Psychopharmacology

, Volume 182, Issue 4, pp 475–484 | Cite as

A comparison of chlordiazepoxide, bretazenil, L838,417 and zolpidem in a validated mouse Vogel conflict test

Original Investigation

Abstract

Rationale

GABAA receptors containing an α2 subunit are proposed to mediate the anxiolytic effect of benzodiazepines (BZ) based on studies in transgenic mice using unconditioned models of anxiety. Conditioned models of anxiety were not assessed and are rarely encountered in phenotyping of genetically modified animals. The novel benzodiazepine site ligand L838,417 is a partial agonist at GABAA receptors containing an α2, α3 or α5 subunit and an antagonist at α1 receptors, giving an anxiolytic profile devoid of sedation. However, this compound has not previously been assessed in mice.

Objectives

(1) Establish the Vogel conflict test (VCT) in C57BL/6J mice and validate it with a range of pharmacological tools and (2) compare the full and partial GABAA receptor positive modulators chlordiazepoxide (CDP) and bretazenil (BRZ), respectively, with the subtype selective ligands zolpidem (ZOL; α1 selective) and L838,417.

Results

(1) enhanced thirst (water deprivation or isoproterenol administration), analgesia (lamotrigine) or cognitive impairment (MK-801) did not generate false positives in the VCT; (2) CDP and BRZ engendered linear dose-related anti-conflict effects and also increased unpunished drinking; (3) L838,417 engendered a bell-shaped anti-conflict effect and did not increase unpunished drinking; (4) the anti-conflict effect of CDP and L838,417 were antagonised by flumazenil, whereas BRZ's effect was insensitive to this antagonist; and (5) ZOL induced motoric deficits and no anti-conflict effect.

Conclusion

We have established the VCT in C57BL/6J mice and validated this test behaviourally, physiologically and pharmacologically. The novel GABAA receptor ligand L838,417 was anxiolytic in this mouse model, and unlike the non-selective compounds, had no effect on unpunished drinking.

Keywords

Anxiety Benzodiazepine Bretazenil Chlordiazepoxide GABAA Isoproterenol L838,417 Mouse Vogel conflict test Zolpidem 

Abbreviations

5-HT

5-hydroxytryptamine

8-OH-DPAT

8-hydroxy-2-(di-n-propylamino)tetralin

ANOVA

Analysis of variance

BRZ

bretazenil

BZ

benzodiazepine

CDP

chlordiazepoxide

FLU

flumazenil

GABA

gamma-aminobutyric acid

i.p.

intraperitoneal

ISO

isoproterenol

mA

milliampere

NS

not significant

NMDA

Nmethyl-d-asparic acid

Veh

vehicle

VCT

Vogel conflict test

ZOL

zolpidem

Introduction

The rat Vogel conflict test (VCT) is a well-established conflict model in which thirsty animals can gain water reward through a water spout but at the expense of receiving a mild electric shock delivered to the tongue (Vogel et al. 1971). Conflict behaviour in animals has relevance to human anxiety disorders (Shekhar et al. 2001; Rodgers 1997; Treit 1985), and the VCT is argued to have characteristics relevant to generalised anxiety disorder (Millan and Brocco 2003). The VCT has played a key role in the characterisation of drugs acting on GABAergic, glutamatergic and monoaminergic neurotransmitter systems and, more recently, those acting at neuropeptide receptors (reviewed in Millan and Brocco 2003). Classical benzodiazepines reduce conflict behaviour in rats in the VCT, as measured by an increase in water intake in the presence of shock delivery (Millan and Brocco 2003; Loiseau et al. 2003; Griebel et al. 1999, 2002; Sorbera et al. 2001; Flores and Pellon 2000; Nazar et al. 1997; Brocco et al. 1990). Although similar effects of benzodiazepines have been shown in mice in the VCT, this species has not been routinely used (Liao et al. 2003; Maruyama et al. 2001; van Gaalen and Steckler 2000; Umezu 1995, 1999; Kuribara et al. 1989, 1990). However, the simplicity of the original VCT in rats and the increased use of transgenic mice in anxiety research (Anagnostopoulos et al. 2001) suggest that conditioned mouse models of anxiety are needed to give a more complete picture of the anxiety phenotype of genetically modified mice.

In the present study, we first established a mouse VCT and, secondly, pharmacologically characterised the test. As part of the first aim, we tested C57BL/6J mice at various shock intensities to determine the most appropriate shock level to suppress behavioural response indicating anxiety. Thereafter, at the shock intensity selected for the C57BL/6J strain, we assessed the level of behavioural suppression in two other strains of mice, the outbred NMRI and inbred DBA/2J, since the literature is replete with examples of strain differences in anxiety tests (Crawley et al. 1997; Griebel et al. 2000; Trullas and Skolnick 1993; Rodgers et al. 2002).

