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Subtype-selective GABAergic drugs facilitate extinction of mouse operant behaviour
Neuropharmacology 46 (2004) 171–178 www.elsevier.com/locate/neuropharm
Subtype-selective GABAergic drugs facilitate extinction of mouse operant behaviour Ciara McCabe a, David Shaw a, John R. Atack b, Leslie J. Street b, Keith A. Wafford b, Gerard R. Dawson b, David S. Reynolds b, Julian C. Leslie a,∗ a
School of Psychology, University of Ulster, Shore Road, Newtownabbey BT37 0QB, Northern Ireland UK b Merck Sharp & Dohme Research Laboratories, Terlings Park, Harlow, Essex CM20 2QR, UK Received 7 July 2003; received in revised form 15 August 2003; accepted 3 September 2003
Abstract Several recent studies have shown that reducing g-aminobutyric acid (GABA)-mediated neurotransmission retards extinction of aversive conditioning. However, relatively little is known about the effect of GABA on extinction of appetitively motivated tasks. We examined the effect of chlordiazepoxide (CDP), a classical benzodiazepine (BZ) and two novel subtype-selective BZs when administered to male C57Bl/6 mice during extinction following training on a discrete-trial fixed-ratio 5 (FR5) food reinforced leverpress procedure. Initially CDP had no effect, but after several extinction sessions CDP significantly facilitated extinction, i.e. slowed responding, compared with vehicle-treated mice. This effect was not due to drug accumulation because mice switched from vehicle treatment to CDP late in extinction showed facilitation immediately. Likewise, this effect could not be attributed to sedation because the dose of CDP used (15 mg/kg i.p.) did not suppress locomotor activity. The two novel subtype-selective BZ partial agonists, L838,417 and TP13, selectively facilitated extinction in similar fashion to CDP. The non-GABAergic anxiolytic buspirone was also tested and found to have similar effects when administered at a non-sedating dose. These studies demonstrate that GABA-mediated processes are important during extinction of an appetitively motivated task, but only after the animals have experienced several extinction sessions. 2003 Elsevier Ltd. All rights reserved. Keywords: Mouse; Operant behaviour; Extinction; GABA; Anxiolytic drugs; Chlordiazepoxide; Buspirone
1. Introduction Recently there has been an upsurge in interest in the neural mechanisms of extinction learning and in particular how they are involved in inhibiting learned fear responses (Myers and Davis, 2002). These neural mechanisms are currently poorly understood, although two recent publications have shed some light on the neurotransmitter systems involved. The first showed that endogenous cannabinoids acting at the cannabinoid receptor 1 facilitate the extinction of aversive memories (Marsicano et al., 2002). The second elegantly indicated that blocking the action of gastrin-releasing peptide, by
Corresponding author: Tel.: +44-2890-366943; fax: +44-2890368471. E-mail address: [email protected] (J.C. Leslie). ∗
0028-3908/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2003.09.004
genetically removing its receptor, retards extinction of learned fear responses (Shumyatsky et al., 2002). These cannabinoid and gastrin-releasing peptide receptors are both located on g-aminobutyric acid (GABA)-containing interneurones, suggesting that the GABAergic system is critically involved in extinction learning. Direct modulation of GABAergic neurones, through the benzodiazepine (BZ) binding site, also affects learned fear responses. The BZ inverse agonist FG7142, which attenuates the effect of GABA at its receptor, retards extinction of conditioned fear (Harris and Westbrook, 1998; Stowell et al., 2000), whereas potentiation of GABA by the BZ agonist chlordiazepoxide (CDP) facilitates extinction (Stowell et al., 2000). A number of BZ agonists are widely used as anxiolytics (Lydiard, 2003) and clinical trials with inverse agonists clearly identified them as being anxiogenic (Dorow et al., 1983). However, BZ agonists are also known to
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be amnesic (e.g., Matthews et al., 2002), a property which is generally considered to be adverse rather than beneficial. Clearly potentiation of GABA inhibits glutamate-dependent long-term potentiation and memory formation (Luscher and Frerking, 2001), but as recent publications show extinction learning requires strengthening of GABAergic neurotransmission (Myers and Davis, 2002; Shumyatsky et al., 2002), and this may be a distinct process. Most studies on extinction processes have used aversive conditioning (with electric shock as the unconditioned stimulus or reinforcer), probably because it is simple to carry out and gives robust results. However, aversive conditioning effects can be more difficult to demonstrate with inbred strains of mice (Falls et al., 1997, 2000), and the role of GABA is largely unknown in extinction processes following other forms of learning, e.g. appetitive learning. If the GABAergic system mediates extinction processes in general then pharmacological potentiation of this system should also affect the rate of extinction of operant responding for food reinforcement. The present study used C57Bl/6 mice, a strain widely used as background in transgenic studies, in a simple appetitive operant procedure in which a number of training sessions requiring lever pressing for a food reinforcer on a fixed-ratio (FR) schedule are then followed by a number of sessions of extinction in which no food is presented. During these extinction sessions the rate of lever pressing slowly decreases as extinction learning takes place. If extinction learning involves the GABAergic system then potentiation of GABA using a BZ agonist should facilitate this process; and exactly this effect was obtained using CDP with rats by Williams et al. (1990). We have studied the effect of the classical BZ CDP, the atypical anxiolytic buspirone, and also two novel agents, L-838,417 and TP13, that show BZ-subtype selective efficacy. McKernan et al. (2000) reported that L-838,417 is a selective partial agonist at the a2, a3 and a5 subtypes of the GABAA receptor and an antagonist at the a1. TP13, which has not been previously studied, has similar a2 and a3 efficacy to L838,417, but also possesses some efficacy at the a1 subtype. Both of these compounds share the anxiolytic effects of CDP (McKernan et al., 2000; personal communication), so it was reasoned that they may also share the effects on extinction learning. Based on findings of the first two experiments, the third experiment reported here included a condition where CDP was administered only for the last 5 of 15 extinction sessions in order to investigate the time of action of the drug. Because it is possible, although unlikely, that the specific effects obtained here with GABAergic drugs are due to locomotor impairment or sedation, our final experiment looked at the effect of doses of CDP, or the non-GABAergic drug buspirone, on a simple test of locomotor activity.
