Figures
Abstract
A previous study comparing non-emotive mice from the strain C57BL/6/ByJ with ABP/Le mice showed ABP/Le to be more anxious in an open-field situation. In the present study, several compounds affecting anxiety were assayed on ABP/Le and C57BL/6/ByJ mice using three behavioural models of anxiety: the elevated plus-maze, the light-dark discrimination test and the free exploratory paradigm. The compounds used were the full benzodiazepine receptor agonist, chlordiazepoxide, and the antagonist, flumazenil, the GABAA antagonist, bicuculline, the full 5-HT1A agonist 8-OH-DPAT, and the mixed 5-HT1A/5-HT1B agonist, RU 24969. Results showed the effect of the compounds to be dependent on both the strain and the behavioural task. Several compounds found to be anxiolytic in ABP/Le mice had an anxiogenic effect on C57BL/6/ByJ mice. More behavioural changes were observed for ABP/Le in the elevated plus-maze, but the clearest findings for C57BL/6/ByJ mice were observed in the light-dark discrimination apparatus. These data demonstrate that anxious behaviour is a complex phenomenon which cannot be described by a single behavioural task nor by the action of a single compound.
Citation: Clément Y, Le Guisquet A-M, Venault P, Chapouthier G, Belzung C (2009) Pharmacological Alterations of Anxious Behaviour in Mice Depending on Both Strain and the Behavioural Situation. PLoS ONE 4(11): e7745. https://doi.org/10.1371/journal.pone.0007745
Editor: Thomas Burne, University of Queensland, Australia
Received: April 30, 2009; Accepted: August 6, 2009; Published: November 11, 2009
Copyright: © 2009 Clément et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by CNRS France. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Anxiety is a widespread phenomenon occurring in response to various stressors. In humans, there is not one single syndrome, but several which may explain different anxiety conditions reported and could provide evidence for hypotheses on the involvement of certain biological substrates [1], [2]. Anxiety in animals is less clear, given the obvious difficulty in assessing psychological components, but it has been suggested that anxiety is not a single phenomenon. Studies of rodents have assessed anxiety using animal models of fear, e.g. the light/dark test and the elevated plus-maze paradigm to measure state anxiety, and the free exploratory test to measure trait anxiety [3]–[10].
It has been suggested that several factors, environmental and genetic, can be seen in the aetiology of anxiety, with genetic factors in both humans and animals regulating the physiological processes involved in anxiety [11], [12]. Studies have shown phenotypic differences in inbred strains of mice [13]–[19] and many loci have been associated with an increase in the behavioural expression of anxiety [11], [20]–[22]. When the genetic factors involved are located on eight or more chromosomes, the behaviour patterns are said to depend on a multigenic system and the genetic background [1], [23]. The strain ABP/Le (hereafter ABP) strain was found to be more anxious than C57BL/6/ByJ (hereafter B6) and the 4th, 7th and 9th murine chromosomes were found to be associated with anxiety [13], [14]. Since two of the three chromosomes (7th and 9th chromosomes) putatively involved in anxiogenic processes contain loci encoding for either the GABAA receptor subunits (α5, β3, γ3) or for the 5-HT1B receptor, the hypothesis of a biochemical correlate with anxiogenic behaviour patterns was tested. Binding studies were conducted and the anxious phenotype was found to be present with modifications caused by the BZ antagonist [3H]flumazenil and the 5-HT1B receptor agonist [I125]cyanolopindolol [24], [25].
Many previous studies have found clear evidence of the anxiolytic effects of GABAA-BZ receptor ligands [26]–[28]. Benzodiazepine (BZ) agonists have anxiolytic properties, whereas BZ inverse agonists have an anxiogenic effect. Studies of in vivo administration of serotoninergic (5-HT) ligands have failed to find clear evidence of either an anxiolytic or an anxiogenic effect when administered to animals, including effects dependent on the behavioural model, the dose or the 5-HT receptor subtype.
Full 5-HT1A agonists have, however, often been seen to produce anxiolytic effects in animals, yet in humans partial agonists are used to relieve anxiety [29]–[32]. Activation of the 5-HT1B receptor can also increase anxiety [33], [34]. A number of pharmacological studies have been confirmed by experimental studies using knockout animals. Decreased GABAA-receptor clustering, and inactivation of the gene coding for the 5-HT1A receptor have been seen to induce anxiety-like behaviour [35]–[39]. Observations of 5-HT1B receptor knockout mice, however, did not find any consistent modification in anxiety levels [40], [41].
The present study set out to investigate the hypothesis that the GABAA-BZ and 5-HT neurotransmission systems may be involved in an animal model of anxiety, and that genetic factors may determine differential sensitivity to specific drugs. In line with previous studies [4], [10], [42], the behavioural analysis was conducted after in vivo administration of one of a number of compounds: the full BZ agonist, chlordiazepoxide (5 mg/kg), the BZ antagonist, flumazenil (3 mg/kg), the GABAA antagonist, bicuculline (1 mg/kg), the 5-HT1A agonist 8-OH-DPAT (0.3 mg/kg) and the mixed 5-HT1A/5-HT1B agonist, RU 24969 (2.5 mg/kg). Three animal models of anxiety were used: the elevated plus-maze, the light-dark test procedure and the free exploratory paradigm. The study was designed as a pilot study to analyse two different transmitter systems (GABA-BZ and 5-HT). For ethical reasons to minimise the number of animals used, only one dose of each compound was administered. The compounds were selected for their relevance as reported in the literature.