It is commonly suggested that drugs affecting primary drives (thirst, hunger, etc.), impairing memory, affecting pain sensitivity or general motor function can lead to erroneous conclusions regarding a drug effect in the VCT (Millan and Brocco 2003; Patel and Malick 1980). Since this study assessed the effects of various benzodiazepine site ligands and it has been shown that benzodiazepines increase primary drives such as thirst and hunger (Witkin et al. 2004; Cooper 1991; Soubrie et al. 1976; Maickel and Maloney 1973), we sought to determine if this was a confound in our mouse VCT. First, we compared mice that had been deprived of water for different lengths of time to induce a thirst drive. Such an approach has been used previously, for example, in the rat's conditioned emotional response test where different levels of food deprivation do not lead to an “anxiolytic profile” (Stanhope and Dourish 1996). In addition to this control study, we also tested isoproteronol, which enhances thirst by increasing levels of angiotesin II as a consequence of elevated blood rennin levels (Stocker et al. 2000; Czech and Vander Zanden 1991; Carli and Samanin 1982; Patel and Malick 1980), to determine if it was a false positive in our test. Finally, as outlined below, we also tested benzodiazepine site ligands in both the absence and presence of shock.

However, in considering the various potential confounding variables highlighted above, it should be considered that it is difficult to set up control experiments for locomotor activity, nociception, learning and memory, etc., because the conditions and the underlying motivation in the “control” situation may be different from that of the test situation (Millan and Brocco 2003). Thus, a compound may not have the same effect on motor activity in chambers utilised for the VCT compared to standard motility cages. Similarly, the effect of drugs on pain threshold may be difficult to transfer from, for example, the tail-flick test to the VCT context (Millan and Brocco 2003). For these reasons, we have not concentrated exhaustively on obviating all potential confounding variables, although some are inherently addressed by the pharmacological agents tested as well as by previous studies (Liao et al. 2003; Maruyama et al. 2001; van Gaalen and Steckler 2000; Umezu 1995, 1999; Kuribara et al. 1989, 1990).

Our primary focus has been to compare standard and novel pharmacological agents in this model. For example, we have compared the classical benzodiazepine chlordiazepoxide (CDP), a full-efficacy non-selective GABAA receptor positive modulator, with the non-selective partial agonist bretazenil (BRZ) and the subtype selective ligands zolpidem (ZOL) and L838,417 to ascertain if these compounds can be differentiated in their ability to engender an anti-conflict effect in the VCT (Vanover et al. 1999; Nazar et al. 1997; Depoortere et al. 1986). ZOL is a sedative-hypnotic which is selective for GABAA receptors containing an α1 subunit (Crestani et al. 2000), whereas L838,417 is selective for GABAA receptors containing either α2, α3 or α5 subunits relative to receptors containing an α1 subunit (McKernan et al. 2000). The selectivity profile of L838,417 is purported to give an anxiolytic profile without sedation (McKernan et al. 2000). The four GABAA receptor modulators were also tested under no-shock conditions to determine any differences in drinking behaviour per se. Moreover, we assessed the sensitivity of any anti-conflict effects observed with these benzodiazepine site ligands to the antagonist flumazenil.

In addition to compounds acting at the benzodiazepine site, we have tested the 5-hydroxytryptamine (5-HT)1A receptor agonists buspirone (partial) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the N-methyl-d-asparic acid (NMDA) antagonist MK-801 and the nicotinic receptor agonist (−)nicotine, to determine if our VCT model is sensitive to drugs shown to be anxiolytic in other test procedures (Dekeyne et al. 2000; Umezu 1999; File et al. 1998; Xie et al. 1995; Soderpalm et al. 1995b; Plaznik et al. 1994). Moreover, whether this version of the VCT is sensitive to anxiogenic agents was assessed using 1-(m-chlorophenyl)piperazine (mCPP; Kennett et al. 1998), N-methyl-β-carboline-3-carboxamide (FG-7142; Agmo et al. 1991; Uyeno et al. 1990) and yohimbine (Umezu 1999; Soderpalm et al. 1995a) as well as acute administration of the monoamine re-uptake inhibitors citalopram and duloxetine (Borsini et al. 2002). Finally, we tested the anticonvulsant lamotrigine and the voltage-gated potassium channel (Kv7) opener retigabine, which we have shown to be anxiolytic in other anxiety tests (Mirza et al. 2005; Korsgaard et al. 2005).