2. Methods and materials 2.1. Subjects All experiments used adult male mice of the C57Bl/6 inbred strain (supplied by Harland UK Ltd., Bicester, England). They were of average weight 25–30 g and aged at least 11 weeks old at the start of an experiment. They were housed alone under temperature-controlled conditions and an alternating light/dark cycle (lights on from 8:00 a.m. to 8:00 p.m.). The mice were fed on a cereal-based chow (Dixon’s Formula FFG (M)). In Experiments 1–3, all mice were given ad libitum access to water and were maintained between 80 and 90% of their free-feeding weight by providing 4–8 g of laboratory chow once daily. In the training phase of each experiment, sessions were only conducted five days a week and ad libitum food was available at the weekends. This was done to prevent the mice becoming hypoglycemic which in itself may be fatal. All animal procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act (1986) and its associated guidelines. 2.2. Apparatus Experiments 1–3 used 12 operant chambers (Med. Associates model No. /ENV 307A). They were enclosed in sound-attenuating boxes with electric fans and were equipped with a retractable response lever and an overhead house light. Reinforcers (20 mg Noyes food pellets) were delivered to a recessed tray. A computer programmed in Med.-PC controlled the lever presentation and recorded presses on the retractable lever to the nearest 20 ms. In Experiment 4, spontaneous locomotor activity was measured using individual perspex activity chambers (20 × 30 cm) equipped with a matrix of 8 × 16 infrared beams (Linton Instruments, Diss, UK). 2.3. Procedure In Experiments 1–3, daily free-operant acquisition sessions were run each with 20–30 reinforcers. During these sessions the retractable lever was permanently extended and the house light was off. Pressing the retractable lever caused pellet delivery. Following freeoperant acquisition, the mice were shifted to a fixed ratio (FR) 5 schedule of food reinforcement. Once on the FR5 schedule, completion of the lever-pressing requirement also caused lever retraction and a buzzer to sound. The inter-trial interval (ITI) prior to lever re-insertion was 60 s and each experimental session had six discrete trials. FR5 acquisition sessions were run on a daily basis until the mice reached asymptotic performance. This took between 10 and 15 days. They then received 12 further sessions of FR5 training. The last four days of this train-
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ing are referred to as the acquisition days of the experiment. On the first two of these four acquisition days vehicle injections were given prior to the sessions, and on the last two acquisition days drug injections were given prior to the sessions. All injections were by the i.p. route, the injected volume was 4 ml/kg, and injections were carried out 15–20 mins before the session. There followed 15 daily extinction sessions in which the same drug administration regime was maintained for each group but extinction was in effect. That is, on each occasion that the FR5 requirement was completed, the lever was withdrawn but no pellet was presented. In these sessions, if a mouse failed to press the lever within the pre-set extinction criterion of 600 s, then its current trial and session were terminated and it was allocated an average response latency of 600 s for each trial not completed. This procedure had the effect of allocating a weighted average inter-response time (IRT), with a maximum value of 600 s, to sessions in which the extinction criterion was met. In Experiment 1, mice received saline (0.9% saline solution; n = 12), CDP (15 mg/kg, in 0.9% saline solution; n = 12), or buspirone (4 mg/kg, in deionised water; n = 13). In Experiment 2, mice received vehicle (methylcellulose 0.5% in deionised water; n = 12), L838,417 (3, 10 or 30 mg/kg in methylcellulose 0.5% in deionised water; n = 12 for each dose), CDP (15 mg/kg in 0.9% saline solution; n = 11) or buspirone (3 mg/kg in deionised water; n = 12). In Experiment 3, mice received vehicle (0.5% methylcellulose in deionised water; n = 12), vehicle followed by CDP (methylcellulose 0.5% in deionised water as vehicle up until tenth extinction session, and CDP 15 mg/kg in 0.9% saline solution for the last five extinction sessions; n = 12), TP13 (0.3, 1 or 3 mg/kg in methylcellulose 0.5% in deionised water; n = 12 for each dose) or CDP (15 mg/kg in 0.9% saline solution; n = 12). The dose of CDP used was determined as effective but not sedative in this procedure in a series of prior experiments with C57/Bl6 mice (McCabe, 2002). The doses of L-838,417 and TP13 used both produced low, medium and close to full receptor occupancy for the three doses respectively, as determined by in vivo receptor occupancy (data not shown). For each session, the average overall response latencies, or inter-response times (IRTs) were calculated and log-transformed, to improve homogeneity of variance. The log-transformed IRT data were submitted to analysis of variance (ANOVA) with repeated measures using a general statistical package (SPSS). Where a main effect was detected, one-way ANOVAs were then conducted to identify the days on which the groups differed. Posthoc comparisons were then made for those days using independent t-tests. Only those main effects and pairwise differences that reached significance (p ⬍ 0.01) are reported. For reasons of clarity, statistical details of pairwise differences are not reported here. The percentage
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of trials completed on each session was also calculated. Because the large proportion of 100% scores skewed these data, they were not subjected to statistical analysis, but summary data are reported. In Experiment 4, naı¨ve mice were dosed with CDP (10 mg/kg, n = 9 or 15 mg/kg, n = 8), buspirone (3 or 4 mg/kg, n = 9) or vehicle (0.9% saline i.p., n = 9) and 30 mins later were placed in the activity cages for 30 mins. The duration of locomotor activity (time spent breaking beams at least 50 mm apart) was recorded. The data were analysed using a one-way ANOVA followed by a Student-Newman-Keuls post-hoc to determine between-group differences. 2.4. Radioligand binding and electrophysiology Electrophysiological determinations of efficacy for L838,417 and TP13 were performed on Ltk-cells expressing human cDNA combinations a1b3g2s, a2b3g2s, a3b3g2s and a5b3g2s as described previously (Whiting et al., 1997)). Data were normalized to the reference benzodiazepine CDP. Displacement radioligand binding of [3H]Ro15-1788 by L-838,417 and TP13 was performed using membranes prepared from Ltk- cells expressing the human cDNA combinations listed above as described previously (Hadingham et al., 1993).
