. 2E) (Student t-test; t¼ 7:069, p¼ 0:001, 14 df). 3.3. Evaluation of hypothalamic-pituitary-adrenal axis (HPA) function To investigate the HPA axis function in CB1 (/) and CB1 (+/+), we examined the basal concentrations of corticosterone in the plasma, basal POMC gene expression in anterior and intermediate lobes of the pituitary gland, gross morphology of adrenal gland and changes in corticosterone secretion induced by restraint stress. 3.3.1. Basal corticosterone plasma concentrations As shown in Fig. 3A, basal corticosterone concentrations in the plasma are 30% lower (p¼ 0:028) in CB1 (/) mice compared to CB1 (+/+) (Student t-test; t¼ 2:564, p¼ 0:028, 10 df). 3.3.2. POMC gene expression Similarly, a 34% lower POMC gene expression was found in the anterior but not in the intermediate lobe of the pituitary gland (Fig. 3B,C) of CB1 (/), as shown by a lower hybridisation signal in the anterior lobe (Fig. 3D). (Student t-test; t¼ 2:297, p¼ 0:039, 13 df for anterior lobe, and t¼ 1:324, ns for intermediate lobe of pituitary gland). Data in Fig. 3B are represented as percentage (%) of control. Histology of the adrenal gland revealed no gross anatomical abnormalities in CB1 (/) mice, as shown in Fig. 3E. 3.3.3. Effect of stress on corticosterone plasma concentrations Data are represented (Fig. 3F) as percentage (%) of control (non-stressed mice). Restraint stress significantly increased corticosterone concentrations in CB1 (+/+) and CB1 (/) when compared with their respective controls (non-stressed) (Student t-test; t¼ 5:188, p< 0:001, 13 df for CB1 (+/+) mice and t¼ 11:975, p< 0:001, 12 df for CB1 (/)). As shown in Fig. 3F, 10 min of restraint stress produces an approximately 200% increase in plasma corticosterone concentrations in the plasma of CB1 (+/+) mice, whereas a 400% increase was observed in CB1 (/). 3.4. Effects of anxiolytic drugs in the light/dark box test 3.4.1. Bromazepam To investigate whether the state of anxiety induced by deletion of CB1 receptors may affect the action of
Fig. 3. Evaluation of the HPA axis function under basal and restraint stress conditions. Columns represent the means and vertical lines +SEM of A (corticosterone (ng/ml)), B and C (POMC RNA levels, % of control) in anterior and intermediate lobes of pituitary, F corticosterone (% of control) in 6–8 mice; , values from CB1 (/) mice that are significantly different (p< 0:05, Student t-test) from CB1 (+/+) mice; D, representative autoradiograms for POMC mRNA in anterior (AL) and intermediate (IL) lobes of the pituitary gland of CB1 (+/+) and CB1 (/) mice; bar represents 1 mm; E, haematoxylin–eosine staining of adrenal gland in CB1 (+/+) and CB1 (/) mice; c, cortex; m, medulla; bar represents 1 mm; lower panels show 5 magnification; F, on the day of the experiment mice were removed from their home cages and immediately decapitated (non-stress), or were restrained in an acrylic tube for 10 min and then decapitated (stress); , values from stress-treated mice that are significantly different (p< 0:05, Student t-test) from non-stressed control mice.
anxiolytic drugs, we examined the effects of the anxiolytic benzodiazepine bromazepam in CB1 (/) and CB1 (+/+) mice. Doses of 50 and 100 lg/kg of bromazepam produced a considerable increase ðp< 0:001Þ in the time CB1 (+/+) spent in the light box ðF ð2; 23Þ ¼ 14:364, p< 0:001) and the number of transitions,
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Fig. 4. Behavioural assessment of anxiolytic action of bromazepam (A–D) and buspirone (E–H) in light/dark box in CB1 (+/+) and CB1 (/) animals. Mice were injected with bromazepam (50, 100 lg/kg i.p.) or buspirone (1, 2 mg/kg i.p.) or its saline vehicle, and 30 min later were exposed to light/dark box for 5 min. Columns represent the means and vertical lines +SEM of A, B, E, F (time (s)) and C, D, G, H (number of transitions) in 6–8 mice; , values from drug-treated CB1 (/) or CB1 (+/+) mice that are significantly different (ANOVA, Student Newman Keul, p< 0:05) from corresponding vehicle-treated CB1 (/) or CB1 (+/+) mice.
(F ð2; 23Þ ¼ 7:746, p< 0:005) (Fig. 4A,C), though no anxiolytic actions were observed in CB1 (/) mice at either of the doses (Fig. 4B,D). 3.4.2. Buspirone Similarly, animals were administered with buspirone (1 or 2 mg/kg i.p.) and exposed to the light/dark box test. Time in the light box significantly increased in CB1 (+/+) mice treated with buspirone (Fig. 4E) (F ð2; 22Þ ¼ 63:90, p< 0:001) and in CB1 (/) (Fig. 4F) (F ð2; 23Þ ¼ 116:673, p< 0:001). Number of transitions between light and dark box (Fig. 4G) increased significantly in CB1 (+/+) after administration of buspirone (F ð2; 23Þ ¼ 5:850, p¼ 0:010). 4. Discussion The results of this study provide clear evidence that cannabinoid CB1 receptor plays a key role in the regulation and treatment of anxiety-like behaviours. This is shown by the fact that mice lacking cannabinoid CB1
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receptors display high anxiety-like behaviours, alterations in the HPA axis under basal conditions, hypersensitivity to stress and impaired action of anxiolytic drugs such as bromazepam and buspirone. To our knowledge, three types of cannabinoid CB1 mice have been developed to date (Zimmer et al., 1999; Ledent et al., 1999; Marsicano et al., 2002), which differ in the way they were generated genetically and with regard to the strain of animals that were finally backcrossed. These differences may account for subtly different behavioural, endocrine and neurochemical responses. In addition, slight changes in certain experimental conditions (light, time of day, temperature, etc.) may significantly alter spontaneous or drug-induced behaviour. The analysis of motor activity in the open field suggests that in our specific conditions and in this type of mice, deletion of cannabinoid CB1 receptors produced altered responses (increases in peripheral areas, decreases in central areas) that seem closer to anxiety-related behaviours than to impaired motor activity. In fact, overall motor activity during the whole period of 30 min reveals no differences between mutant and wildtype mice. These results obtained in the whole period of analysis of motor activity are consistent with those reported by (Marsicano et al., 2002). In contrast, in our study we found increases in peripheral areas and decreases in central areas in the first 10-min interval, whereas Marsicano et al. (2002) did not observe differences in motor activity in 5-min interval. These subtle differences between the two studies may be related to the fact that Marsicano et al. (2002) used mice generated in a different genetic background (C57 strain) from the mice analysed in our study (sixth backcrossed generation on CD1 mice) and to the precise method for evaluating motor behaviour (open field equipped with two infrared sensor rings for measurement of horizontal and vertical locomotion); in our study we used a SMART program that measured distance travelled by the mice in peripheral and central areas of the open field. Maccarrone et al. (2002), using the same strain of mice, found differences in the locomotor activity, although these alterations are probably due to the age of the mice used (young and old mice) and to the different evaluation of motor activity. Indeed, these authors used an open field with different dimensions (70 90 60 cm) from the open field of the present study (25 25 25 cm). The size of the open field may account for the differences in the exploratory and motor behaviour between the two studies. In contrast to these studies, Steiner et al. (1999) showed that mice lacking CB1 receptors were extremely hypoactive. These discrepancies may be explained by differences in the specific method used to evaluate motor activity or the strain of mice in which the mutation was carried out. Our findings show that mice lacking cannabinoid CB1 receptors display a high level of anxiety-like beha-