Materials and methods

Subjects

Male C57BL/6J Bom Tac, DBA/2J and NMRI mice (age 6–7 weeks) were used (M&B, Denmark). Animals were housed in groups of ten in cages measuring 21×38×15 cm and kept under 12:12 h dark:light cycle (lights on at 6 a.m.), with food and tap water available ad libitum except when water deprivation took place. All experiments were performed according to the guidelines of the Danish Committee for Experiments on Animals.

Vogel conflict test: apparatus and method

Habituation and testing took place during the light period of the dark–light cycle in white plastic chambers (14.5×16×16 cm), with a metal grid floor connected to a shocker (NeuroSearch technical department) delivering an electric shock to a water spout located in the center of one wall and 2.5 cm above the grid floor. Experimental contingencies and measures were controlled and recorded by an IBM computer running software developed in-house. The light level in the room was 15 lx during habituation and test sessions.

In preliminary studies, C57BL/6J mice were water-deprived for 24 h prior to being placed in the experimental chambers for 6 min and allowed to drink from the water spout (habituation session). Once the session finished mice were allowed 30 min free access to water, they were then water-deprived for another 24 h. Following this second deprivation period, mice were again placed in the chambers for 6 min for a test session, where every completion of 20 consecutive licks on the water spout was punished by a 0.14-mA shock (0.5 s) to the tongue. This suppressed drinking behaviour. In a subsequent study, naive mice were habituated to the chambers and then allocated to four treatment groups that were matched for mean±standard error of mean (SEM) number of licks. Mice showing <200 licks in the habituation session were excluded. The different groups were administered either placebo or different doses of CDP (5–20 mg/kg; see Drugs and solutions) 30 min prior to the test session, with the outcome that CDP dose-dependently increased the number of licks in the test session indicative of an anti-conflict effect (data not shown). In subsequent studies, including repeating the CDP experiment described above, the procedure was refined such that the test session took place 4 h after the habituation session, i.e. a 1-day procedure. In this 1-day procedure, animals did not get 30 min access to water between the habituation and test sessions. The response rate of animals during the habituation session varied between experiments from 694±42 and 1,461±101 and, as noted above for the 2-day procedure, animals completing <200 licks in the habituation session were excluded prior to matching groups with respect to mean±SEM number of licks.

Experiments

For experiments where drugs were used, see Drugs and solutions for doses tested, route of administration, time of administration prior to test and the vehicle used.
  1. 1.

    Influence of shock intensity. The effect of shock intensity (0.14–0.98 mA, 0.5 s) was assessed in separate groups of C57BL/6J mice using the 1-day procedure described above.

     
  2. 2.

    Influence of mouse strain. C57BL/6J, DBA/2J and NMRI mice were compared using the 1-day procedure described above at a single-shock intensity level (0.14 mA, 0.5 s). All experiments described below (experiments 3–5) were conducted in C57BL/6J mice.

     
  3. 3.

    Influence of a primary thirst drive. Since the VCT entails water deprivation, it is important to ascertain if a drug with an apparent anti-conflict effect does not increase drinking behaviour per se, i.e. a false positive. To tackle this issue, we first determined if length of water deprivation (3, 6, 24 or 48 h) would itself influence licking rate in the unpunished session. We also assessed animals deprived for these same time intervals in the punished session to ascertain if water deprivation could lead to a false-positive response per se. To conform to the Danish Committee for Experiments on Animals, mice deprived of water for 48 h had to be allowed a 30-min access to water after the initial 24 h of deprivation.

    In a second experiment, we tested various doses of isoproteronol, a dipsogenic agent that has previously been used to validate the rat VCT (Carli and Samanin 1982; Patel and Malick 1980) in groups of mice that were placed in the chambers with the shock generator either on or off. This pharmacologically induced thirst was a control experiment notably for the benzodiazepine site ligands assessed, which have been shown to enhance thirst per se (see Introduction).

     
  4. 4.
    Effect of acute drug administration
    1. (a)

      Benzodiazepine site ligands. CDP, ZOL, BRZ and L838,417 were assessed at various doses under both shock and no-shock conditions. If significant anti-conflict effects were seen, subsequent studies determined the flumazenil sensitivity of these effects.

       
    2. (b)

      Non-benzodiazepine site ligands. The 5-HT1A agonists buspirone and 8-OH-DPAT, the NMDA antagonist MK-801 and the nicotinic agonist (−)nicotine were assessed at various doses under shock conditions.

       
    3. (c)

      Anxiogenic agents. Drugs that have been shown to induce anxiogenesis in man and animals were also assessed to determine if the mouse VCT was sensitive to such effects, i.e. an anticipated further reduction in licking rate during the test session. The drugs tested for anxiogenic properties in this model were mCPP, yohimbine and FG-7142. Additionally, we tested the antidepressants duloxetine and citalopram since in the clinic acute administration of monoamine re-uptake inhibitor, antidepressants induces an anxiogenic effect.