3. Results The structures of L-838,417 and TP13 are shown in Fig. 1. Both compounds show nanomolar affinity at the four classical BZ-sensitive GABAA receptor subtypes (a1, a2, a3 and a5; Table 1) and much lower affinity at the two classical BZ-insensitive subtypes (a4 and a6). The efficacy of these two compounds to potentiate a GABA EC20 response in Ltk-cells expressing human recombinant receptors was normalized to that of the reference benzodiazepine CDP (Table 1). L-838,417 has no efficacy at the a1 subtype and is a partial agonist at the
Fig. 1.
Structures of L-838,417 and TP13.
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Table 1 Affinity and efficacy values of L-838,417 and TP13 at human recombinant GABAA receptors Receptor combination
a1b3g2 a2b3g2 a3b3g2 a4b3g2 a5b3g2 a6b3g2
Efficacy relative to CDP (CDP = 1)
Binding Ki(nM) L-838,417
TP13
L-838,417
TP13
0.79 ± 0.18 0.67 ± 0.24 0.67 ± 0.15 267 ± 19 2.25 ± 0.75 2183 ± 65
0.20 ± 0.06 0.18 ± 0.06 0.11 ± 0.03 11.0 ± 4.0 0.09 ± 0.03 209 ± 85
0.00 0.34 0.39 ND 0.36 ND
0.23 0.35 0.43 ND 0.19 ND
Data are mean ± sem. A competition binding assay with [3H]Ro15-1788 was used to determine binding affinity. Electrophysiological determination of efficacy was carried out in Ltk cells stably expressing human recombinant GABAA receptors. Data were normalised to the standard benzodiazepine CDP. “ND” indicates value not determined.
a2, a3 and a5 subtypes, with efficacy of approximately 35% of CDP. TP13 has similar efficacy at a2 and a3 receptors compared to L-838,417, but also has some efficacy at α1 and lower efficacy at α5. 3.1. CDP and buspirone effects on operant extinction Fig. 2 compares the effects in Experiment 1 of CDP and buspirone with saline on overall mean IRTs across all injection days. A two-way ANOVA with repeated measures of these data showed a main effect of days (F[18,612] = 66.290), of group (F[2,34] = 41.602), and
an interaction between days and group (F[36, 612] = 4.890). As indicated in Fig. 2, the CDP group responded faster (i.e., had shorter IRTs) than the saline group on days 1 and 2 of extinction and responded slower (i.e., had longer IRTs) on extinction days 11 to 15; the buspirone group responded slower than the saline group on days 1 and 2 of acquisition (drug), and on extinction days 1 to 7, and 11 to 15. All trials (100%) were completed by all groups on acquisition days and the first four extinction days. Over the last three extinction days (13–15), the saline group completed an average of 97.7% trials, the CDP group completed an average of 41.2% trials, and the buspirone group completed an average of 50.9 % trials. 3.2. CDP and buspirone effects compared with those of novel compound L-838,417
Fig. 2. Log-transformed overall group mean IRTs in Experiment 1 for the last four sessions of acquisition and 15 extinction sessions. Values plotted are log-transformed means of the 30 IRTs (measured in centiseconds) that occurred in each daily session (2.0 log units = 10 s). Buspirone dose was 4 mg/kg and CDP was 15mg/kg.
Fig. 3 compares the effects in Experiment 2 of CDP, a lower dose of buspirone and L-838,417 with a vehicle on overall mean IRTs across all injection days. A twoway ANOVA with repeated measures of these data showed a main effect of days (F[18, 1170] = 245.51), of group (F[5, 65] = 10.987), and an interaction between days and group (F[90, 1170] = 6.195). As indicated in Fig. 3, there was a dose-related effect of L-838,417: the 3 mg/kg group responded slower than the vehicle group on extinction days 9, 10 and 11; the 10 mg/kg group responded slower than the vehicle group on extinction days 9, 10, 11, 13 and 15; and the 30 mg/kg group responded slower than vehicle on extinction days 9 to 13 and 15. Additionally, the 30 mg/kg L-838,417 group responded slower than the 3 mg/kg L-838,417 group on extinction days 10 to 14, and slower than the 10 mg/kg L-838,417 group on extinction days 10, 12 and 13; and the 10 mg/kg L-838,417 group was slower than the 3 mg/kg L-838,417 group on extinction days 10 and 13. This is further evidence of the dose-related effect of L838,417. The CDP group responded slower than all the L-838,417 groups on extinction days 10 to 15.
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Fig. 3. Log-transformed overall group mean IRTs in Experiment 2 for the last four days of acquisition and 15 extinction days. Values plotted are log-transformed means of the 30 IRTs (measured in centiseconds) that occurred in each daily session (2.0 log units = 10 s). Buspirone dose was 3 mg/kg; CDP was 15mg/kg; doses of L838,417 are indicated on the graph.