Materials and Methods
Animals
The animals were male and female mice bred in the laboratory from two parent strains: ABP/Le (n = 151) and C57BL/6ByJ (n = 185). They were reared under standard conditions: temperature 23.5±0.5°C. A 12:12 h photoperiod with lights on at 8:00 am, tap water and Souriffarat (IM UAR) feed available ad libitum, and dust-free softwood sawdust bedding. Litters were culled to 7 subjects at birth. From birth to weaning, the animals were kept with their mothers only; the sires were removed from the mating cages one or two days before parturition. Male and female offspring were separated when weaned at 30+2 days. The animals were 10 weeks old ± 2 weeks when tested. All experiments complied with the ethical guidelines laid down by the French Ministry of Agriculture and with the European Council Directive 86/609/EEC.
Behavioural testing
The experiments were conducted in a room outside the breeding room between 13.00 h and 17.00 h. Data were recorded using a hand-held computer (Psion Organiser). An independent group of mice was used for each behavioural test. The tables give details of the number of animals in each. Mice were naive to the test apparatus.
Light-dark apparatus
Two polyvinyl chloride boxes (20×20×14 cm) covered with Plexiglas were connected via a semi-opaque plastic tunnel (5×7×10 cm). One box was dark, while the other box was lit by a 100 W desk lamp 20 cm above the box; this was the only light in the room. The subjects were individually tested in 5-min sessions. The mouse was placed in the lit box to start the test session. The parameters recorded were the time spent in the lit box, the number of transitions (i.e. the mouse crossing and placing all four paws in the opposite box) and the number of entries into the tunnel after the first entry (an entry being defined as more than 2 seconds spent in the tunnel).
Elevated plus-maze
The apparatus was a polyvinyl chloride plus-maze with two lit open arms (27×5 cm) and two closed arms (27×5×15 cm) covered with cardboard to block out the light; all four arms radiated from a central platform (5×5 cm). The apparatus was mounted on a base raising the arms to a height of 38.5 cm above the floor. To initiate the test session, the mouse was placed on the central platform, facing an open arm, and was observed for 5 min. The mouse was considered to be on the central platform whenever two paws were on it, and in one of the arms when all four paws were inside. The following behavioural variables were recorded, counting both number and duration: entries into an open arm, entry into a closed arm and unprotected head-dipping (the animal extending its head into the open, below the open arm).
Free exploratory paradigm (Hughes Box)
The apparatus consisted of a polyvinyl chloride box (30×20×20 cm) covered with Plexiglas and subdivided into six identical square exploration units, all interconnected by small doors. A temporary partition divided the apparatus in half lengthwise. Approximately 24 h before testing, each subject was placed in one half of the apparatus, with the temporary partition in place, to be familiarised with it. The floor was covered with sawdust and the animal was given unlimited access to food and water. The next day, the same mouse was exposed to both the familiar and novel environments after the temporary partition was removed, but without the animal being removed from the box. The subject was then observed under red light for 10 min. Parameters recorded were the number of episodes of avoidance behaviour in response to the novel environment (attempts), the number of units entered (locomotion), the number of rearings and the time spent in the novel side.
Drugs
Chlordiazepoxide HCl (Sigma, L'Isle D'Abeau, France) (5 mg/kg), suspended in a vehicle, was administered 30 min before testing. RU 24969 (Tocris, Illkirch, France) (2.5 mg/kg), dissolved in a vehicle, was administered 40 minutes before testing. 8-OH-DPAT (Sigma, L'Isle D'Abeau, France) (0.3 mg/kg) and flumazenil (donated by Hoffmann-La Roche, Basle, Switzerland) (3 mg/kg) were dissolved in saline and injected 20 minutes before testing. Bicuculline (Sigma, St. Louis, MO) (1 mg/kg) was dissolved in hot saline and mixed with acetic acid to produce a final concentration of approximately 0.01 M; this was done because bicuculline is unstable in physiological pH. The cooled solution was injected 10 minutes before testing. All the drugs were administered by intraperitoneal injection; the volume injected was 10 ml/kg body weight. The vehicle injection was saline with one drop of Tween 80 and one drop of acetic acid, and was injected 20 minutes before testing (i.e. the approximate harmonic mean of the intervals before behavioural testing). Each animal was given one injection, either an active drug or saline solution.
Statistical analysis
A multivariate analysis of variance was performed with “Strain”, “Treatment” and “Gender” as the main components, plus their interactions using a GLM SAS procedure followed by planned contrast comparisons. Partial comparisons were done using the adjusted means with the Least Squares Means (LSMeans) statement of GLM (SAS). Strain * Treatment interaction was also tested.
Results
Light-dark apparatus (Table 1)
A Strain effect was observed, but only for the number of entries into the tunnel, F1,98 = 8.09, p = 0.005; it also showed a Treatment effect which was significant for time spent in the lit box and the number of transitions, F5,98 = 3.29, p = 0.009; F = 4.45, p = 0.001, respectively.
The Strain * Treatment interaction was significant for the number of transitions, F5,98 = 4.51, p = 0.0001. Neither the Strain * Treatment * Gender interaction, nor the Strain * Gender or Treatment * Gender interactions were significant. As no Gender effect was observed, male and female data were pooled for the partial comparisons.
Partial comparisons after:
- Saline solution and 8-OH-DPAT treatment: no treatment or strain effect was observed.
- Chlordiazepoxide (CDZ)
The number of transitions and the number of entries into the tunnel were lower for ABP than for B6, t = 6.97, p = 0.017, t = 10.51, p = 0.005, respectively.
CDZ increased the number of entries into the tunnel for B6 but not for ABP, t = 1.97, p = 0.05.