       
    4. (d)

      Other pharmacological agents. We tested the anticonvulsant lamotrigine and the Kv7 opener retigabine at various doses under shock conditions.

       
     

Data analysis

All experiments assessing the effect of drugs were analysed by one-way ANOVA for both (a) the number of licks and (b) latency to the 20th lick measures. The comparison of different strains (factor 1) and their response rate in the presence/absence (factor 2) of a 0.14-mA shock was analysed by two-way ANOVA. Likewise, the effect of water deprivation level on licking rate was analysed by two-way repeated measures ANOVA with condition (unpunished or punished) as the between-groups factor and deprivation level (3, 6, 24 and 48 h) as the within-subject factor. Analysis of variance was followed by post hoc Dunnett's/Tukey's test or Fisher's least significant difference test where appropriate. Data are presented as mean number of licks during the test session±SEM. The latency to complete the first 20 licks on the water spout was taken as a measure of a drug effect on general sensorimotor ability.

Drugs and solutions

(+-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), N-methyl-β-carboline-3-carboxamide (FG-7142), CDP (Sigma-Aldrich), lamotrigine, buspirone (RBI), citalopram (a gift from H. Lundbeck A/S), flumazenil, duloxetine, L838,417 (synthesised in the Department of Medicinal Chemistry, NeuroSearch A/S), MK-801 and ZOL (Tocris) were dissolved in 5% cremophore (Basis Kemi) in deionised water and subjected to ultrasound for 20–30 min (Branson Sonifier 250, G. Heinemann, Untraschall- und Labortechnik, Germany). Nicotine, 1-(m-chlorophenyl)piperazine (mCPP) and yohimbine were dissolved in 0.9% saline. Retigabine (synthesised in the Department of Medicinal Chemistry, NeuroSearch A/S) was dissolved in 5% Tween 80, whereas isoproterenol (Sigma-Aldrich) was dissolved in deionised water. All compounds were administered in a volume of 10 ml/kg intraperitoneally (i.p.) 30 min prior to test with the exception of nicotine (15 min, i.p.), isoproterenol and citalopram (30 min, subcutaneously).

Results

Generally, if not otherwise stated, none of the experimental conditions or drugs had a significant effect on the latency to complete the first 20 licks.

Influence of shock intensity and mouse strain

In C57BL/6J mice, shock intensities between 0.14 and 0.98 mA significantly suppressed drinking behaviour compared to unpunished controls [F(6,58)=6.4, P<0.001; Table 1]. Since the multiple comparison test showed no difference between shock intensities in the level of behavioural suppression, the drug studies described below utilised the lowest shock intensity (0.14 mA).
Table 1

The effect of different shock intensities on number of licks in C57BL/6J mice

Shock (mA)

Lick rate (mean±SEM)

0

763±100.2

0.14

353.9±77.3*

0.38

348.0±132.3**

0.47

149.5±18.4***

0.68

146.4±20.2***

0.8

162.8±24.9***

0.98

306.4±117.3**

Data shown are mean±SEM. *P<0.05, **P<0.01 and ***P<0.001 indicate significant difference vs. unpunished controls (0 mA)

Using the 0.14-mA shock level, we compared C57BL/6J, DBA/2J and NMRI mice in the Vogel test (Fig. 1). There was no main effect of strain [F(2,42)=0.4, NS], whereas there was a main effect of shock, indicating a suppression of drinking behaviour in each of the strains [F(1,42)=30.4, P≤0.001]. There was no Strain × Shock interaction [F(2,42)=0.3, NS], suggesting that behavioural suppression was equivalent in the three strains after 0.14-mA shock and that the strains did not differ in response rate under the no-shock condition.
Fig. 1

The effect of 0.14-mA shock punishment on drinking behaviour in NMRI, C57BL/6J and DBA/2J mice. Data shown are mean±SEM. **P<0.01 and ***P<0.001 indicate significant difference vs unpunished group of same strain