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viours in three different types of experimental paradigm (light/dark box, elevated plus maze and social interaction). These data are in line with previous findings supporting the role of cannabinoid CB1 receptor function in the regulation of emotional responses (Martin et al., 2002; Haller et al., 2002). In contrast, Marsicano et al. (2002) failed to detect changes in the elevated plus maze between mutant and wild-type mice. Our results provide for the first time evidence of ‘‘generalised anxiety related-behaviours’’, since CB1 knockout mice show a high level of anxiety with different types of anxiogenic stimuli, such as light, open area and elevated position and social encounter under unfamiliar conditions. These profound anxiety-like behavioural alterations occurring in mice deficient in cannabinoid CB1 receptors are probably associated with neuroendocrine and molecular neuroadaptations in key elements that regulate anxiety. HPA axis alterations are closely related to anxietyand mood-related behaviours in animals and humans (Arborelius et al., 1999). Considering the behavioural alterations found in our study, it is possible to speculate that the absence of cannabinoid receptors may affect this neuroendocrine axis. Indeed, basal corticosterone concentrations in the plasma were lower in mutant mice compared to wild-type animals. The reduced secretion of HPA axis hormones was also detected in patients with stress-related disorders. Indeed, hypocortisolism was observed in patients with post-traumatic stress disorder, fatigue syndrome, fibromyalgia and other somatoform disorders (Heim et al., 2000). Potential mechanisms may include dysfunctions on several levels of HPA axis, in addition to genetic vulnerability and personality styles that may determine this neuroendocrine effect. In accordance with the hypocorticosteronemia found in mice lacking CB1 receptors, POMC gene expression in the anterior lobe of the pituitary gland was lower in mutant compared to wild-type mice. However, the gross morphology of the adrenal gland is not dependent upon the presence or absence of cannabinoid receptors. These functional basal differences detected in the HPA axis suggest altered responses to stressful situations in mice lacking CB1 receptors. In fact, our study reveals that restraint stress induced a greater increase in plasma corticosterone concentrations in mutant compared to wild-type animals. These results once more suggest hypersensitivity to stress in mice deficient in CB1 receptors, further supporting a homeostatic function for cannabinoid receptors in the control of anxiety- and mood-related behaviours. Although the precise mechanisms that determine lower levels of corticosterone and POMC gene expression in the anterior lobe of the pituitary and hypersensitivity to stress remain to be elucidated, it is tempting to speculate that impaired feedback regulation of relevant stress responses at the level of gluco-
corticoid receptors may contribute, at least in part, to this neuroendocrine and behavioural abnormality. If deletion of cannabinoid receptors produces neuroendocrine and molecular adaptations that are associated with strong anxiety-like behaviours, it is possible to speculate that treatment with anxiolytic drugs may produce different responses in mutant and wild-type mice. The results of our study clearly demonstrated that bromazepam, a well-known anxiolytic benzodiazepine, failed to modify anxiety-like behaviour of mutant mice. In contrast, both doses of bromazepam used in our study markedly increased the time in the light area in wild-type mice. The nature of the precise molecular alterations produced in mice deficient in cannabinoid CB1 receptors that completely eliminate the action of bromazepam remains to be elucidated. However, considering that benzodiazepines produce their anxiolytic action by acting on GABAA receptors, it is tempting to speculate that deletion of cannabinoid receptors may functionally disrupt any of the different types of a, b or c subunits most closely related to the anxiolytic actions of benzodiazepines (Lo¨w et al., 2000). Further studies on GABAA receptor autoradiography and in situ hybridisation of GABAA receptor subunits are in progress to clarify this hypothesis. Moreover, it is interesting to note that CB1 receptors are presynaptically located in GABAergic interneurons in key regions regulating anxiety (Tsou et al., 1999; Hoffman and Lupica, 2000), and activation of these cannabinoid receptors reduces GABA release from presynaptic terminals. Therefore, deletion of cannabinoid receptors probably modifies GABA neuronal activity or, alternatively, may produce functional alterations at the GABAA receptor in brain areas controlling emotional responses. On the other hand, an alternative explanation may be that in CB1 knockout mice cholecystokinin release is enhanced, since this anxiogenic peptide is co-stored with GABA in a subpopulation of GABAergic interneurons of the ‘‘emotion circuits’’, suggesting that a possible interaction between this peptide and endocannabinoids might participate in the control of anxiety. The administration of the anxiolytic buspirone increased, at both doses, the time spent in the light area in wild-type animals. In contrast, only the highest dose of buspirone produced an anxiolytic effect in mice deficient in cannabinoid CB1 receptors. Since buspirone exerts its anxiolytic activity by acting as a partial agonist at the 5-HT1A receptor, the impairment of this compound to decrease the anxiety-like behaviour of mice lacking CB1 receptors may be related to decreased function of 5-HT1A in brain areas closely related to the control of emotional responses. This hypothesis, however, remains to be clarified. In summary, the results presented here show that cannabinoid receptors play a pivotal role in the regulation
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of the neurobiology and treatment of anxiety-like behaviours. Deletion of cannabinoid receptors results in pronounced alterations in the HPA axis (reduced basal corticosterone secretion and POMC gene expression in the anterior pituitary gland and hypersensitivity to stress). Interestingly, it appears that the presence of cannabinoid receptors is necessary for bromazepam and buspirone to achieve complete anxiolytic action. Overall, these findings strongly suggest that functional alterations in cannabinoid receptors may affect the efficacy of anxiolytic drugs in the treatment of moodrelated disorders. Acknowledgements This research was supported by a grant from the ‘‘Fondo de Investigaciones Sanitarias’’ (FISS 03/0216) to J. Manzanares. Leyre Urigu¨en is a recipient of a predoctoral fellowship from the Basque Country Government. References Arborelius, L., Owens, M.J., Plotsky, P.M., Nemeroff, C.B., 1999. The role of corticotropin-releasing factor in depression and anxiety disorders. J. Endocrinol. 160, 1–12. Berrendero, F., Maldonado, R., 2002. Involvement of the opioid system in the anxiolytic-like effects induced by Delta(9)-tetrahydrocannabinol. Psychopharmacology 163, 111–117. Corchero, J., Fuentes, J.A., Manzanares, J., 1999. Chronic administration with the cannabinoid receptor agonist CP-55,940 regulates corticotropin releasing factor and proopiomelanocortin gene expression in the hypothalamus and pituitary gland of the rat. Life Sci. 64, 905–911. Haller, J., Bakos, N., Szirmay, M., Ledent, C., Freund, T.F., 2002. The effects of genetic and pharmacological blockade of the CB1 cannabinoid receptor on anxiety. Eur. J. Neurosci. 16, 1395–1398. Heim, C., Ehlert, U., Hellhammer, D.H., 2000. The potential role of hypocortisolism in the pathophysiology of stress-related bodily disorders. Psychoneuroendocrinology 25, 1–35. Hoffman, A.F., Lupica, C.R., 2000. Mechanisms of cannabinoid inhibition of GABA(A) synaptic transmission in the hippocampus. J. Neurosci. 20, 2470–2479. Kathuria, S., Gaetani, S., Fegley, D., Valin˜o, F., Duranti, A., Tontini, A., Mor, M., Tarzia, G., La Rana, G., Calignano, A., Giustino, A., Tattoli, M., Palmery, M., Cuomo, V., Piomelli, D., 2003. Modulation of anxiety through blockade of anandamide hydrolysis. Nat. Med. 9, 76–81. Ledent, C., Valverde, O., Cossu, G., Petitet, F., Aubert, J.F., Beslot, F., Bohme, G.A., Imperato, A., Pedrazzini, T., Roques, B.P., Vassart, G., Fratta, W., Parmentier, M., 1999. Unresponsiveness to cannabinoids and reduced addictive effects of opiates in CB1 receptor knockout mice. Science 283, 401–404. Low, K., Crestani, F., Keist, R., Benke, D., Brunig, I., Benson, J.A., Fritschy, J.M., Rulicke, T., Bluethmann, H., Mohler, H., Rudolph, U., 2000. Molecular and neuronal substrate for the selective attenuation of anxiety. Science 290, 131–134. Maccarrone, M., Valverde, O., Barbaccia, M.L., Castane, A., Maldonado, R., Ledent, C., Parmentier, M., Finazzi-Agro, A., 2002. Age-related changes of anandamide metabolism in CB1 cannabinoid receptor knockout mice: correlation with behaviour. Eur. J. Neurosci. 15, 1178–1186.