- Bicuculline
There was no difference in behaviour between ABP and B6. Bicuculline treatment when compared to saline did, however, reduce the time spent in the lit box and the number of transitions by B6 but not by ABP: t = 3.03, p = 0.003; t = 2.94, p = 0.006, respectively. The treatment (bicuculline vs saline) reduced the number of entries into the tunnel by both strains: t = 3.63, p = 0.007; t = 2.80,p = 0.006, respectively.
- Flumazenil
No strain effect on behaviour was observed between ABP and B6, but a treatment effect (flumazenil vs saline) was observed, with both B6 and ABP spending less time in the lit box: t = 1.92, p = 0.05; t = 1.76, p = 0.08, respectively.
- RU 24969
The number of transitions and the number of entries into the tunnel were higher for ABP than for B6: t = 2.96, p = 0.009; t = 5.60, p = 0.0003, respectively.
Compared to saline treatment, RU 24969 treatment caused a decrease in time spent in the lit box but only by B6, not by ABP, while the number of transitions and the number of entries into the tunnel increased in ABP, but not in B6: t = 2.30, p = 0.02; t = 3.66, p = 0.004, respectively.
Elevated plus-maze (Table 2)
A Strain effect was observed for the time spent in the open arms: F 1.79 = 71.98, p = 0.0001.
The Treatment factor was significant for the duration head-dipping, F = 3.27, p = 0.010. A Strain * Treatment interaction was observed for head-dipping, F = 2.66, p = 0.028, respectively.
Partial comparisons after:
- Saline treatment
The number of entries into the open arms and the number of head-dippings were higher in ABP than in B6: t = 2.39, p 0.019; t = 1.98, p = 0.050 respectively.
- 8-OH-DPAT
The number of entries into the open arms was higher in ABP strain than in B6, t = 2.47, p = 0.016.
The treatment caused a decrease in head-dippings and entries in the closed arms in ABP but not in B6: t = 1.75, p = 0.08; t = 2.00, p = 0.05, respectively.
- Chlordiazepoxide
The ABP mice spent longer and recorded more entries into the open arms as well as more head-dippings than B6: t = 3.57, p = 0.0006; t = 3.31, p = 0.002; t = 2.43, p = 0.017, respectively.
CDZ caused an increase in the time spent in the open arms by ABP, but not by B6: t = 1.95, p = 0.05.
- Bicuculline
The duration and number of entries into the open arms and the duration and number of head dippings were higher in ABP than in B6: t = 3.67, p = 0.0005; t = 4.80, p = 0.0001; t = 3.59, p = 0.0006, t = 3.01, p = 0.004, respectively.
Bicuculline caused an increase in the time spent in the open arms by ABP but not by B6, t = 2.84, p = 0.006.
- Flumazenil
The time spent in the open arms, and the number and duration of head-dippings were higher in ABP than in B6: t = 3.89, p = 0.0002; t = 4.15, p = 0.0001; t = 2.10, p = 0.039, respectively.
Flumazenil caused an increase in the time spent in the open arms by ABP but not by B6: t = 2.05, p = 0.043.
- RU 24969
The time spent and the number of entries into the open arms were higher in ABP than in B6, t = 3.34, p = 0.001; t = 4.45, p = 0.000, respectively. The number and duration of head-dippings were also higher in ABP strain than in B6; t = 4.59, p = 0.0001; t = 2.05, p = 0.042, respectively.
RU 24969 caused an increase in the number of entries and duration in the open arms and the number and duration of head-dippings, but only in ABP mice: t = 2.62, p = 0.01; t = 2.75, p = 0.005; t = 2.81, p = 0.006; t = 1.86, p = 0.067, respectively.
Free Exploratory Paradigm, Hughes Box (table 3)
Three-way ANOVA showed Treatment x Strain interactions for rearing, F5,123 = 5.70, p = 0.001, and for time spent in the novel area, F5,123 = 5.46, p = 0.001. A Strain x Gender interaction was found to be significant for rearing, F1,123 = 4.66, p = 0.03. This appears to be the only Gender interaction with another factor, and was only small in magnitude; the interaction was therefore considered fortuitous and the factor was not included in the analysis. The Treatment factor was significant for locomotion, rearing and time spent in the novel area: F5,123 = 13.53, p = 0.001; F = 3.56, p = 0.005; F = 2.93, p = 0.01, respectively. A Strain effect was observed for the same parameters: F1,123 = 25.58, p = 0.001; F = 8.59, p = 0.004; F = 14.61, p = 0.001, respectively. The Gender effect reached significance for rearing and the time in the novel area: F1,123 = 7.26, p = 0.008; F = 5.84, p = 0.01, respectively. The number of attempts was not different.
Effects of Treatments
- Saline treatment
Novelty preference (time in novel side) was lower in B6 than in ABP: t = 4.28, p = 0.001.
- 8-OH-DPAT
Locomotion and rearing were lower for ABP than for B6: t = 3.11, p = 0.005; t = 3.18, p = 0.005, respectively.
No effect of 8-OH-DPAT was seen in either strain.
- Chlordiazepoxide
Rearing decreased in ABP mice compared to B6: t = 2.04, p = 0.05.
No treatment effect was detected.
- Bicuculline
The two strains recorded different times spent in the novel side, with B6 mice displaying a lower novelty preference: t = 2.85, p = 0.01.
Bicuculline, compared with controls, caused a decrease in novelty preference, but only in B6: t = 2.08, p = 0.05.
- Flumazenil
The two strains had different results: locomotion and rearing were lower in ABP than in B6: t = 2.61, p = 0.01; t = 3.85, p = 0.001, respectively.
The treatment effect was not significant.