Effect of a primary thirst drive

Depriving C57BL/6J mice of water for 3, 6, 24 and 48 h clearly led to a “deprivation length” dependent increase in licking behaviour notably in animals tested in the shock-off rather than the shock-on condition (Fig. 2a). There was a main effect of condition [F(1,73)=48.5, P<0.001], deprivation length [F(3,73)=24.5, P<0.001] as well as a significant interaction [F(3,73)=17.3, P<0.001], indicating that the lick rate was greater in animals deprived of water and subjected to the shock-off condition compared to animals subjected to the shock-on condition. Specifically, there was a significant difference between the two sets of animals at the 24- and 48-h deprivation levels (Fig. 2a). Moreover, the number of licks increased from ∼10 licks after 3- to 6-h deprivation to ∼1,500 after 48-h deprivation when the shock was off (Fig. 2a). Further analysis confirmed a significant increase in lick rate after 24 and 48 h of deprivation compared to 3 and 6 h (P<0.001) in animals tested in the absence of shock. By contrast, a smaller and non-significant increase in lick rate at 24- to 48-h deprivation compared to 3- to 6-h deprivation was noted in animals tested in the presence of shock (Fig. 2a). The number of licks increased from ∼25 at 3–6 h to ∼310 licks at 24–48 h in animals tested with the shock on.
Fig. 2

The effect of a different lengths of water deprivation and b different doses of isoproterenol on the lick rate under punished and unpunished conditions. Data shown are mean±SEM. (a, b) P<0.001 indicates significantly difference compared to 3/6-h deprivation, respectively. ***P<0.001 indicates significant difference compared to unpunished animals at the equivalent deprivation level (water deprivation experiment). *P<0.05 refers to significant difference compared to vehicle control group (isoproterenol experiment)

Isoproterenol was used to pharmacologically enhance thirst in a second experiment. From Fig. 2b, it is clear that isoproteronol dose-dependently enhanced unpunished drinking [F(3,108)=8.6, P<0.001], with a significant increase compared to vehicle-treated animals at 15–30 μg/kg. By contrast, over the same dose range (7.5–30 μg/kg), isoproteronol failed to increase drinking behaviour in the punished condition [F(3,34)=0.5, NS; Fig. 2b]. Clearly, this would indicate that drugs that pharmacologically enhance a primary thirst drive do not necessarily have an anti-conflict profile in the Vogel test.

Effect of benzodiazepine site ligands

Punished drinking

A significant increase in punished licking was seen after administration of CDP [F(3,102)=5.7, P=0.001], BRZ [F(4,70)=5.5, P<0.001] and L838,417 [F(3,41)=6.5, P=0.001; Fig. 3a–c]. Whilst both CDP and BRZ showed dose-dependent increases in licking response, the effect of L838,417 was bell-shaped (Fig. 3c). Post hoc analysis confirmed that 10 and 20 mg/kg CDP, 10 mg/kg BRZ and 3 mg/kg L838,417 significantly increased punished drinking compared to the vehicle-treated group. In contrast, the sedative-hypnotic ZOL dose-dependently and significantly reduced the number of punished licks [F(6,103)=19.8, P<0.001; Fig. 3d]. However, ZOL also significantly increased the latency to complete the first 20 licks [F(6,103)=37.8, P<0.001] (data not shown). This effect on latency was clearly a consequence of a motoric deficit at these doses of ZOL.
Fig. 3

ah The effect of CDP, BRZ and L838,417 on punished (ad) and unpunished (eh) drinking in the VCT. Data shown are mean±SEM. *P<0.05, **P<0.01 and ***P<0.001 indicate significant difference compared to the vehicle control group

Flumazenil sensitivity

The anti-conflict effects of CDP [F(2,83)=11.0, P<0.001] and L838,417 [F(2,34)=5.76, P=0.007] were antagonised by co-administration of 10 mg/kg flumazenil. As can be seen from Fig. 4a–c, CDP (20 mg/kg) and L838,417 (3 mg/kg) induced a significant increase in punished licking compared to the vehicle-treated group, but in the presence of flumazenil, this effect was blocked. Surprisingly, whilst BRZ (10 mg/kg) engendered an anti-conflict effect [F(2,49)=10.8, P<0.001], this effect was not blocked by co-administration of 10 mg/kg flumazenil (Fig. 4b).
Fig. 4

The effect of 10 mg/kg flumazenil on the anti-conflict effect of CDP (a), BRZ (b) and L838,417 (c). Data shown are mean±SEM. **P<0.01 and ***P<0001 indicate significant difference compared to veh./veh. control group. ++P<0.05 indicates significant difference compared to drug alone group

Unpunished drinking

In addition to its anti-conflict effect described above, CDP significantly [F(3,88)=6.8, P<0.001] increased unpunished responding (Fig. 3e). Dunnett's test confirmed that the number of licks was significantly increased after 10–20 mg/kg CDP. Figure 3f shows that an increase in unpunished licking responses was also apparent after BRZ (10 mg/kg), although this was only marginally significant [F(1,17)=4.2, P=0.06]. In contrast, L838,417 (Fig. 3g) had no effect on unpunished drinking behaviour [F(3,34)=0.8, NS]. Finally, at non-sedative doses, ZOL (0.03–0.3 mg/kg) had no effect on unpunished drinking behaviour [F(3,37)=0.01, NS; Fig. 3h].