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Manzanares, J., Corchero, J., Romero, J., Fernandez-Ruiz, J.J., Ramos, J.A., Fuentes, J.A., 1999a. Pharmacological and biochemical interactions between opioids and cannabinoids. Trends Pharmacol. Sci. 20, 287–294. Manzanares, J., Corchero, J., Fuentes, J.A., 1999b. Opioid and cannabinoid receptor-mediated regulation of the increase in adrenocorticotropin hormone and corticosterone plasma concentrations induced by central administration of delta(9)-tetrahydrocannabinol in rats. Brain Res. 21, 173–179. Marsicano, G., Wotjak, C.T., Azad, S.C., Bisogno, T., Rammes, G., Cascio, M.G., Hermann, H., Tang, J., Hofmann, C., Zieglgansberger, W., Di Marzo, V., Lutz, B., 2002. The endogenous cannabinoid system controls extinction of aversive memories. Nature 418, 530–534. Martin, M., Ledent, C., Parmentier, M., Maldonado, R., Valverde, O., 2002. Involvement of CB1 cannabinoid receptors in emotional behaviour. Psychopharmacology 159, 379–387. Navarro, M., Hernandez, E., Munoz, R.M., del Arco, I., Villanua, M.A., Carrera, M.R., Rodriguez de Fonseca, F., 1997. Acute administration of the CB1 cannabinoid receptor antagonist SR 141716A induces anxiety-like responses in the rat. Neuroreport 20, 491–496. Navarro, M., Carrera, M.R.A., Fratta, W., Valverde, O., Cossu, G., Fattore, L., Chowen, J.A., Go´mez, R., del Arco, I., Villanua, M.A., Maldonado, R., Koob, G.F., Rodrı´guez de Fonseca, F., 2001. Functional interaction between opioid and cannabinoid receptors in drug self-administration. J. Neurosci. 21, 5344–5350. Oliva, J.M., Ortiz, S., Palomo, T., Manzanares, J., 2003. Behavioural and gene transcription alterations induced by spontaneous cannabinoid withdrawal in mice. J. Neurochem. 85, 94–104. Rodriguez de Fonseca, F., Rubio, P., Menzaghi, F., Merlo-Pich, E., Rivier, J., Koob, G.F., Navarro, M., 1996. Corticotropin-releasing factor (CRF) antagonist [D-Phe12,Nle21,38,C alpha MeLeu37]CRF attenuates the acute actions of the highly potent cannabinoid receptor agonist HU-210 on defensive-withdrawal behavior in rats. J. Pharmacol. Exp. Ther. 276, 56–64. Rodriguez de Fonseca, F., Carrera, M.R., Navarro, M., Koob, G.F., Weiss, F., 1997. Activation of corticotropin-releasing factor in the limbic system during cannabinoid withdrawal. Science 276, 2050–2054. Steiner, H., Bonner, T.I., Zimmer, A.M., Kitai, S.T., Zimmer, A., 1999. Altered gene expression in striatal projection neurons in CB1 cannabinoid receptor knockout mice. Proc. Natl. Acad. Sci. USA 96, 5786–5790. Tsou, K., Mackie, K., Sanudo-Pena, M.C., Walker, J.M., 1999. Cannabinoid CB1 receptors are localized primarily on cholecystokinin-containing GABAergic interneurons in the rat hippocampal formation. Neuroscience 93, 969–975. Valverde, O., Ledent, C., Beslot, F., Parmentier, M., Roques, B.P., 2000. Reduction of stress-induced analgesia but not of exogenous opioid effects in mice lacking CB1 receptors. Eur. J. Neurosci. 12, 533–539. Weidenfeld, J., Feldman, S., Mechoulam, R., 1994. Effect of the brain constituent anandamide, a cannabinoid receptor agonist, on the hypothalamo-pituitary-adrenal axis in the rat. Neuroendocrinology 59, 110–112. Young, S., Bonner, T., Brann, 1986. Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc. Natl. Acad. Sci. USA 83, 9827–9831. Zimmer, A., Zimmer, A.M., Hohmann, A.G., Herkenham, M., Bonner, T.I., 1999. Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice. Proc. Natl. Acad. Sci. USA 96, 5780–5785. Zuardi, A.W., Shirakawa, I., Finkelfarb, E., Karniol, I.G., 1982. Action of cannabidiol on the anxiety and other effects produced by delta 9-THC in normal subjects. Psychopharmacology (Berl) 3, 245–250.