- RU 24969
Between-strain differences were observed for all variables tested: locomotion was lower, t = 2.02, p = 0.05; time in the novel side, attempts and rearing were higher in ABP than in B6, t = 5.03, p = 0.001; t = 2.02, p = 0.05; t = 2.73, p = 0.01, respectively.
RU 24969 increased locomotion in both ABP and B6: t = 4.00, p<0.001; t = 2.98; p = 0.007, respectively. Drug treatment decreased the time spent in the novel side in B6, but not in ABP: t = 3.84, p = 0.001.
Discussion
An animal is usually considered anxious if it spends less time in the lit box of a light-dark apparatus, does less exploration of the open arms of a plus-maze apparatus and spends less time in the novel side of the free exploratory apparatus [4], [8], [15], [36], [43]. Using these three tests, recognised as models of anxiety in rodents, (tables 1, 2 & 3), and a comparative design with and without pharmacological treatment, we studied the anxiety behaviour of two inbred strains, ABP and B6. Saline treated controls showed strain-dependent differences in behaviour, most significantly in the plus-maze model and the free exploratory test. In the plus-maze, ABP mice recorded more entries into the open arms and more head-dippings (table 2), more rearing and grooming (data not included). In the free exploratory test, ABP mice spent more time in the novel side, suggesting they are less anxious (table 3). The behaviour cannot be linked to any difference in the level of locomotion, as not only were there no strain-related differences for entries into the closed arms, but in the free exploratory test, the general activity of B6 mice was higher than ABP mice.
It must be noted that these results do not appear to tally with previous findings obtained in open-field testing, where ABP mice were found to be more active than B6 [25]. Differences in experimental situations may account for this discrepancy and clear out a complex interaction between genetic and environmental factors [44], [45].
The pharmacological action of selected compounds was reported using the same 3 tests and 2 strains, showing, for example, that the effects on ABP mice in the elevated plus-maze were always anxiolytic, and that the effects on B6 mice in the light/dark apparatus were always anxiogenic.
Two comments can be made at this point. First, it could be argued that the ABP strain may be a better murine model for studying the anxiolytic effects of drugs, while the B6 strain would be better suited to uncovering anxiogenic effects. But some compounds were also seen to have anxiolytic effects on B6 mice [46]–[49]. Secondly, it could be argued that when experimenting with mice, the light/dark choice test may be more relevant for detecting anxiogenic effects, while the plus-maze may be better suited to measuring anxiolytic effects, even though anxiolytic effects have been clearly observed using the same version of the light/dark apparatus ([42], [50]–[52] 52 Griebel et al., 1992), and anxiogenic actions have been observed in the elevated plus-maze [53]–[56].
The administration of chlordiazepoxide (benzodiazepine receptor agonist) induced anxiolytic effects in ABP mice tested in the elevated plus-maze, confirming the anti-anxiety effects of benzodiazepine agonists which have been extensively reported. However, the same effect was not observed in ABP mice in the light/dark test, providing further evidence for the argument that the two tests assess different behaviour patterns. This is not a new discovery; diazepam has been shown to produce anxiolytic effects in Swiss, BALB/c and C3HeOuJIco mice in either the elevated plus-maze test or the light/dark choice test; yet the same compound, at the same dose, produced anxiolytic effects in C57BL/6JIco, DBA/2JIco, NMRI and NZB/Ola/Hsd mice in the elevated plus-maze, but not in the light/dark choice test [47], [57].
Flumazenil (benzodiazepine receptor antagonist) is usually described as devoid of intrinsic action in rodent models of anxiety, such as conditioned conflict paradigms [58], [59], the elevated plus-maze [60], [61], the light/dark choice test [62], the staircase [62], [63], the burying test [64] and ultrasonic vocalisations [65]. Yet flumazenil has also been described as an anxiogenic agent for testing in the elevated plus-maze [66], [67], the social interaction model [68], [69] and the mouse defence test battery [58], [70]; it has been shown to produce an inverse agonist-like promnesic effect in a learning task [71]. In some cases it has even been described as agonistic [72], [73]; e.g. rats trained to discriminate clorazepate from saline extend the cue to include flumazenil [74]. In some situations, flumazenil has been seen to induce agonist or inverse agonist-like effects, depending on the level of threat or stress [75]–[77], and on the strain used [47]. In the present study, the benzodiazepine antagonist was anxiolytic in ABP mice in the elevated plus-maze but had an anxiogenic effect on the B6 mice in the light/dark apparatus. As pharmacological reactions were induced in both strains, it is difficult to implicate pharmacokinetic differences in any explanation of behavioural differences. The treatment*strain interaction confirms that the effects of flumazenil depend upon environmental and genetic factors.
The behavioural effects of the GABAA receptor antagonist, bicuculline, are also strain-dependent. Bicuculline induced anxiety in B6 mice in both the free exploratory test (a decrease in the time spent in the novel environment) and the light/dark test, but induced an anxiolytic effect on ABP mice in the elevated plus-maze. As ABP and B6 mice were both sensitive to bicuculline, the differences observed should not be related to pharmacokinetic differences. The anxiogenic effects of the GABAA receptor antagonist are not surprising given that benzodiazepines are believed to produce their anxiolytic effect by increasing GABAergic neurotransmission. Another experiment obtained similar results using very high doses (up to 8 mg/kg) [78]. But no consistent evidence of an anxiogenic profile has been found [79] and it has been suggested that when anxiogenesis is observed after bicuculline administration, it may be attributed to behavioural suppression rather than to any effect on anxiety; for example, a dose of 1 mg/kg produced behavioural suppression in Swiss mice in the free exploratory test [80]. This was not found in the present study; in the free exploratory test, bicuculline had no effect on locomotion or rearing in either strain. The anxiolytic effect of the GABAA receptor antagonist on the ABP strain is surprising and may be related to dysfunction of the GABAA receptor.