Effect of non-benzodiazepine site ligands

The 5-HT1A partial agonist buspirone significantly reduced punished drinking [F(4,71)=2.7, P=0.039] and increased latency to complete the first 20 licks [F(4,65)=9.3, P<0.001]. The effect was significant on both measures at 3 mg/kg compared to the vehicle-treated group (Table 2). Likewise, the 5-HT1A full agonist 8-OH-DPAT dose-dependently decreased the number of punished licks [F(4,98)=6.2, P<0.001] and increased the latency to the 20th lick [F(4,98)=16.5, P<0.001]. The effect on licking rate and latency after 8-OH-DPAT was significant compared to the vehicle group at doses of 3 and 1–3 mg/kg for the two measures, respectively (Table 2).
Table 2

Pharmacological characterisation of the VCT

Compound (mg/kg)

Licks

Latency to 20th lick

Non-benzodiazepine

 Buspirone

  Veh.

303.4±38.7

40.09±4.31

  0.1

292.1±36.8

49.55±17.41

  0.3

308.5±41.9

32±5.69

  1

238.4±20.8

59.54±9.34

  5

157.5±39.9*

179.1±46.27***

 8-OH-DPAT

  Veh.

345.6±48.6

51.83±8.84

  0.1

357.3±62.6

62.17±29.08

  0.3

327.3±28.0

70.46±14.26

  1

174.9±63.4

122.48±23.47*

  3

76.4±24.2***

275.54±25.59***

 MK-801

  Veh.

452±131.24

32.3±8.93

  0.0025

309.2±63.04

49.3±12.65

  0.05

290.7±40.57

34±9.05

  0.1

295±57

53.5±8.82

 Nicotine

  Veh.

397±75.9

28.25±8.46

  0.03

445.3±99.8

32.78±7.03

  0.3

335.3±46.3

32.3±8.21

  1

246.2±45.8

38.67±12.65

Other pharmacological agents

 Lamotrigine

  Veh.

303.4±50

95.5±33.42

  3

309.2±53.5

46±8.37

  10

344.4±82.9

59.5±16.49

  30

317.5±78

92.1±33.88

 Retigabine

  Veh.

247.2±54.2

30.8±6.52

  3

333.7±46.5

27.5±6.95

  10

305.1±30.3

25.9±5.89

  30

80.9±32.7*

226.3±54.63***

“Anxiogenic” agents

 mCPP

  Veh.

276±52.6

77.78±35.99

  1

163.5±38.9

156.2±47.11

  2

191.6±46.5

139.5±48.35

  4

93.1±28.9*

172±47.53

 FG-7142

  Veh.

218.375±25.676

31.89±6.54

  3

241.444±41.798

65.2±14.14

  10

189.778±27.504

50±10.04

  30

100.111±16.32*

71.11±14.28

 Yohimbine

  Veh.

276±52.6

77.78±36

  3

427.1±55.4

34.64±5.84

  10

431.7±98.9

37.82±10.3

  30

395±61

25.9±5.05

 Citalopram

  Veh.

385.7±67.935

32.3±8.93

  3

229.111±32.522

31.6±7.1

  10

261.5±23.715

54.9±13.84

  30

222.2±21.095*

92.3±17.51**

 Duloxetine

  Veh.

397.33±111.53

64.13±16.3

  1

429.8±84.07

67.5±33.12

  3

448.1±179.13

32±6.06

  10

301.3±64.02

78±19.31

Effect of various pharmacological agents on licking rate and response latency (s). Data shown are mean±SEM

In the studies with lamotrigine and buspirone, a technical fault resulted in the loss of the latency measure for six animals in either study, although data for licking rate shown is for all animals tested

*P<0.05, **P<0.01 and ***P<0.001 indicate significant difference compared to control for each drug

Neither MK-801 [F(3,38)=1.1, NS] nor nicotine [F(3,35)=1.5, NS], at doses that have been shown to be anxiolytic in other studies, increased punished responding. Indeed, both compounds tended to dose-dependently reduce response rate and engender an increase in latency to complete the first 20 licks (Table 2).

“Anxiogenic” agents

The 5-HT2C agonist mCPP and the GABAA receptor negative modulator FG-7142 significantly decreased punished licking behaviour [mCPP F(3,37)=3.0, P=0.045; FG-7142 F(3,34)=4.5, P=0.01] with no significant effect on latency to complete 20 licks [mCPP F(3,37)=0.8, NS; FG-7142 F(3,34)=2.0, NS], suggesting an anxiogenic profile (Table 2). By contrast, the α2 adrenoceptor antagonist yohimbine had no significant effect on either the number of punished responses or latency [F(3,40)=1.0 and 1.7, respectively, NS; Table 2].