Impaired action of anxiolytic drugs in mice deficient in cannabinoid CB1 receptors Leyre Urigu¨en a, Sandra Pe´rez-Rial a, Catherine Ledent b, Toma´s Palomo a, Jorge Manzanares a, a
Servicio de Psiquiatrı´a y Unidad de Investigacio´n, Pabello´n de Medicina Comunitaria, Hospital Universitario 12 de Octubre, Avda Co´rdoba s/n, 28041 Madrid, Spain b IRIBHN, Universite´ Libre de Bruxelles, Brussels, Belgium Received 20 June 2003; received in revised form 11 November 2003; accepted 7 January 2004
Abstract The role of cannabinoid CB1 receptors in the action of anxiolytics was examined. Deletion of CB1 receptors resulted in increased anxiety-like behaviours in light/dark box, elevated plus maze and social interaction tests. Mutant mice presented basal low corticosterone concentrations and low proopiomelanocortin gene expression in the anterior lobe of the pituitary gland compared to wildtype mice. Ten minutes of restraint stress resulted in a twofold increase in corticosterone concentrations in the plasma of mutant mice, compared to wild-type mice. Bromazepam (50 or 100 lg/kg) markedly increased the time spent in light area in wild-type animals, though both doses were without effect in mutant mice. Administration of buspirone (1 or 2 mg/kg) produced anxiolytic effects in wild-type mice. In contrast, only the highest dose of buspirone had anxiolytic results in mutant mice. Our findings reveal that CB1 receptors are involved in the regulation of emotional responses, and play a pivotal role in the action mechanism of anxiolytics. They suggest that alterations in the functional activity of the CB1 receptor may be related to the emergence of anxiety disorders, and may affect treatment with anxiolytics. # 2004 Elsevier Ltd. All rights reserved. Keywords: Cannabinoid CB1 receptor; Anxiety; Knockout mice; Anxiety-like behaviour; Benzodiazepine; Buspirone
1. Introduction The control of emotional states in the brain is subject to multiheterogenous regulation by several neurotransmitters and hormones. Increases or decreases in the functional activity of many of these neuromodulators represent the rationale underlying the molecular mechanisms of anxiolytic drugs. Thus, the discovery of neurochemical elements that alter the state of anxiety may help to identify new therapeutic targets and contribute to an understanding of differences in the response of anxiolytics in certain clinical situations. The role played by cannabinoid CB1 receptors in the action mechanism or the efficacy of anxiolytic drugs is largely unknown. However, a large body of evidence Corresponding author. Tel.: +34-91-390-8252; fax: +34-91-3908538. E-mail address: [email protected] (J. Manzanares).
0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.01.003
indicates the participation of cannabinoid receptors in the regulation of key elements affecting anxiety-like behaviours. It has been reported that acute administration of cannabinoids may cause anxiogenic responses in humans (Zuardi et al., 1982). Animal studies reveal that cannabinoids can induce both anxiolytic and anxiogenic-like responses depending on the doses and the familiarity of the environment (Rodriguez de Fonseca et al., 1996). In contrast, blockade of CB1 cannabinoid receptors with SR 141716A induced anxiety-like responses in the elevated plus maze and in the defensive withdrawal test in rats (Navarro et al., 1997). Furthermore, recent studies suggest that blockade of anandamide hydrolysis by inhibition of FAAH may result in anxiolytic action in two different validated animal models of anxiety (Kathuria et al., 2003). THC or cannabinoid receptor agonists produced pronounced alterations in the endogenous opioid system
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(Manzanares et al., 1999a; Valverde et al., 2000; Navarro et al., 2001), which has recently been proposed as being involved in the anxiolytic-like effects induced by THC (Berrendero and Maldonado, 2002). In addition, the endogenous cannabinoid system plays an important physiological role in the control of the hypothalamic-pituitary-adrenal (HPA) axis function: (1) THC or anandamide increases plasma corticotropin and corticosterone concentrations (Manzanares et al., 1999b; Weidenfeld et al., 1994), (2) THC or cannabinoid receptor agonists increase corticotropin releasing factor gene expression in the paraventricular nucleus of the hypothalamus (Corchero et al., 1999), (3) an increase in the release of CRF or in the secretion of corticosterone in the plasma occurred after spontaneous (Oliva et al., 2003) or antagonist-precipitated cannabinoid withdrawal syndrome (Rodrı´guez de Fonseca et al., 1997), and (4) administration of SR-141716A (at doses of 12.5 and 50 lg/rat, icv) produced a marked increase in the secretion of both ACTH and corticosterone (Manzanares et al., 1999b), suggesting that under basal conditions endogenous cannabinoid ligands might be tonically inhibiting the release of both hormones. Cannabinoid CB1 knockout mice represent an excellent tool for exploring the role of cannabinoid receptors in the neurobiology of anxiety and their involvement in the action mechanism of anxiolytic drugs. Recent findings supporting the role of cannabinoid CB1 receptors in the regulation of anxiety include studies showing that mutant mice exhibited an anxiogenic-like response in the light/dark box, increased anhedony in chronic unpredictable mild stress procedure (Martin et al., 2002), and reduced exploration in the elevated plus maze (Haller et al., 2002). In contrast, Marsicano et al. (2002) found no alterations in the time on open arms in the elevated maze between mutant and wild-type animals. In this study, we examined behavioural and neuroendocrinological alterations induced by deletion of cannabinoid CB1 receptors, its effect on restraint stressmediated regulation of plasma corticosterone secretion, and the differential response of anxiolytic drugs (bromazepam, buspirone) in the light/dark box test between mutant and wild-type mice. Our results led us to speculate that deletion of cannabinoid CB1 receptors produces a high state of anxiety-like behaviours, basal alterations in the function of the pituitary adrenal axis, hypersensitivity to stress and impaired action of anxiolytic drugs.
2. Materials and methods 2.1. Animals Male cannabinoid receptor (CB1) double mutant CB1 (/) mice and wild-type CB1 (+/+) littermates
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were used in all the experiments. All mice were born at the Complutense University vivarium from breeding pairs CB1 (+/). The generation of mice lacking the CB1 receptor was previously described by Ledent et al. (1999). Heterozygote mice from the sixth generation on a CD1 (Charles River, France) background were bred at our animal facilities and homozygotes from the same generation were selected for our experiments. Therefore, wild-type and CB1 mutant mice used in a given experiment originated from the same breeding series and were matched for age and weight (age ¼ 2 3 months; weight ¼ 25 35 g). Mice were maintained v at a constant temperature of 23 2 C and in a 12-h dark/light cycle (light from 7:00 to 19:00 h), with free access to food (commercial diet for rodents A04 Panlab, Barcelona, Spain) and water. Animals were housed in standard laboratory cages, each one containing groups of 8–10 individuals. All experimental procedures were carried out between 09:00 and 13:00 h. Behavioural tests were carried out under the same conditions. All the experiments in this study were performed in compliance with the Royal Decree 223/1988 of 14 March (BOE.8 18) and the Ministerial Order of 13 October 1989 (BOE 18) regarding protection of experimental animals, as well as with the European Council Directive of 24 November 1986 (86/609/EEC).
2.2. Drugs N-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro [4,5]decane-7,9-dione hydrobromide-hemicarbonate salt (buspirone hydrochloride) and 7-bromo-5-(2-pyridyl)3H-1,4-benzodiazepin-2(1H)-one (bromazepam) were obtained from Sigma–Aldrich (Madrid, Spain) and dissolved in NaCl. Drugs were administered as indicated in the figure legends.
2.3. Motor activity-open field test The open field consists of a transparent square cage 25 25 25 cm with a white plexiglas floor. The base of the cage is divided by lines into peripheral squares (12) and central squares (4). A lamp directed at the centre of the field provides illumination for the floor. Testing was conducted in a silent room with constant light. Mice were individually placed in the centre of the apparatus to initiate a 30-min test session. Each session was recorded with a video camera and analysed directly with the SMART (Spontaneous Motor Activity Recording & Tracking) v. 2.0 software system (Panlab, Barcelona, Spain) on a computer. Total, central and peripheral distance covered (m), divided into three 10-min intervals, was analysed.