As shown in Fig. 3, the CDP group responded faster than the vehicle group on extinction day 1 and slower on extinction days 9 to 15. The buspirone group responded slower than the vehicle group on extinction days 3, 9 to 11, and 13. All trials (100%) were completed by all groups on acquisition days and the first six extinction days. Over the last three extinction days (13 to 15), the saline group completed 100% of trials, the CDP group completed an average of 34.3% trials, the buspirone group completed an average of 89.8 % trials, and the three groups given L-838,417 completed 97.2% (3mg/kg), 96.3% (10 mg/kg), and 88.7% (30 mg/kg) of trials respectively. 3.3. CDP effects compared with those of novel compound TP13 Fig. 4 compares the effects in Experiment 3 of CDP and TP13 with vehicle on the overall mean IRTs across all injection days. A two-way ANOVA with repeated measures showed a main effect of days (F[18, 936] = 317.8), of group (F[4, 52] = 82.48), and an interaction between days and group (F[72, 936] = 11.42; p ⬍ 0.001). As indicated in Fig. 4, the groups given CDP or the any of the doses of TP13 responded slower then the vehicle group on extinction days 6 to 15. The effect of
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Fig. 4. Log-transformed overall group mean IRTs in Experiment 3 for the last four days of acquisition and 15 extinction days. Values plotted are log-transformed means of the 30 IRTs (measured in centiseconds) that occurred in each daily session (2.0 log units = 10 s). Vehicle/CDP group received CDP from extinction session 11. CDP dose was 15mg/kg, doses of TP13 are indicated on the graph.
TP13 was dose-related: the 10 mg/kg TP13 group responded slower than the 1 mg/kg TP13 group on extinction days 8, 9, and 11 to 15, and slower than the 3 mg/kg TP13 group on extinction days 8 and 11 to 15. The 3 mg/kg TP13 group responded slower than the 1 mg/kg TP13 group on extinction days 8 and 11 to 15. There was also a dose-related difference in the effect of TP13 from that of CDP: the CDP group showed no differences from the 1 mg/kg TP13 group, but responded faster than the 3 mg/kg TP13 group on extinction days 8 and 11 to 15, and faster than the 10 mg/kg TP13 group on extinction days 6 to 9 and 11 to 15. As shown in Fig. 4, the group given vehicle followed by CDP showed no differences from the vehicle group until extinction days 11 to 15 on which CDP was administered. On all those days, the group given vehicle followed by CDP responded slower than the vehicle group. There was no significant difference in mean IRT between the group given vehicle followed by CDP and the group given CDP on extinction days 11 to 14. All trials (100%) were completed by all groups on acquisition days and the first six extinction days. Over the last three extinction days (13 to 15), the saline group completed 100% of trials, the CDP group completed an
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average of 66.0% trials, the lowed by CDP completed an and the three groups given (1mg/kg), 26.9% (3 mg/kg), trials respectively.
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group given vehicle folaverage of 91.9 % trials, TP13 completed 58.1% and 4.0% (10 mg/kg) of
3.4. Locomotor effects of CDP and buspirone A one-way ANOVA on the total time in Experiment 4 spent mobile during the 30 minute locomotor test period showed significant variation between the drug groups (F[4,39] = 7.27) (see Fig. 5). Post-hoc analysis showed that CDP-treated mice (10 mg/kg or 15 mg/kg) did not show a significant decrease in activity (p ⬎ 0.05). Indeed, the 10 mg/kg CDP group showed a trend towards increased activity, although this did not reach statistical significance. Both groups treated with buspirone showed a decrease in activity, although only 4 mg/kg buspirone produced a significant decrease (p ⬍ 0.05). Analysis of the number of beam broken during the test yielded similar results (F[4,39] = 6.08) and posthoc analysis indicated that only 4 mg/kg buspirone significantly altered activity (p ⬍ 0.05; data not shown). L838,417 is not sedating in mice and nor is TP13 (McKernan et al., 2000).
4. Discussion The present series of studies provides evidence that GABAergic anxiolytic compounds selectively facilitate extinction of previously food-reinforced operant behaviour in at least one of the in-bred strains (C57Bl/6) of mice typically used as background in genetic manipulation studies. Using C57Bl/6 mice in the present series of experiments, we found that buspirone at a higher dose (4 mg/kg) had an effect that could be interpreted as seda-
Fig. 5. Total locomotor activity in 30 minute session of mice treated with CDP (10 or 15 mg/kg), buspirone (3 or 4 mg/kg) or vehicle (0.9% saline i.p.). Mice treated with 4 mg/kg buspirone showed a significant decrease in locomotor activity, whereas as all other groups did not significantly differ from vehicle. ∗p ⬍ 0.05 compared with vehicle.