In a previous study [25], [3H]flumazenil binding in brain homogenates of ABP and B6 mice was measured after exposure to a novel situation. Scatchard analysis showed greater affinity in the BZR binding sites of the ABP strain. Kd changes may indicate that certain animals considered as “more anxious” have fast adaptive cellular mechanisms, causing an increase in BZR affinity in response to novelty-induced stress. The different behaviour patterns observed in the present report after administering flumazenil or bicuculline may be explained by differential qualitative changes in both strains (i.e. changes in the molecular stoechiometry of the GABAA receptors) or by a rapid post-transcriptional regulatory mechanism, such as phosphorylation of the receptor protein. The possibility, however, that different allelic forms encoding for GABAA receptors may be correlated with the pharmacological profiles observed cannot be excluded. The ABP linkage-testing strain is interesting as it contains a genetic marker (pink-eyed dilution, 7th chromosome) close to loci encoding for the α5 and β3 GABAA protein receptor [81]. Many mRNAs encoding for these proteins are found in the cortex (α5 and β3) and in the hippocampus (mainly α5). As these loci co-segregate in intercrossed F2 Mendelian populations (easily identifiable animals) and since the α5 subunit has been associated with the pharmacological effects of benzodiazepines [82], further pharmacological experiments on these populations (p/p F2), using specific and high affinity ligands for these receptors, could clarify the putative role of these GABA and BZ binding sites in anxiety.
Administration of 8-OH-DPAT (full 5-HT1A agonist) had no effect in the light-dark apparatus and there was no strain difference in drug sensitivity, confirming previous data [24], [83]. In the plus-maze and the free exploratory paradigm we observed some minor effects which also corroborated the findings of previous studies [4], [43], [61].
RU 24969, a mixed 5-HT1A/5-HT1B agonist, produced anxiolytic effects in ABP mice in the elevated plus-maze, while it produced anxiogenic effects in B6 mice in the elevated plus-maze and the free exploratory test. A review of the literature shows RU 24969 to have either anxiolytic or anxiogenic effects, depending on the behavioural test used; it is usually anxiogenic in the elevated plus-maze test and has been reported as being anxiolytic in a modified Vogel test and in the four plate test [84]. The interaction observed in the present study is therefore not surprising and contributes new data. The administration of RU 24969 stimulated locomotion in the free exploratory test in both strains, but RU 24969 produced opposite effects on anxiety in the two strains, suggesting that while the drug affects locomotion, it may not affect the expression of anxiety. Assuming that the effects of RU 24969 on locomotion can be linked to 5-HT1B within the striatum, it may be that the two strains differ in their expression of 5-HT1B in other brain areas, e.g. the limbic system. The 5-HT1B receptors are mainly found in extrapyramidal neural pathways, and these are mainly presynaptic terminal autoreceptors which inhibit the release of 5-HT in the cortex and substantia nigra. The 5-HT1B receptors are also heteroreceptors and modulate the release of other neurotransmitters; for example, 5-HT inhibits ACh release in the hippocampus. In the globus pallidus and the substantia nigra, GABA release is inhibited by 5-HT1B activation [57], [85]. In a previous study, quantitative autoradiography [I125] was used to measure cyanolopindolol binding sites in different areas of the brain in ABP and B6 mice [24]. An increase was observed in the density of 5-HT1B in the globus pallidus and substantia nigra of the more “anxious” and more active ABP mice, confirming the involvement of striatal 5-HT1B receptors in locomotion. Unfortunately no data are available on binding sites in the limbic system of the mice. The ABP strain also has a genetic marker which is close to a locus encoding for the 5-HT1B subtype. The short-ear (se) locus (9th chromosome), expresses itself in an easily identifiable phenotype [86]. It is thus possible to make segregating populations homozygous for the se gene, and consequently cosegregate for the 5-HT1B gene. This population can be used for measuring differential mRNA 5-HT1B and/or 5-HT1B protein expression in areas of the limbic system such as the hippocampus. It may then be assumed that the 5-HT1B gene could be mutated in the ABP strain and correlated with a differential pharmacological pattern.
The present data challenge the conventional view that the anxiolytic effect of benzodiazepines is the same regardless of the behavioural situation, although this may still be the case for certain specific strains of mice. When applied to another mouse strain, as evidenced the present study, several compounds known to be anxiolytic, displayed a clearly anxiogenic profile. Furthermore, the anxious phenotype also depends on characteristics of the behaviour test used. Finally, more data with more than one dose are indeed necessary before concluding in anxiogenic or anxiolytic effect of such a ligand, mainly because interaction between Strain X Environment X Treatment is complex.
Acknowledgments
We wish to thank Raymond Jegat who built the testing devices, Thierry Papin who bred the mice and Shan Benson for improving the English text.
Author Contributions
Conceived and designed the experiments: YC CB. Performed the experiments: YC AMLG. Analyzed the data: YC CB. Wrote the paper: YC CB. Edited the manuscript: PV. Critical input on all versions of the manuscript: GC.
References
- 1. Clément Y, Chapouthier G (1998) Biological bases of anxiety. Neurosci Biobehav Rev 22: 623–633.
- 2.
Diagnostic and statistical manual of mental disorders, 4th edition. 886 p. DSM IV. (1994) American Psychiatric Association. Washington.
- 3.
Belzung C (1999) Rodent exploratory behavior. In: Crusio WE, Gerlai R, editors. Molecular genetic techniques for behavioral neuroscience. Elsevier: Amsterdam. pp. 738–749.