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2.4. Assessment of anxiety-like behaviours
2.6. Corticosterone radioimmunoassay
2.4.1. Light/dark box The light/dark box test makes use of rodents’ natural aversion to bright areas compared with darker ones. In a two-compartment box, rodents will prefer dark areas, whereas anxiolytics should increase the time spent in the light compartment. The apparatus consisted of two metacrilate boxes (20 20 15 cm) one transparent and one black and opaque, separated by an opaque tunnel (4 cm). Light from a 60 W desk lamp placed 25 cm above the light box provided room illumination. Mice were individually tested in 5-min sessions. The floor of each box was cleaned between sessions. At the beginning of the session, mice were placed in the tunnel facing the dark box. The time spent by mice in the light area was recorded over 5-min periods. A mouse whose four paws were in the new box was considered as having changed boxes.
Blood samples were centrifuged (3000 rpm for 15 min) v and plasma was stored at 80 C. Plasma corticosterone concentrations were measured using a radioimmunoassay kit (Coat-A-Count rat/mice corticosterone, Diagnostic Product Corporation, Los Angeles, CA, USA). Lower sensitivity limit of the assay was 5.7 ng/ml. Intra-assay variability was 5.8%.
2.4.2. Elevated plus maze The elevated plus maze consisted of two open arms and two enclosed horizontal perpendicular arms 50 cm above the floor. The junction of four arms formed a central squared platform (5 5 cm). The test began with the animal being placed in the centre of the apparatus facing one of the enclosed arms and allowed to explore freely for 5 min. We considered arm entries as entry of four paws into the arm. The time spent in open arms and the number of open-arm entries were recorded. Number of entries and time spent in open arms were expressed as percentages of total entries and total test time, respectively.
2.8. In situ hybridisation histochemistry (ISHH)
2.4.3. Social interaction When two mice from separate cages are placed together in a small chamber in which neither has established territory, they engage in social interaction which includes a variety of behavioural patterns: sniffing, following, grooming, kicking, crawling under or over the partner, and touching or nearly touching their faces. On the day of the experiment, pairs of mice from different home cages were placed together in a small plastic cage (20 40 10 cm) with a cardboard lid and fresh wood litter on the floor (no change in the light level). The time that mice socially interacted was measured for 5 min. 2.5. Restraint stress procedure Unanaesthetised mice were confined in an acrylic cylindrical tube (inner size 10 3 3 cm) for 10 min. After this period of restraint stress, the animals were removed from the tubes and rapidly decapitated. Control animals remained in their home cages until decapitation.
2.7. Histology of adrenal glands Adrenal gland tissues were fixed in neutral-buffered formalin for 48 h, dehydrated in 70% ethanol, and embedded in paraffin. Tissues were sectioned at 8 lm thickness, deparaffinised, and stained with haematoxylin and eosine (H&E).
Pituitary gland sections were mounted onto gelatinv coated slides and stored at 80 C until the day of the assay. ISHH was performed as described previously (Young et al., 1986) using synthetic oligonucleotide probes complementary to proopiomelanocortin (POMC), nucleotides 96–134 (CTT CTT GCC CAC CGG CTT GCC CCA GCG CCA GCG GAA GTG CTC CAT GGA GTA GGA) of the rat POMC gene mRNA. Oligonucleotide probe was labelled using terminal deoxytransferase (Amersham, Madrid, Spain) to add 35Slabelled deoxyATP (1000 Ci mmol1; Amersham, Madrid, Spain) tail to the 30 end. The probe (in 50 ll of hybridisation buffer) was applied to each section and v left overnight at 37 C for hybridisation. Following hybridisation, sections were washed four times for 15 min each in 0.15 M NaCl, 0.015 M sodium citrate, pH v 7.2 (1X saline sodium citrate, SSC) at 55 C, followed by two 30-min washes in 1X SSC at room temperature, one brief water dip and blow-dry with air. The dried slides were apposed to Kodak BioMax MR-1 film (Amersham, Madrid, Spain) for 4 days. Autoradiograms were analysed with a PC computer using the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). Optical densities were calculated from the uncalibrated mode by subtracting from each measurement its corresponding background, and expressed in grey scale values. The background measurement was taken from an area of the slice with the lowest non-specific hybridisation signal and subtracted from the hybridisation signal measurement in the same slice. Results were presented considering mean control values as 100%.
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2.9. Statistical analyses Statistical analyses were performed using the Student t-test when comparing two groups, one-way analysis of variance followed by the Student Newman Keul test when comparing three or four groups, and repeatedmeasures ANOVA followed by the Student Newman Keul test when comparing groups at different time points. Differences were considered significant if the probability of error was less than 5%. 3. Results 3.1. Motor activity-open field
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wild-type and mutant mice were found at 10–20 and 20–30-min periods. Speed in central areas significantly increased at 1–10 and 10–20-min periods in CB1 (/) (data not shown). 3.2. Assessment of anxiety-like behaviours 3.2.1. Light/dark box test In the light/dark box test, CB1 (/) spent significantly (p < 0:001) less time in the light area (Fig. 2A) and displayed a pronounced (p < 0:001) reduction in the number of transitions between compartments (Fig. 2B) (Student t-test; t¼ 7:522, p < 0:001, 18 df, and t¼ 7:672, p < 0:001, 10 df).
To determine whether deletion of the cannabinoid CB1 receptor alters the spontaneous motor activity, we compared CB1 (+/+) and CB1 (/) mice in the open field for a period of 30 min. No differences were found between mutant and wild-type animals when considering the whole period of activity (distance) in central and peripheral areas of the open field (data not shown). However, subtle changes were found when considering this time period divided into three phases of 10 min each and separating central and peripheral areas. The results revealed that distance in peripheral areas of the open field significantly increased at 0–10min period (Fig. 1A) (RM ANOVA followed by Student Newman Keul test: F ð55; 71Þ ¼ 19:752, p¼ 0:015); no differences were found at 10–20-min or 20–30-min periods, whereas distance in central areas significantly decreased (Fig. 1B) in the first 10-min period in CB1 (/) mice compared to CB1 (+/+) mice (RM ANOVA followed by Student Newman Keul test: F ð55; 71Þ ¼ 6:297, p< 0:001); no differences between
3.2.2. Elevated plus maze In the elevated plus maze, the percentage (%) of time spent in the open arms, as well as percentage (%) of entries into the open arms, significantly decreased in CB1 (/) (Fig. 2C,D (Student t-test; t¼ 6:698, p< 0:001, 12 df, and t¼ 7:929, p< 0:001, 12 df).
Fig. 1. Assessment of spontaneous locomotor activity in CB1 (+/+) and CB1 (/) mice in the open field. Motor activity (peripheral and central activities) was measured in a 30-min session, divided into three periods of 10 min (n¼ 12). Columns represent the means and vertical lines +SEM of distance (m) in 12 mice; , values from CB1 (/) mice that are significantly different (p< 0:001, RM ANOVA in each 10-min period) from CB1 (+/+) mice; (A) peripheral and (B) central distance in the open field.