tive or due to locomotor impairment, while at a lower dose (3 mg/kg) effects on extinction were similar to those found with the GABAergic compounds. Parallel findings were obtained with the same doses in an activity test, with the higher does of buspirone producing a marked reduction in activity. This suggests that the FR5 operant procedure may also be selectively affected by buspirone (although we have obtained lack of effect with a range of other 5-HTergic compounds, see McCabe, 2002), and this may occur because buspirone indirectly increases GABA by acting through neuronal networks (Siemiatkowski et al., 2000; Soderpalm et al., 1997). In these experiments, FR5 training in mice results in sustained responding during extinction. In vehicletreated mice responding slowed across 15 extinction sessions, with mean IRTs typically increasing by a factor greater than 10 fold. However, close to 100% of trials were still being completed during the 15th extinction session. Compared to this baseline, we found that CDP significantly slowed responding in late, but not early, extinction sessions. We obtained separate evidence (Experiment 4) that at the dose used in the extinction experiments, CDP did not reduce activity, suggesting that the effects on extinction were not due to sedation. Furthermore, subtype-selective compounds, which are devoid of sedative-like effects in animals (McKernan et al., 2000), similarly slowed responding in the late stages of extinction. The possibility that the facilitation of extinction by CDP was due to accumulation of the drug is not supported by the finding in Experiment 3. In this experiment, mice were switched from vehicle to CDP from extinction session 11 onwards and an immediate increase in the mean IRT was observed. This suggests that CDP interacts with a time-dependent extinction process. One hypothesis regarding the effects of anxiolytic-like drugs in extinction of appetitively-motivated behaviours is that they act to reduce frustrative non-reward (Gray, 1977; see Gray & McNaughton, 2000 for further discussion). The present results do not support this interpretation of the data. It might be expected that frustration would be highest at the onset of extinction and would decrease with subsequent sessions. Thus the effects of an acutely acting anxiolytic drug, such as CDP, would be expected to act immediately to slow responding from the onset of extinction by reducing frustration (Gray, 1977). In the current study, as in Williams et al. (1990), a clearly different pattern is observed. One theory of extinction is that ‘new learning’ is required to change to behaviour (Myers and Davis, 2002). In addition, it has recently been suggested that the changes in behaviour observed in extinction are timeand GABA-dependent. In recent studies, reducing GABAergic tone produced no difference in the amount of conditioned freezing observed initially in extinction, but then slowed extinction of conditioned freezing over
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several trials (Harris and Westbrook, 1998; Marsicano et al., 2002; Shumyatsky et al., 2002). Thus one possible role for the inhibitory effects of GABA during extinction is that it facilitates the inhibition of a behaviour that is no longer required by the new contingencies (Myers and Davis, 2002). However, as learning about new contingencies is experience- or time-dependent, there is a delay in the ability of GABA to influence inappropriate behaviour. Once this has occurred GABA may facilitate the inhibition of the inappropriate behaviour. Our data are consistent with this hypothesis, as CDP administered throughout extinction does not have an immediate effect on the mean IRT, but CDP treatment from extinction day 11 onwards does. Clearly further studies are required to demonstrate that a GABA-independent learning process is occurring in the initial extinction sessions. The precise brain region(s) responsible for GABAmediated potentiation of extinction is not known. However, previous work with this model in rats has shown the septo-hippocampal system to be important in facilitating extinction (Williams et al., 1990). The amygdala is also known to be important in appetitively-motivated behaviour (Baxter and Murray, 2002; Balleine et al., 2003) as well and in extinction processes of fear conditioned responses (for review see Myers and Davis, 2002). Both the amygdala and hippocampus show a high density of GABAergic neurones. Detailed mapping studies using both in situ hybridisation (Wisden et al., 1992) and receptor subunit-specific antibodies (Pirker et al., 2000; Sperk et al., 1997) have shown a highly heterogeneous distribution of these subunits. Cell bodies and dendritic processes express a1 subunits throughout the amygdala, whereas a3-expressing cell bodies are largely confined to the central nucleus (Pirker et al., 2000). Other amygdalar nuclei only show diffuse staining for a2 and a3, and very little a5 (Pirker et al., 2000). In contrast, the hippocampus expresses mostly a1, a2 and a5 subunits, with very little a3 expression (Pirker et al., 2000; Sperk et al., 1997). A further layer of complexity is that GABA receptor subtypes are differently expressed on different cell types within any one brain region. Elegant studies in the hippocampus have shown that different populations of GABAergic interneurones express discrete combinations of GABA receptors and form synapses in a spatially-controlled manner with glutamatergic projection neurones (Fritschy and Brunig, 2003). Although the highly complex expression patterns of GABA receptor subtypes makes it difficult to determine exactly which brain regions or neuronal populations are responsible for extinction processes, the differing pharmacology of the two novel GABAergic drugs does give us some clues as to which subtypes are involved. Whilst both drugs show equivalent effects in the rat plus maze and fear-potentiated startle anxiolytic models (McKernan et al., 2000; personal communication), L838,417 was less efficacious than CDP or TP13 in this
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model. Both TP13 and L-838,417 share similar efficacy at the a2 and a3 subtypes of the GABAA receptor and it is this efficacy at the amygdala a2/a3 and/or the hippocampal a2 subunits that is likely to underlie the majority of extinction-facilitating effects seen here. Interestingly, TP13 had a greater extinction-facilitating effect than L-838,417 or CDP (note that more-or-less complete extinction occurred with the group given the highest dose of TP13). The reason for this perhaps relates to its differing efficacy profile at a1 and a5 subtypes. In contrast to L-838,417 and CDP, TP13 is a partial agonist at the a1 subtype. This subtype has been shown to be responsible for the sedative, anti-convulsant and amnesic effects of BZs (Crestani et al., 2000; McKernan et al., 2000; Rudolph et al., 1999). The locomotor activity data shows clearly that a direct sedative action is not mediating the facilitation of extinction seen here. However, there may be other functional consequences of efficacy at a1 that do play a role in facilitating extinction and further investigation will be required to clarify these. Genetically removing or reducing expression of the a5 subtype is known to improve performance in hippocampal-dependent learning and memory tasks (Collinson et al., 2002; Crestani et al., 2002), which may have some impact in this procedure. Certainly CDP and L-838,417 have higher a5 efficacy than TP13, and TP13 produces a greater facilitatory effect than either of these drugs, which would be consistent with this hypothesis. A final point to note is that both novel drugs are partial agonists at those receptor subtypes, indicating that full BZ agonism is not necessary to produce extinction-facilitating effects. In summary, the data presented here indicate that operant procedures first developed for rats can be transferred for use in mice. This is of particular importance in recent years with the explosion in genetic techniques for creating mice with specific mutations. Detailed behavioural analysis of such mice has been somewhat hampered by the lack of operant procedures, where many parameters of these models can be tightly controlled. The present findings show that both non-selective BZs and novel subtype-selective compounds facilitate the extinction of prior learning in a mouse operant model. This has been extensively demonstrated with aversive conditioning (Myers and Davis, 2002) and is demonstrated here for the first time following foodreinforced operant conditioning.
Acknowledgements We are grateful to Maree Holmes, Helen Sharkey, Sarah Strange and Gillian O’Meara for their assistance in running experiments.