- 4. Belzung C, Berton F (1997) Further pharmacological validation of the BALB/c neophobia in the free exploratory paradigm as an animal model of trait anxiety. Behav Pharmacol 8: 541–548.
- 5. Belzung C, Griebel G (2001) Measuring normal and pathological anxiety-like behaviour in mice: a review. Behav Brain Res 125: 141–149.
- 6. Beuzen A, Belzung C (1995) Link between emotional memory and anxiety states: a study by principal component analysis. Physiol Behav 58: 111–118.
- 7. Bourin M, Petit-Demoulière B, Dhonnchadha BN, Hascöet M (2007) Animal models of anxiety in mice. Fundam Clin Pharmacol 21: 567–574.
- 8. Carobrez AP, Bertoglio LJ (2005) Ethological and temporal analyses of anxiety-like behavior: the elevated plus-maze model 20 years on. Neurosci Biobehav Rev 29: 1193–1205.
- 9. Carola V, D'Olimpio F, Brunamonti E, Mangia F Renzi P (2002) Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behaviour in inbred mice. Behav Brain Res 134: 49–57.
- 10. Griebel G, Belzung C, Misslin R, Vogel E (1993) The free-exploratory paradigm: an effective method for measuring neophobic behaviour in mice and testing potential neophobia-reducing drugs. Behav Pharmacol 4: 637–644.
- 11. Gershenfeld HK, Paul SM (1998) Towards a genetics of anxious temperament: from mice to men. Acta Psychiatr Scand suppl 393: 56–65.
- 12. Lau JY, Gregory AM, Goldwin MA, Pine DS, Eley TC (2007) Assessing gene-environment interactions on anxiety symptom subtypes across childhood and adolescence. Dev Psychopathol 19: 1129–1146.
- 13. Clément Y, Adelbrecht C, Martin B, Chapouthier G (1994) Association of autosomal loci with the grooming activity in mice observed in open-field. Life Sci 55: 1725–1734.
- 14. Clément Y, Martin B, Venault P, Chapouthier G (1995) Involvement of regions of the 4th and 7th chromosomes in the open-field activity of mice. Behav Brain Res 70: 51–57.
- 15. Clément Y, Joubert C, Kopp C, Lepicard EM, Venault P, et al. (2007) Anxiety in mice: a principal component analysis study. Neural Plast 35457.
- 16. Mathis C, Neumann PE, Gershenfeld H, Paul SM, Crawley JN (1995) Genetic analysis of anxiety-related behaviors and responses to benzodiazepine-related drugs in AXB and BXA recombinant inbred mouse strains. Behav Genet 25: 557–568.
- 17. Rodgers RJ, Davies B, Shore R (2002) Absence of anxiolytic response to chlordiazepoxide in two common background strains exposed to the elevated plus-maze: importance and implications of behavioural baseline. Genes Brain Behav 1: 242–251.
- 18. Vadasz C, Kobor G, Lajtha A (1992) Motor activity and the mesotelencephalic dopamine function. I. High resolution temporal and genetic analysis of open-field behavior. Behav Brain Res 48: 29–39.
- 19. Van Gaalen MM, Steckler T (2000) Behavioural analysis of four mouse strains in an anxiety test battery. Behav Brain Res 115: 95–106.
- 20. Flint J, Corley R, DeFries JC, Fulker DW, Gray JA, et al. (1995) A simple genetic basis for a complex psychological trait in laboratory mice. Science 269: 1432–1435.
- 21. Talbot CJ, Nicod A, Cherny SS, Fulker DW, Collins AC, et al. (1999) High-resolution mapping of quantitative trait loci in outbred mice. Nat Genet 21: 305–308.
- 22. Wise J (1996) Gene may code for anxiety. Brit Med J 313: 1353.
- 23. Flint J, Goodwin G (1999) Psychiatric genetics: a genetic basis for health? Curr Biol 9: R326–328.
- 24. Clément Y, Kia KH, Daval G, Vergé D (1996) An autoradiographic study of serotonergic receptors in a murine genetic model of anxiety-related behaviors. Brain Res 709: 229–242.
- 25. Clément Y, Proeschel MF, Bondoux D, Girard F, Launay JM, et al. (1997) Genetic factors regulate processes related to anxiety in mice. Brain Res 752: 127–135.
- 26. Möhler H (2006) GABAA receptors in central nervous system disease: anxiety, epilepsy, and insomnia. J Recept Signal Transduct Res 26: 731–740.
- 27. Nutt D (2006) GABAA receptors: subtypes, regional distribution, and function. J Clin Sleep Med 2: S7–11.
- 28. Rupprecht R, Eser D, Zwanzger P, Möller HJ (2006) GABAA receptors as targets for novel anxiolytic drugs. World J Biol Psychiatry 7: 231–237.
- 29. Hoyer D, Clarke DE, Fozard Hartig PR JR, Martin GR, et al. (1994) VII. International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin). Pharmacol Rev 46: 157–203.
- 30. Menard J, Treit D (1999) Effects of centrally administered anxiolytic compounds in animal models of anxiety. Neurosci Biobehav Rev 23: 591–613.
- 31. Peroutka SJ (1994) Molecular biology of serotonin (5-HT) receptors. Synapse 18: 24–260.
- 32. Söderpalm B, Lundin B, Hjorth S (1993) Sustained 5-hydroxytryptamine release-inhibitory and anxiolytic-like action of the partial 5-HT1A receptor agonist, buspirone, after prolonged chronic administration. Eur J Pharmacol 239: 69–73.