Fig. 2. Assessment of anxiety-like behaviours in CB1 (+/+) and CB1 (/) mice in the light/dark box, elevated plus maze and social interaction tests. Behaviour in each test was evaluated for a period of 5 min. Columns represent the means and vertical lines +SEM of A (time (s)), B (number of transitions), C (% time in open arms), D (% entries), E (time (s)) in 6–8 mice; , values from CB1 (/) mice (black columns) that are significantly different (p< 0:05, Student ttest) from CB1 (+/+) mice (white columns).
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3.2.3. Social interaction test In the social interaction test, the time in which mice from separate cages (unfamiliar situation) engaged in social interaction was significantly lower (p< 0:001) in CB1 (/) (Fig. 2E) (Student t-test; t¼ 7:069, p¼ 0:001, 14 df). 3.3. Evaluation of hypothalamic-pituitary-adrenal axis (HPA) function To investigate the HPA axis function in CB1 (/) and CB1 (+/+), we examined the basal concentrations of corticosterone in the plasma, basal POMC gene expression in anterior and intermediate lobes of the pituitary gland, gross morphology of adrenal gland and changes in corticosterone secretion induced by restraint stress. 3.3.1. Basal corticosterone plasma concentrations As shown in Fig. 3A, basal corticosterone concentrations in the plasma are 30% lower (p¼ 0:028) in CB1 (/) mice compared to CB1 (+/+) (Student t-test; t¼ 2:564, p¼ 0:028, 10 df). 3.3.2. POMC gene expression Similarly, a 34% lower POMC gene expression was found in the anterior but not in the intermediate lobe of the pituitary gland (Fig. 3B,C) of CB1 (/), as shown by a lower hybridisation signal in the anterior lobe (Fig. 3D). (Student t-test; t¼ 2:297, p¼ 0:039, 13 df for anterior lobe, and t¼ 1:324, ns for intermediate lobe of pituitary gland). Data in Fig. 3B are represented as percentage (%) of control. Histology of the adrenal gland revealed no gross anatomical abnormalities in CB1 (/) mice, as shown in Fig. 3E. 3.3.3. Effect of stress on corticosterone plasma concentrations Data are represented (Fig. 3F) as percentage (%) of control (non-stressed mice). Restraint stress significantly increased corticosterone concentrations in CB1 (+/+) and CB1 (/) when compared with their respective controls (non-stressed) (Student t-test; t¼ 5:188, p< 0:001, 13 df for CB1 (+/+) mice and t¼ 11:975, p< 0:001, 12 df for CB1 (/)). As shown in Fig. 3F, 10 min of restraint stress produces an approximately 200% increase in plasma corticosterone concentrations in the plasma of CB1 (+/+) mice, whereas a 400% increase was observed in CB1 (/). 3.4. Effects of anxiolytic drugs in the light/dark box test 3.4.1. Bromazepam To investigate whether the state of anxiety induced by deletion of CB1 receptors may affect the action of
Fig. 3. Evaluation of the HPA axis function under basal and restraint stress conditions. Columns represent the means and vertical lines +SEM of A (corticosterone (ng/ml)), B and C (POMC RNA levels, % of control) in anterior and intermediate lobes of pituitary, F corticosterone (% of control) in 6–8 mice; , values from CB1 (/) mice that are significantly different (p< 0:05, Student t-test) from CB1 (+/+) mice; D, representative autoradiograms for POMC mRNA in anterior (AL) and intermediate (IL) lobes of the pituitary gland of CB1 (+/+) and CB1 (/) mice; bar represents 1 mm; E, haematoxylin–eosine staining of adrenal gland in CB1 (+/+) and CB1 (/) mice; c, cortex; m, medulla; bar represents 1 mm; lower panels show 5 magnification; F, on the day of the experiment mice were removed from their home cages and immediately decapitated (non-stress), or were restrained in an acrylic tube for 10 min and then decapitated (stress); , values from stress-treated mice that are significantly different (p< 0:05, Student t-test) from non-stressed control mice.
anxiolytic drugs, we examined the effects of the anxiolytic benzodiazepine bromazepam in CB1 (/) and CB1 (+/+) mice. Doses of 50 and 100 lg/kg of bromazepam produced a considerable increase ðp< 0:001Þ in the time CB1 (+/+) spent in the light box ðF ð2; 23Þ ¼ 14:364, p< 0:001) and the number of transitions,
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Fig. 4. Behavioural assessment of anxiolytic action of bromazepam (A–D) and buspirone (E–H) in light/dark box in CB1 (+/+) and CB1 (/) animals. Mice were injected with bromazepam (50, 100 lg/kg i.p.) or buspirone (1, 2 mg/kg i.p.) or its saline vehicle, and 30 min later were exposed to light/dark box for 5 min. Columns represent the means and vertical lines +SEM of A, B, E, F (time (s)) and C, D, G, H (number of transitions) in 6–8 mice; , values from drug-treated CB1 (/) or CB1 (+/+) mice that are significantly different (ANOVA, Student Newman Keul, p< 0:05) from corresponding vehicle-treated CB1 (/) or CB1 (+/+) mice.