The selective serotonin re-uptake inhibitor citalopram decreased punished licking [F(3,38)=3.4, P=0.027] and increased latency to complete 20 licks [F(3,39)=5.2, P=0.005]. The effect on both measures was significant compared to the vehicle-treated group at the highest dose of 30 mg/kg (Table 2). By contrast, the antidepressant duloxetine did not affect punished responding [F(3,35)=1.6, NS] nor latency [F(3,35)=0.8, NS].

Other pharmacological agents

Lamotrigine was without effect in the mouse VCT on either the number of licks [F(3,39)=0.1, NS] or latency [F(3,33)=0.9, NS] measures. However, retigabine significantly decreased the licking rate [F(3,39)=7.2, P<0.001] at a dose (30 mg/kg) which also significantly increased latency to the 20th lick [F(3,39)=12.6, P<0.001]; see Table 2.

Discussion

In the present study, we have (1) established a 1-day mouse VCT procedure in C57BL/6J mice, (2) demonstrated that the commonly used strains C57BL/6J, DBA/2J and NMRI do not differ in the level of behavioural suppression under specific shock conditions, (3) verified that an anti-conflict effect cannot be induced by either physiologically (extended water deprivation) or pharmacologically (isoproterenol) induced enhancement of thirst, (4) shown that non-selective (CDP and BRZ) and a novel subtype selective (L838,417) GABAA receptor positive modulator has anti-conflict effects that are not necessarily secondary to effects on drinking per se and (5) characterised the model using a wide range of pharmacological tools, which suggest that analgesic (see lamotrigine data; Table 2; Blackburn-Munro and Ericksen 2004), cognition impairing or motor stimulant (see MK-801 data; Table 2; Itoh et al. 1991) effects of drugs do not necessarily induce false-positive outcomes.

The VCT was originally established in rats, and there have been few cited literature demonstrating robust data in mice (see Introduction and references therein). Here, we demonstrate that it is possible to establish the VCT in mice and, at least for the three strains assessed, we encountered no major obstacles in demonstrating behavioural suppression (Fig. 1), although we only pharmacologically characterised the C57BL/6J strain. However, a release of behavioural suppression may not necessarily be a consequence of a drug-induced anxiolytic effect. Benzodiazepines, for example, increase drinking behaviour in rats (Cooper 1982). However, our results show that increasing thirst by water deprivation (up to 48 h.) or pharmacologically by administering mice the dipsogenic agent isoproterenol did not release punished suppression of drinking (Table 2).

In this study, we show that the benzodiazepine site positive modulators CDP and BRZ have anti-conflict effects, whereas this was not seen with ZOL, a range of effects previously demonstrated in other shock-based models of anxiety (Millan and Brocco 2003; Stanhope and Dourish 1996; De Vry et al. 1993; Conti et al. 1990; Davis 1979; Boissier et al. 1968). However, doses of CDP (10–20 mg/kg) that increased punished drinking also increased unpunished drinking (Fig. 3a,e). As discussed above, an increase in unpunished drinking does not necessarily lead to an increase in punished drinking, suggesting that CDP's anti-conflict effect is probably a composite of an anxiolytic effect and an effect on a primary thirst drive (Cooper 1982). With BRZ, there was also an increase, albeit non-significant, of unpunished drinking (Fig. 3b,f). However, interestingly, L838,417 showed a clear separation between punished and unpunished drinking at the doses assessed (Fig. 3c,g). This compound is a partial agonist at GABAA receptors containing an α2, α3 or α5 subunit, but does not have any efficacy at GABAA receptors containing an α1 subunit (McKernan et al. 2000). Potentially, L838,417's lack of efficacy at GABAA receptors containing a α1 subunit and/or its partial agonist profile at select GABAA receptors may account for its lack of effect on unpunished drinking. However, the α1 selective drug ZOL did not increase unpunished drinking at the doses tested (Fig. 3h).