- 33. Morelli N, Gori S, Choub A, Maluccio MR, Orlandi G, et al. (2002) Do 5HT1B/1D receptor agonists have an effect on mood and anxiety disorders? Cephalalgia 27: 471–472.
- 34.
Wilkinson LO, Dourish CT (1991) Serotonin and animal behaviour. In: Peroutka SJ, editor. Serotonin receptor subtypes, basic and clinical aspects. New York: pp. 147–210.
- 35. Bruening S, Oh E, Hetzenauer A, Escobar-Alvarez S, Westphalen RI, et al. (2006) The anxiety-like phenotype of 5-HT receptor null mice is associated with genetic background-specific perturbations in the prefrontal cortex GABA-glutamate system. J Neurochem 99: 892–899.
- 36. Crawley J, Goodwin FK (1980) Preliminary report of a single animal behavior model for the anxiolytic effects of benzodiazepines. Pharmacol Biochem Behav 13: 167–170.
- 37. Parks CL, Robinson PS, Sibille E, Shenk T, Toth M (1998) Increased anxiety of mice lacking the serotonin 1A receptor. Proc Natl Acad Sci USA 95: 10734–10739.
- 38. Ramboz S, Oosting R, Amara DA, Kung HF, Blier P, et al. (1998) Serotonin receptor 1A knockout: an animal model of anxiety-related disorder. Proc Natl Acad Sci USA 95: 14476–14481.
- 39. Rudolph U, Crestani F, Benke D, Brünig I, Benson JA, et al. (1999) Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes. Nature 401: 796–80040.
- 40. Lesch KP (2005) Serotonergic gene inactivation in mice: models for anxiety and aggression? Novartis Found Symp 268: 111–140.
- 41. Ramboz S, Saudou F, Amara DA, Belzung C, Segu L, et al. (1996) 5-HT1B receptor knock out - behavioral consequences. Behav Brain Res 73: 305–302.
- 42. Belzung C, Misslin R, Vogel E, Dodd RH, Chapouthier G (1987) Anxiogenic effects of methyl-beta-carboline-3-carboxylate in a light/dark choice situation. Pharmacol Biochem Behav 28: 29–33.
- 43. Lister RG (1987) The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology 92: 180–185.
- 44. Belzung C (2001) The genetic basis of the pharmacological effects of anxiolytics: a review based on rodent models. Behav Pharmacol 12: 451–460.
- 45. Crabbe JC, Wahlsten D, Dubek BC (1999) Genetics of mouse behavior: interactions with laboratory environment. Science 284: 1670–1672.
- 46. Ågmo A, Belzung C, Deloire X, Grassin M, Lewis S (1999) Blockade of anxiolytic-like actions of chlordiazepoxide by naloxone in the elevated plus-maze: Comparisons between SWISS, C57BL/6 and BALB/c mice. Psychobiology 27: 105–113.
- 47. Belzung C, Le Guisquet AM, Crestani F (2000) Flumazenil induces benzodiazepine partial agonist-like effects in BALB/c but not C57BL/6 mice. Psychopharmacology 148: 24–32.
- 48. Belzung C, Dubreuil D (1998) Naloxone potentiates the anxiolytic but not the amnesic action of chlordiazepoxide in C57BL/6 mice. Behav Pharmacol 9: 691–698.
- 49. Lepicard EM, Joubert C, Hagneau I, Perez-Diaz F, Chapouthier G (2000) Differences in anxiety-related behavior and response to diazepam in BALB/cByJ and C57BL/6J strains of mice. Pharmacol Biochem Behav 67: 739–748.
- 50. Belzung C, Ågmo A (1997) Naloxone blocks anxiolytic-like effects of benzodiazepines in Swiss but not in Balb/c mice. Psychopharmacology 132: 195–201.
- 51. Bourin M, Hascoët M (2003) The mouse light/dark box test. Eur J Pharmacol 463: 55–65.
- 52. Griebel G, Misslin R, Pawlowski M, Guardiola-Lemaitre B, Guillaumet G, et al. (1992) Anxiolytic-like effects of a selective 5-HT1A agonist; S20244, and its enantiomers in mice. NeuroReport 3: 84–86.
- 53. Dolu N (2007) Dose-related anxiogenic effect of glycine in the elevated plus maze and in electrodermal activity. J Basic Clin Physiol Pharmacol 18: 141–147.
- 54. Florio C, Prezioso A, Papaioannou A, Vertua R (1998) Adenosine A1 receptors modulate anxiety in CD1 mice. Psychopharmacology 136: 311–319.
- 55. Rodgers RJ, Cole JC, Aboualfa K, Stephenson LH (1995) Ethopharmacological analysis of the effects of putative ‘anxiogenic’ agents in the mouse elevated plus-maze. Pharmacol Biochem Behav 52: 805–813.
- 56. Teixeira RM, Santos AR, Ribeiro SJ, Calixto JB, Rae GA, et al. (1996) Effects of central administration of tachykinin receptor agonists and antagonists on plus-maze behavior in mice. Eur J Pharmacol 311: 7–14.
- 57. Griebel G, Belzung C, Perrault G, Sanger DJ (2000) Differences in anxiety-related behaviours and in sensitivity to diazepam in inbred and outbred strains of mice. Psychopharmacology 148: 164–170.
- 58. Blanchard RJ, Griebel G, Henrie JA, Blanchard DC (1997) Differentiation of anxiolytic and panicolytic drugs by effects on rat and mouse defense test batteries. Neurosci Biobehav Rev 21: 783–789.
- 59. Söderpalm B, Engel JA (1990) Serotonergic involvement in conflict behaviour. Eur Neuropsychopharmacol 1: 7–13.