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Subtype-selective GABAergic drugs facilitate extinction of mouse operant behaviour Ciara McCabe a, David Shaw a, John R. Atack b, Leslie J. Street b, Keith A. Wafford b, Gerard R. Dawson b, David S. Reynolds b, Julian C. Leslie a,∗ a
School of Psychology, University of Ulster, Shore Road, Newtownabbey BT37 0QB, Northern Ireland UK b Merck Sharp & Dohme Research Laboratories, Terlings Park, Harlow, Essex CM20 2QR, UK Received 7 July 2003; received in revised form 15 August 2003; accepted 3 September 2003
Abstract Several recent studies have shown that reducing g-aminobutyric acid (GABA)-mediated neurotransmission retards extinction of aversive conditioning. However, relatively little is known about the effect of GABA on extinction of appetitively motivated tasks. We examined the effect of chlordiazepoxide (CDP), a classical benzodiazepine (BZ) and two novel subtype-selective BZs when administered to male C57Bl/6 mice during extinction following training on a discrete-trial fixed-ratio 5 (FR5) food reinforced leverpress procedure. Initially CDP had no effect, but after several extinction sessions CDP significantly facilitated extinction, i.e. slowed responding, compared with vehicle-treated mice. This effect was not due to drug accumulation because mice switched from vehicle treatment to CDP late in extinction showed facilitation immediately. Likewise, this effect could not be attributed to sedation because the dose of CDP used (15 mg/kg i.p.) did not suppress locomotor activity. The two novel subtype-selective BZ partial agonists, L838,417 and TP13, selectively facilitated extinction in similar fashion to CDP. The non-GABAergic anxiolytic buspirone was also tested and found to have similar effects when administered at a non-sedating dose. These studies demonstrate that GABA-mediated processes are important during extinction of an appetitively motivated task, but only after the animals have experienced several extinction sessions. 2003 Elsevier Ltd. All rights reserved. Keywords: Mouse; Operant behaviour; Extinction; GABA; Anxiolytic drugs; Chlordiazepoxide; Buspirone
1. Introduction Recently there has been an upsurge in interest in the neural mechanisms of extinction learning and in particular how they are involved in inhibiting learned fear responses (Myers and Davis, 2002). These neural mechanisms are currently poorly understood, although two recent publications have shed some light on the neurotransmitter systems involved. The first showed that endogenous cannabinoids acting at the cannabinoid receptor 1 facilitate the extinction of aversive memories (Marsicano et al., 2002). The second elegantly indicated that blocking the action of gastrin-releasing peptide, by
Corresponding author: Tel.: +44-2890-366943; fax: +44-2890368471. E-mail address: [email protected] (J.C. Leslie). ∗
0028-3908/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2003.09.004
genetically removing its receptor, retards extinction of learned fear responses (Shumyatsky et al., 2002). These cannabinoid and gastrin-releasing peptide receptors are both located on g-aminobutyric acid (GABA)-containing interneurones, suggesting that the GABAergic system is critically involved in extinction learning. Direct modulation of GABAergic neurones, through the benzodiazepine (BZ) binding site, also affects learned fear responses. The BZ inverse agonist FG7142, which attenuates the effect of GABA at its receptor, retards extinction of conditioned fear (Harris and Westbrook, 1998; Stowell et al., 2000), whereas potentiation of GABA by the BZ agonist chlordiazepoxide (CDP) facilitates extinction (Stowell et al., 2000). A number of BZ agonists are widely used as anxiolytics (Lydiard, 2003) and clinical trials with inverse agonists clearly identified them as being anxiogenic (Dorow et al., 1983). However, BZ agonists are also known to
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be amnesic (e.g., Matthews et al., 2002), a property which is generally considered to be adverse rather than beneficial. Clearly potentiation of GABA inhibits glutamate-dependent long-term potentiation and memory formation (Luscher and Frerking, 2001), but as recent publications show extinction learning requires strengthening of GABAergic neurotransmission (Myers and Davis, 2002; Shumyatsky et al., 2002), and this may be a distinct process. Most studies on extinction processes have used aversive conditioning (with electric shock as the unconditioned stimulus or reinforcer), probably because it is simple to carry out and gives robust results. However, aversive conditioning effects can be more difficult to demonstrate with inbred strains of mice (Falls et al., 1997, 2000), and the role of GABA is largely unknown in extinction processes following other forms of learning, e.g. appetitive learning. If the GABAergic system mediates extinction processes in general then pharmacological potentiation of this system should also affect the rate of extinction of operant responding for food reinforcement. The present study used C57Bl/6 mice, a strain widely used as background in transgenic studies, in a simple appetitive operant procedure in which a number of training sessions requiring lever pressing for a food reinforcer on a fixed-ratio (FR) schedule are then followed by a number of sessions of extinction in which no food is presented. During these extinction sessions the rate of lever pressing slowly decreases as extinction learning takes place. If extinction learning involves the GABAergic system then potentiation of GABA using a BZ agonist should facilitate this process; and exactly this effect was obtained using CDP with rats by Williams et al. (1990). We have studied the effect of the classical BZ CDP, the atypical anxiolytic buspirone, and also two novel agents, L-838,417 and TP13, that show BZ-subtype selective efficacy. McKernan et al. (2000) reported that L-838,417 is a selective partial agonist at the a2, a3 and a5 subtypes of the GABAA receptor and an antagonist at the a1. TP13, which has not been previously studied, has similar a2 and a3 efficacy to L838,417, but also possesses some efficacy at the a1 subtype. Both of these compounds share the anxiolytic effects of CDP (McKernan et al., 2000; personal communication), so it was reasoned that they may also share the effects on extinction learning. Based on findings of the first two experiments, the third experiment reported here included a condition where CDP was administered only for the last 5 of 15 extinction sessions in order to investigate the time of action of the drug. Because it is possible, although unlikely, that the specific effects obtained here with GABAergic drugs are due to locomotor impairment or sedation, our final experiment looked at the effect of doses of CDP, or the non-GABAergic drug buspirone, on a simple test of locomotor activity.