(F ð2; 23Þ ¼ 7:746, p< 0:005) (Fig. 4A,C), though no anxiolytic actions were observed in CB1 (/) mice at either of the doses (Fig. 4B,D). 3.4.2. Buspirone Similarly, animals were administered with buspirone (1 or 2 mg/kg i.p.) and exposed to the light/dark box test. Time in the light box significantly increased in CB1 (+/+) mice treated with buspirone (Fig. 4E) (F ð2; 22Þ ¼ 63:90, p< 0:001) and in CB1 (/) (Fig. 4F) (F ð2; 23Þ ¼ 116:673, p< 0:001). Number of transitions between light and dark box (Fig. 4G) increased significantly in CB1 (+/+) after administration of buspirone (F ð2; 23Þ ¼ 5:850, p¼ 0:010). 4. Discussion The results of this study provide clear evidence that cannabinoid CB1 receptor plays a key role in the regulation and treatment of anxiety-like behaviours. This is shown by the fact that mice lacking cannabinoid CB1
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receptors display high anxiety-like behaviours, alterations in the HPA axis under basal conditions, hypersensitivity to stress and impaired action of anxiolytic drugs such as bromazepam and buspirone. To our knowledge, three types of cannabinoid CB1 mice have been developed to date (Zimmer et al., 1999; Ledent et al., 1999; Marsicano et al., 2002), which differ in the way they were generated genetically and with regard to the strain of animals that were finally backcrossed. These differences may account for subtly different behavioural, endocrine and neurochemical responses. In addition, slight changes in certain experimental conditions (light, time of day, temperature, etc.) may significantly alter spontaneous or drug-induced behaviour. The analysis of motor activity in the open field suggests that in our specific conditions and in this type of mice, deletion of cannabinoid CB1 receptors produced altered responses (increases in peripheral areas, decreases in central areas) that seem closer to anxiety-related behaviours than to impaired motor activity. In fact, overall motor activity during the whole period of 30 min reveals no differences between mutant and wildtype mice. These results obtained in the whole period of analysis of motor activity are consistent with those reported by (Marsicano et al., 2002). In contrast, in our study we found increases in peripheral areas and decreases in central areas in the first 10-min interval, whereas Marsicano et al. (2002) did not observe differences in motor activity in 5-min interval. These subtle differences between the two studies may be related to the fact that Marsicano et al. (2002) used mice generated in a different genetic background (C57 strain) from the mice analysed in our study (sixth backcrossed generation on CD1 mice) and to the precise method for evaluating motor behaviour (open field equipped with two infrared sensor rings for measurement of horizontal and vertical locomotion); in our study we used a SMART program that measured distance travelled by the mice in peripheral and central areas of the open field. Maccarrone et al. (2002), using the same strain of mice, found differences in the locomotor activity, although these alterations are probably due to the age of the mice used (young and old mice) and to the different evaluation of motor activity. Indeed, these authors used an open field with different dimensions (70 90 60 cm) from the open field of the present study (25 25 25 cm). The size of the open field may account for the differences in the exploratory and motor behaviour between the two studies. In contrast to these studies, Steiner et al. (1999) showed that mice lacking CB1 receptors were extremely hypoactive. These discrepancies may be explained by differences in the specific method used to evaluate motor activity or the strain of mice in which the mutation was carried out. Our findings show that mice lacking cannabinoid CB1 receptors display a high level of anxiety-like beha-
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viours in three different types of experimental paradigm (light/dark box, elevated plus maze and social interaction). These data are in line with previous findings supporting the role of cannabinoid CB1 receptor function in the regulation of emotional responses (Martin et al., 2002; Haller et al., 2002). In contrast, Marsicano et al. (2002) failed to detect changes in the elevated plus maze between mutant and wild-type mice. Our results provide for the first time evidence of ‘‘generalised anxiety related-behaviours’’, since CB1 knockout mice show a high level of anxiety with different types of anxiogenic stimuli, such as light, open area and elevated position and social encounter under unfamiliar conditions. These profound anxiety-like behavioural alterations occurring in mice deficient in cannabinoid CB1 receptors are probably associated with neuroendocrine and molecular neuroadaptations in key elements that regulate anxiety. HPA axis alterations are closely related to anxietyand mood-related behaviours in animals and humans (Arborelius et al., 1999). Considering the behavioural alterations found in our study, it is possible to speculate that the absence of cannabinoid receptors may affect this neuroendocrine axis. Indeed, basal corticosterone concentrations in the plasma were lower in mutant mice compared to wild-type animals. The reduced secretion of HPA axis hormones was also detected in patients with stress-related disorders. Indeed, hypocortisolism was observed in patients with post-traumatic stress disorder, fatigue syndrome, fibromyalgia and other somatoform disorders (Heim et al., 2000). Potential mechanisms may include dysfunctions on several levels of HPA axis, in addition to genetic vulnerability and personality styles that may determine this neuroendocrine effect. In accordance with the hypocorticosteronemia found in mice lacking CB1 receptors, POMC gene expression in the anterior lobe of the pituitary gland was lower in mutant compared to wild-type mice. However, the gross morphology of the adrenal gland is not dependent upon the presence or absence of cannabinoid receptors. These functional basal differences detected in the HPA axis suggest altered responses to stressful situations in mice lacking CB1 receptors. In fact, our study reveals that restraint stress induced a greater increase in plasma corticosterone concentrations in mutant compared to wild-type animals. These results once more suggest hypersensitivity to stress in mice deficient in CB1 receptors, further supporting a homeostatic function for cannabinoid receptors in the control of anxiety- and mood-related behaviours. Although the precise mechanisms that determine lower levels of corticosterone and POMC gene expression in the anterior lobe of the pituitary and hypersensitivity to stress remain to be elucidated, it is tempting to speculate that impaired feedback regulation of relevant stress responses at the level of gluco-
corticoid receptors may contribute, at least in part, to this neuroendocrine and behavioural abnormality. If deletion of cannabinoid receptors produces neuroendocrine and molecular adaptations that are associated with strong anxiety-like behaviours, it is possible to speculate that treatment with anxiolytic drugs may produce different responses in mutant and wild-type mice. The results of our study clearly demonstrated that bromazepam, a well-known anxiolytic benzodiazepine, failed to modify anxiety-like behaviour of mutant mice. In contrast, both doses of bromazepam used in our study markedly increased the time in the light area in wild-type mice. The nature of the precise molecular alterations produced in mice deficient in cannabinoid CB1 receptors that completely eliminate the action of bromazepam remains to be elucidated. However, considering that benzodiazepines produce their anxiolytic action by acting on GABAA receptors, it is tempting to speculate that deletion of cannabinoid receptors may functionally disrupt any of the different types of a, b or c subunits most closely related to the anxiolytic actions of benzodiazepines (Lo¨w et al., 2000). Further studies on GABAA receptor autoradiography and in situ hybridisation of GABAA receptor subunits are in progress to clarify this hypothesis. Moreover, it is interesting to note that CB1 receptors are presynaptically located in GABAergic interneurons in key