- 60. Chopin P, Briley M (1993) The benzodiazepine antagonist flumazenil blocks the effects of CCK receptor agonists and antagonists in the elevated plus-maze. Psychopharmacology 110: 409–414.
- 61. Pellow S, File SE (1986) Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated plus-maze: a novel test for anxiety in the rat. Pharmacol Biochem Behav 24: 525–529.
- 62. Belzung C, Vogel E, Misslin R (1988) Benzodiazepine antagonist RO 15-1788 partly reverses some anxiolytic effects of ethanol in the mouse. Psychopharmacology 95: 516–519.
- 63. Emmanouil DE, Quock RM (1990) Effects of benzodiazepine agonist, inverse agonist and antagonist drugs in the mouse staircase test. Psychopharmacology 102: 95–97.
- 64. Fernández-Guasti A, Picazo O (1995) Flumazenil blocks the anxiolytic action of allopregnanolone. Eur J Pharmacol 281: 113–115.
- 65. Gardner CR, Budhram P (1987) Effects of agents which interact with central benzodiazepine binding sites on stress-induced ultrasounds in rat pups. Eur J Pharmacol 134: 275–283.
- 66. Pokk P, Zharkovsky A (1997) The effects of flumazenil, RO 15-4513 and beta-CCM on the behaviour of control and stressed mice in the plus-maze test. J Physiol Pharmacol 48: 253–261.
- 67. Lee C, Rodgers RJ (1991) Effects of benzodiazepine receptor antagonist, flumazenil, on antinociceptive and behavioural responses to the elevated plus-maze in mice. Neuropharmacology 30: 1263–1267.
- 68. File SE, Lister RG (1983) Interactions of ethyl-beta-carboline-3-carboxylate and Ro 15–1788 with CGS 8216 in an animal model of anxiety. Neurosci Lett 39: 91–94.
- 69. File SE, Pellow S, Jensen LH (1986) Actions of the beta-carboline ZK 93426 in an animal test of anxiety and the holeboard: interactions with RO 15–1788. J Neural Transm 65: 103–114.
- 70. Griebel G, Blanchard DC, Blanchard RJ (1996) Predator-elicited flight responses in Swiss-Webster mice: an experimental model of panic attacks. Prog Neuropsychopharmacol Biol Psychiatry 20: 185–205.
- 71. Raffali-Sébille MJ, Chapouthier G (1991) Flumazenil (Ro 15–1788) enhances learning in both negatively and positively reinforced tasks. Drug Dev Res 23: 153–157.
- 72. Grecksch G, Prado de Carvalho L, Venault P, Chapouthier G, Rossier J (1983) Convulsions induced by submaximal dose of pentylenetetrazol in mice are antagonized by the benzodiazepine antagonist Ro 15–1788. Life Sci 32: 2579–2584.
- 73. Prado de Carvalho LP, Grecksch G, Chapouthier G, Rossier J (1983) Anxiogenic and non-anxiogenic benzodiazepine antagonists. Nature 301(5895): 64–66.
- 74. Dantzer R, Perio A (1982) Behavioural evidence for partial agonist properties of RO 15–1788, a benzodiazepine receptor antagonist. Eur J Pharmacol 81: 655–658.
- 75. Ferré P, Fernández-Terruel A, Escorihuela RM, García E, Zapata A, et al. (1994) Struggling and flumazenil effects in the swimming test are related to the level of anxiety in mice. Neuropsychobiology 29: 23–27.
- 76. File SE, Hitchcott PK (1990) A theory of benzodiazepine dependence that can explain whether flumazenil will enhance or reverse the phenomena. Psychopharmacology 101: 525–532.
- 77. Griebel G (1995) 5-Hydroxytryptamine-interacting drugs in animal models of anxiety disorders: more than 30 years of research. Pharmacol Ther 65: 319–395.
- 78. Dalvi A, Rodgers RJ (1996) GABAergic influences on plus-maze behaviour in mice. Psychopharmacology 128: 380–397.
- 79. Ågmo A, Pruneda R, Guzmán M, Guttiérrez M (1991) GABAergic drugs and conflict behavior in the rat: lack of similarities with the actions of benzodiazepines. Naunyn Schmiedebergs Arch Pharmacol 344: 314–322.
- 80. Ågmo A, Belzung C (1998) Interactions between dopamine and GABA in the control of ambulatory activity and neophobia in the mouse. Pharmacol Biochem Behav 59: 239–247.
- 81. Brilliant MH, Williams RW, Conti CJ, Angel JM, Oakey RJ, et al. (1994) Mouse chromosome 7. Mamm Genome 5: S104–S123.
- 82. Möhler H, Lusher B, Fritschy JM, Benke D, Benson J, et al. (1998) GABA(A)-receptor assembly in vivo: lessons from subunit mutant mice. Life Sci 62: 1611–1615.
- 83. Sánchez C (1996) 5-HT(1A) receptors play an important role in modulation of behavior of rats in a two-compartment black and white box. Behav Pharmacol 7: 788–797.
- 84. Griebel G, Blanchard DC, Jung A, Blanchard RJ (1995) A model of “antipredator” defense in Swiss-Webster mice: effects of benzodiazepine receptor ligands with different intrinsic activities. Behav Pharmacol 6: 732–745.
- 85. Pazos A, Palacios JM (1985) Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-1 receptors. Brain Res 346: 205–230.
- 86.
Lyon MF (1989) Rules and guidelines for gene nomenclature. In: Lyon MF, Searle A, editors. Genetic variants and strains of the laboratory Mouse. pp. 1–11. Gustav Fischer Verlag, Stuttgart.