2. Methods and materials 2.1. Subjects All experiments used adult male mice of the C57Bl/6 inbred strain (supplied by Harland UK Ltd., Bicester, England). They were of average weight 25–30 g and aged at least 11 weeks old at the start of an experiment. They were housed alone under temperature-controlled conditions and an alternating light/dark cycle (lights on from 8:00 a.m. to 8:00 p.m.). The mice were fed on a cereal-based chow (Dixon’s Formula FFG (M)). In Experiments 1–3, all mice were given ad libitum access to water and were maintained between 80 and 90% of their free-feeding weight by providing 4–8 g of laboratory chow once daily. In the training phase of each experiment, sessions were only conducted five days a week and ad libitum food was available at the weekends. This was done to prevent the mice becoming hypoglycemic which in itself may be fatal. All animal procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act (1986) and its associated guidelines. 2.2. Apparatus Experiments 1–3 used 12 operant chambers (Med. Associates model No. /ENV 307A). They were enclosed in sound-attenuating boxes with electric fans and were equipped with a retractable response lever and an overhead house light. Reinforcers (20 mg Noyes food pellets) were delivered to a recessed tray. A computer programmed in Med.-PC controlled the lever presentation and recorded presses on the retractable lever to the nearest 20 ms. In Experiment 4, spontaneous locomotor activity was measured using individual perspex activity chambers (20 × 30 cm) equipped with a matrix of 8 × 16 infrared beams (Linton Instruments, Diss, UK). 2.3. Procedure In Experiments 1–3, daily free-operant acquisition sessions were run each with 20–30 reinforcers. During these sessions the retractable lever was permanently extended and the house light was off. Pressing the retractable lever caused pellet delivery. Following freeoperant acquisition, the mice were shifted to a fixed ratio (FR) 5 schedule of food reinforcement. Once on the FR5 schedule, completion of the lever-pressing requirement also caused lever retraction and a buzzer to sound. The inter-trial interval (ITI) prior to lever re-insertion was 60 s and each experimental session had six discrete trials. FR5 acquisition sessions were run on a daily basis until the mice reached asymptotic performance. This took between 10 and 15 days. They then received 12 further sessions of FR5 training. The last four days of this train-
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ing are referred to as the acquisition days of the experiment. On the first two of these four acquisition days vehicle injections were given prior to the sessions, and on the last two acquisition days drug injections were given prior to the sessions. All injections were by the i.p. route, the injected volume was 4 ml/kg, and injections were carried out 15–20 mins before the session. There followed 15 daily extinction sessions in which the same drug administration regime was maintained for each group but extinction was in effect. That is, on each occasion that the FR5 requirement was completed, the lever was withdrawn but no pellet was presented. In these sessions, if a mouse failed to press the lever within the pre-set extinction criterion of 600 s, then its current trial and session were terminated and it was allocated an average response latency of 600 s for each trial not completed. This procedure had the effect of allocating a weighted average inter-response time (IRT), with a maximum value of 600 s, to sessions in which the extinction criterion was met. In Experiment 1, mice received saline (0.9% saline solution; n = 12), CDP (15 mg/kg, in 0.9% saline solution; n = 12), or buspirone (4 mg/kg, in deionised water; n = 13). In Experiment 2, mice received vehicle (methylcellulose 0.5% in deionised water; n = 12), L838,417 (3, 10 or 30 mg/kg in methylcellulose 0.5% in deionised water; n = 12 for each dose), CDP (15 mg/kg in 0.9% saline solution; n = 11) or buspirone (3 mg/kg in deionised water; n = 12). In Experiment 3, mice received vehicle (0.5% methylcellulose in deionised water; n = 12), vehicle followed by CDP (methylcellulose 0.5% in deionised water as vehicle up until tenth extinction session, and CDP 15 mg/kg in 0.9% saline solution for the last five extinction sessions; n = 12), TP13 (0.3, 1 or 3 mg/kg in methylcellulose 0.5% in deionised water; n = 12 for each dose) or CDP (15 mg/kg in 0.9% saline solution; n = 12). The dose of CDP used was determined as effective but not sedative in this procedure in a series of prior experiments with C57/Bl6 mice (McCabe, 2002). The doses of L-838,417 and TP13 used both produced low, medium and close to full receptor occupancy for the three doses respectively, as determined by in vivo receptor occupancy (data not shown). For each session, the average overall response latencies, or inter-response times (IRTs) were calculated and log-transformed, to improve homogeneity of variance. The log-transformed IRT data were submitted to analysis of variance (ANOVA) with repeated measures using a general statistical package (SPSS). Where a main effect was detected, one-way ANOVAs were then conducted to identify the days on which the groups differed. Posthoc comparisons were then made for those days using independent t-tests. Only those main effects and pairwise differences that reached significance (p ⬍ 0.01) are reported. For reasons of clarity, statistical details of pairwise differences are not reported here. The percentage
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of trials completed on each session was also calculated. Because the large proportion of 100% scores skewed these data, they were not subjected to statistical analysis, but summary data are reported. In Experiment 4, naı¨ve mice were dosed with CDP (10 mg/kg, n = 9 or 15 mg/kg, n = 8), buspirone (3 or 4 mg/kg, n = 9) or vehicle (0.9% saline i.p., n = 9) and 30 mins later were placed in the activity cages for 30 mins. The duration of locomotor activity (time spent breaking beams at least 50 mm apart) was recorded. The data were analysed using a one-way ANOVA followed by a Student-Newman-Keuls post-hoc to determine between-group differences. 2.4. Radioligand binding and electrophysiology Electrophysiological determinations of efficacy for L838,417 and TP13 were performed on Ltk-cells expressing human cDNA combinations a1b3g2s, a2b3g2s, a3b3g2s and a5b3g2s as described previously (Whiting et al., 1997)). Data were normalized to the reference benzodiazepine CDP. Displacement radioligand binding of [3H]Ro15-1788 by L-838,417 and TP13 was performed using membranes prepared from Ltk- cells expressing the human cDNA combinations listed above as described previously (Hadingham et al., 1993).