regions regulating anxiety (Tsou et al., 1999; Hoffman and Lupica, 2000), and activation of these cannabinoid receptors reduces GABA release from presynaptic terminals. Therefore, deletion of cannabinoid receptors probably modifies GABA neuronal activity or, alternatively, may produce functional alterations at the GABAA receptor in brain areas controlling emotional responses. On the other hand, an alternative explanation may be that in CB1 knockout mice cholecystokinin release is enhanced, since this anxiogenic peptide is co-stored with GABA in a subpopulation of GABAergic interneurons of the ‘‘emotion circuits’’, suggesting that a possible interaction between this peptide and endocannabinoids might participate in the control of anxiety. The administration of the anxiolytic buspirone increased, at both doses, the time spent in the light area in wild-type animals. In contrast, only the highest dose of buspirone produced an anxiolytic effect in mice deficient in cannabinoid CB1 receptors. Since buspirone exerts its anxiolytic activity by acting as a partial agonist at the 5-HT1A receptor, the impairment of this compound to decrease the anxiety-like behaviour of mice lacking CB1 receptors may be related to decreased function of 5-HT1A in brain areas closely related to the control of emotional responses. This hypothesis, however, remains to be clarified. In summary, the results presented here show that cannabinoid receptors play a pivotal role in the regulation
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of the neurobiology and treatment of anxiety-like behaviours. Deletion of cannabinoid receptors results in pronounced alterations in the HPA axis (reduced basal corticosterone secretion and POMC gene expression in the anterior pituitary gland and hypersensitivity to stress). Interestingly, it appears that the presence of cannabinoid receptors is necessary for bromazepam and buspirone to achieve complete anxiolytic action. Overall, these findings strongly suggest that functional alterations in cannabinoid receptors may affect the efficacy of anxiolytic drugs in the treatment of moodrelated disorders. Acknowledgements This research was supported by a grant from the ‘‘Fondo de Investigaciones Sanitarias’’ (FISS 03/0216) to J. Manzanares. Leyre Urigu¨en is a recipient of a predoctoral fellowship from the Basque Country Government. References Arborelius, L., Owens, M.J., Plotsky, P.M., Nemeroff, C.B., 1999. The role of corticotropin-releasing factor in depression and anxiety disorders. J. Endocrinol. 160, 1–12. Berrendero, F., Maldonado, R., 2002. Involvement of the opioid system in the anxiolytic-like effects induced by Delta(9)-tetrahydrocannabinol. Psychopharmacology 163, 111–117. Corchero, J., Fuentes, J.A., Manzanares, J., 1999. Chronic administration with the cannabinoid receptor agonist CP-55,940 regulates corticotropin releasing factor and proopiomelanocortin gene expression in the hypothalamus and pituitary gland of the rat. Life Sci. 64, 905–911. Haller, J., Bakos, N., Szirmay, M., Ledent, C., Freund, T.F., 2002. The effects of genetic and pharmacological blockade of the CB1 cannabinoid receptor on anxiety. Eur. J. Neurosci. 16, 1395–1398. Heim, C., Ehlert, U., Hellhammer, D.H., 2000. The potential role of hypocortisolism in the pathophysiology of stress-related bodily disorders. Psychoneuroendocrinology 25, 1–35. Hoffman, A.F., Lupica, C.R., 2000. Mechanisms of cannabinoid inhibition of GABA(A) synaptic transmission in the hippocampus. J. Neurosci. 20, 2470–2479. Kathuria, S., Gaetani, S., Fegley, D., Valin˜o, F., Duranti, A., Tontini, A., Mor, M., Tarzia, G., La Rana, G., Calignano, A., Giustino, A., Tattoli, M., Palmery, M., Cuomo, V., Piomelli, D., 2003. Modulation of anxiety through blockade of anandamide hydrolysis. Nat. Med. 9, 76–81. Ledent, C., Valverde, O., Cossu, G., Petitet, F., Aubert, J.F., Beslot, F., Bohme, G.A., Imperato, A., Pedrazzini, T., Roques, B.P., Vassart, G., Fratta, W., Parmentier, M., 1999. Unresponsiveness to cannabinoids and reduced addictive effects of opiates in CB1 receptor knockout mice. Science 283, 401–404. Low, K., Crestani, F., Keist, R., Benke, D., Brunig, I., Benson, J.A., Fritschy, J.M., Rulicke, T., Bluethmann, H., Mohler, H., Rudolph, U., 2000. Molecular and neuronal substrate for the selective attenuation of anxiety. Science 290, 131–134. Maccarrone, M., Valverde, O., Barbaccia, M.L., Castane, A., Maldonado, R., Ledent, C., Parmentier, M., Finazzi-Agro, A., 2002. Age-related changes of anandamide metabolism in CB1 cannabinoid receptor knockout mice: correlation with behaviour. Eur. J. Neurosci. 15, 1178–1186.
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Manzanares, J., Corchero, J., Romero, J., Fernandez-Ruiz, J.J., Ramos, J.A., Fuentes, J.A., 1999a. Pharmacological and biochemical interactions between opioids and cannabinoids. Trends Pharmacol. Sci. 20, 287–294. Manzanares, J., Corchero, J., Fuentes, J.A., 1999b. Opioid and cannabinoid receptor-mediated regulation of the increase in adrenocorticotropin hormone and corticosterone plasma concentrations induced by central administration of delta(9)-tetrahydrocannabinol in rats. Brain Res. 21, 173–179. Marsicano, G., Wotjak, C.T., Azad, S.C., Bisogno, T., Rammes, G., Cascio, M.G., Hermann, H., Tang, J., Hofmann, C., Zieglgansberger, W., Di Marzo, V., Lutz, B., 2002. The endogenous cannabinoid system controls extinction of aversive memories. Nature 418, 530–534. Martin, M., Ledent, C., Parmentier, M., Maldonado, R., Valverde, O., 2002. Involvement of CB1 cannabinoid receptors in emotional behaviour. Psychopharmacology 159, 379–387. Navarro, M., Hernandez, E., Munoz, R.M., del Arco, I., Villanua, M.A., Carrera, M.R., Rodriguez de Fonseca, F., 1997. Acute administration of the CB1 cannabinoid receptor antagonist SR 141716A induces anxiety-like responses in the rat. Neuroreport 20, 491–496. Navarro, M., Carrera, M.R.A., Fratta, W., Valverde, O., Cossu, G., Fattore, L., Chowen, J.A., Go´mez, R., del Arco, I., Villanua, M.A., Maldonado, R., Koob, G.F., Rodrı´guez de Fonseca, F., 2001. Functional interaction between opioid and cannabinoid receptors in drug self-administration. J. Neurosci. 21, 5344–5350. Oliva, J.M., Ortiz, S., Palomo, T., Manzanares, J., 2003. Behavioural and gene transcription alterations induced by spontaneous cannabinoid withdrawal in mice. J. Neurochem. 85, 94–104. Rodriguez de Fonseca, F., Rubio, P., Menzaghi, F., Merlo-Pich, E., Rivier, J., Koob, G.F., Navarro, M., 1996. Corticotropin-releasing factor (CRF) antagonist [D-Phe12,Nle21,38,C alpha MeLeu37]CRF attenuates the acute actions of the highly potent cannabinoid receptor agonist HU-210 on defensive-withdrawal behavior in rats. J. Pharmacol. Exp. Ther. 276, 56–64. Rodriguez de Fonseca, F., Carrera, M.R., Navarro, M., Koob, G.F., Weiss, F., 1997. Activation of corticotropin-releasing factor in the limbic system during cannabinoid withdrawal. Science 276, 2050–2054. Steiner, H., Bonner, T.I., Zimmer, A.M., Kitai, S.T., Zimmer, A., 1999. Altered gene expression in striatal projection neurons in CB1 cannabinoid receptor knockout mice. Proc. Natl. Acad. Sci. USA 96, 5786–5790. Tsou, K., Mackie, K., Sanudo-Pena, M.C., Walker, J.M., 1999. Cannabinoid CB1 receptors are localized primarily on cholecystokinin-containing GABAergic interneurons in the rat hippocampal formation. Neuroscience 93, 969–975. Valverde, O., Ledent, C., Beslot, F., Parmentier, M., Roques, B.P., 2000. Reduction of stress-induced analgesia but not of exogenous opioid effects in mice lacking CB1 receptors. Eur. J. Neurosci. 12, 533–539. Weidenfeld, J., Feldman, S., Mechoulam, R., 1994. Effect of the brain constituent anandamide, a cannabinoid receptor agonist, on the hypothalamo-pituitary-adrenal axis in the rat. Neuroendocrinology 59, 110–112. Young, S., Bonner, T., Brann, 1986. Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc. Natl. Acad. Sci. USA 83, 9827–9831. Zimmer, A., Zimmer, A.M., Hohmann, A.G., Herkenham, M., Bonner, T.I., 1999. Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice. Proc. Natl. Acad. Sci. USA 96, 5780–5785. Zuardi, A.W., Shirakawa, I., Finkelfarb, E., Karniol, I.G., 1982. Action of cannabidiol on the anxiety and other effects produced by delta 9-THC in normal subjects. Psychopharmacology (Berl) 3, 245–250.