The anti-conflict effects of CDP and L838,417 were reversed by co-administration of the benzodiazepine antagonist flumazenil. Surprisingly, the anti-conflict effect of BRZ was not reversed with flumazenil in this study, although others have been able to demonstrate this in rats (Di Scala et al. 1992) and pigeons (Witkin et al. 1997). It has previously been shown that diazepam's anxiolytic effect on some parameters on the plus-maze are insensitive to flumazenil antagonism (Dalvi and Rodgers 1999). Moreover, Gonzalez and File 1997 found that flumazenil was unable to antagonise the anxiolytic effect of midazolam infused into the dorsal raphe nucleus in rats on the elevated plus-maze. Clearly, there are various potential explanations, not the least of the low affinity of flumazenil for the benzodiazepine site and its short half-life in combination with a very high potency ligand such as BRZ (Whitwam and Amrein 1995; Sieghart 1995) In addition, unlike CDP, BRZ has appreciable affinity and efficacy at α4 containing GABAA receptors, at which flumazenil has a positive modulatory effect (Wafford et al. 1996; Benke et al. 1997), and this may account for the flumazenil-insensitive anti-conflict effect of BRZ.

Although control levels of punished licking differed between experiments (Fig. 3), a comparison of the maximal efficacy attained with CDP (20 mg/kg, 81%), BRZ (10 mg/kg, 130%) and L838,417 (3 mg/kg, 194%), relative to their respective control groups, suggests that L838,417 had greater efficacy. However, L838,417 clearly differed from CDP and BRZ since its dose–effect profile was bell-shaped, whereas the latter two drugs had linear dose-related anti-conflict effects. The lack of anti-conflict efficacy at the highest dose of L838,417 was not a consequence of any overt signs of behavioural disruption, and the compound has no sedative effects at these doses (McKernan et al. 2000). In the elevated plus-maze and fear-potentiated startle models, L838,417 shows clear linear dose-related effects, although these studies were in rats (McKernan et al. 2000). However, we have also noted a bell-shaped dose–response relationship, across the dose range used here, in the rat's conditioned emotional response test with L838,417 (data not shown). Regardless, our data confirm the data of McKernan et al. (2000) and extend the anxiolytic profile of L838,417 to a second rodent species and another model of anxiety.

However, our data with L838,417 raise a more general issue regarding the need to assess novel drugs or transgenic animals in a range of anxiety tests. For example, recent studies using different groups of transgenic mice with point mutations in different α subunits of the GABAA receptor, rendering selective receptors insensitive to diazepam (Low et al. 2000; Rudolph et al. 1999; Crestani et al. 2001), came to the conclusion that GABAA receptors with an α2 subunit mediate the anxiolytic effects of diazepam. However, this conclusion was based on data from the light–dark and elevated-plus-maze models of anxiety, both unconditioned models of anxiety. Validating the VCT test in C57BL/6J mice, commonly used in generating transgenic animals, clearly gives an opportunity to ascertain if the role of GABAA receptors with an α2 subunit in anxiety extends to shock-based conditioned models.

In addition to the benzodiazepine site ligands discussed above, this version of the VCT appears sensitive to the anxiogenic effects of some (FG-7142 and mCPP) but not other (citalopram and yohimbine) drugs shown to induce anxiety in man (Burghardt et al. 2004; Brauer et al. 2002; Tam et al. 2001; Teloken et al. 1998). Thus, whereas FG-7142 and mCPP dose-dependently reduced punished responding without affecting latency to complete the first 20 licks, such a dissociation was not seen with citalopram, although clearly the increased latency to make the first 20 licks after the highest dose of citalopram was not due to obvious motoric deficits and could be interpreted as an anxiogenic profile. Regardless, in comparison to the selective serotonin re-uptake inhibitor citalopram, the selective serotonin nonepinephrine inhibitor duloxetine was without effect on either measure. This VCT does not appear to be sensitive to the 5-HT1A agonists 8-OH-DPAT and buspirone, the latter a marketed anxiolytic, nor nicotine or MK-801, both of which have been shown to have anxiolytic effects in some animal models of anxiety (see references in Introduction). However, this is a pharmacological profile of C57BL/6J mice, and it is possible that other strains, such as the DBA/2J and NMRI, may show a different pharmacology and indeed show sensitivity to drugs' inactivity in the C57BL/6J strain.

In conclusion, we have established a VCT in C57BL/6J mice and validated this test to some extent. We show that benzodiazepine site ligands that differ in terms of selectivity and efficacy at GABAA receptors have different profiles in this model. In particular, we show that the subtype selective compound L838,417 is active in this test without non-specifically enhancing drinking like CDP and BRZ. Finally, the data we present is of value in phenotyping of genetically modified mice since conditioned models of anxiety are rarely encountered in these assessments and the C57BL/6J mouse strain is frequently utilised as a parental strain in generating such mice (Anagnostopoulos et al. 2001; Voikar et al. 2001).

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© Springer-Verlag 2005

Authors and Affiliations

  1. 1.Department of in-vivo PharmacologyNeuroSearch A/SBallerupDenmark

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