3. Results The structures of L-838,417 and TP13 are shown in Fig. 1. Both compounds show nanomolar affinity at the four classical BZ-sensitive GABAA receptor subtypes (a1, a2, a3 and a5; Table 1) and much lower affinity at the two classical BZ-insensitive subtypes (a4 and a6). The efficacy of these two compounds to potentiate a GABA EC20 response in Ltk-cells expressing human recombinant receptors was normalized to that of the reference benzodiazepine CDP (Table 1). L-838,417 has no efficacy at the a1 subtype and is a partial agonist at the
Fig. 1.
Structures of L-838,417 and TP13.
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Table 1 Affinity and efficacy values of L-838,417 and TP13 at human recombinant GABAA receptors Receptor combination
a1b3g2 a2b3g2 a3b3g2 a4b3g2 a5b3g2 a6b3g2
Efficacy relative to CDP (CDP = 1)
Binding Ki(nM) L-838,417
TP13
L-838,417
TP13
0.79 ± 0.18 0.67 ± 0.24 0.67 ± 0.15 267 ± 19 2.25 ± 0.75 2183 ± 65
0.20 ± 0.06 0.18 ± 0.06 0.11 ± 0.03 11.0 ± 4.0 0.09 ± 0.03 209 ± 85
0.00 0.34 0.39 ND 0.36 ND
0.23 0.35 0.43 ND 0.19 ND
Data are mean ± sem. A competition binding assay with [3H]Ro15-1788 was used to determine binding affinity. Electrophysiological determination of efficacy was carried out in Ltk cells stably expressing human recombinant GABAA receptors. Data were normalised to the standard benzodiazepine CDP. “ND” indicates value not determined.
a2, a3 and a5 subtypes, with efficacy of approximately 35% of CDP. TP13 has similar efficacy at a2 and a3 receptors compared to L-838,417, but also has some efficacy at α1 and lower efficacy at α5. 3.1. CDP and buspirone effects on operant extinction Fig. 2 compares the effects in Experiment 1 of CDP and buspirone with saline on overall mean IRTs across all injection days. A two-way ANOVA with repeated measures of these data showed a main effect of days (F[18,612] = 66.290), of group (F[2,34] = 41.602), and
an interaction between days and group (F[36, 612] = 4.890). As indicated in Fig. 2, the CDP group responded faster (i.e., had shorter IRTs) than the saline group on days 1 and 2 of extinction and responded slower (i.e., had longer IRTs) on extinction days 11 to 15; the buspirone group responded slower than the saline group on days 1 and 2 of acquisition (drug), and on extinction days 1 to 7, and 11 to 15. All trials (100%) were completed by all groups on acquisition days and the first four extinction days. Over the last three extinction days (13–15), the saline group completed an average of 97.7% trials, the CDP group completed an average of 41.2% trials, and the buspirone group completed an average of 50.9 % trials. 3.2. CDP and buspirone effects compared with those of novel compound L-838,417
Fig. 2. Log-transformed overall group mean IRTs in Experiment 1 for the last four sessions of acquisition and 15 extinction sessions. Values plotted are log-transformed means of the 30 IRTs (measured in centiseconds) that occurred in each daily session (2.0 log units = 10 s). Buspirone dose was 4 mg/kg and CDP was 15mg/kg.
Fig. 3 compares the effects in Experiment 2 of CDP, a lower dose of buspirone and L-838,417 with a vehicle on overall mean IRTs across all injection days. A twoway ANOVA with repeated measures of these data showed a main effect of days (F[18, 1170] = 245.51), of group (F[5, 65] = 10.987), and an interaction between days and group (F[90, 1170] = 6.195). As indicated in Fig. 3, there was a dose-related effect of L-838,417: the 3 mg/kg group responded slower than the vehicle group on extinction days 9, 10 and 11; the 10 mg/kg group responded slower than the vehicle group on extinction days 9, 10, 11, 13 and 15; and the 30 mg/kg group responded slower than vehicle on extinction days 9 to 13 and 15. Additionally, the 30 mg/kg L-838,417 group responded slower than the 3 mg/kg L-838,417 group on extinction days 10 to 14, and slower than the 10 mg/kg L-838,417 group on extinction days 10, 12 and 13; and the 10 mg/kg L-838,417 group was slower than the 3 mg/kg L-838,417 group on extinction days 10 and 13. This is further evidence of the dose-related effect of L838,417. The CDP group responded slower than all the L-838,417 groups on extinction days 10 to 15.
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Fig. 3. Log-transformed overall group mean IRTs in Experiment 2 for the last four days of acquisition and 15 extinction days. Values plotted are log-transformed means of the 30 IRTs (measured in centiseconds) that occurred in each daily session (2.0 log units = 10 s). Buspirone dose was 3 mg/kg; CDP was 15mg/kg; doses of L838,417 are indicated on the graph.
As shown in Fig. 3, the CDP group responded faster than the vehicle group on extinction day 1 and slower on extinction days 9 to 15. The buspirone group responded slower than the vehicle group on extinction days 3, 9 to 11, and 13. All trials (100%) were completed by all groups on acquisition days and the first six extinction days. Over the last three extinction days (13 to 15), the saline group completed 100% of trials, the CDP group completed an average of 34.3% trials, the buspirone group completed an average of 89.8 % trials, and the three groups given L-838,417 completed 97.2% (3mg/kg), 96.3% (10 mg/kg), and 88.7% (30 mg/kg) of trials respectively. 3.3. CDP effects compared with those of novel compound TP13 Fig. 4 compares the effects in Experiment 3 of CDP and TP13 with vehicle on the overall mean IRTs across all injection days. A two-way ANOVA with repeated measures showed a main effect of days (F[18, 936] = 317.8), of group (F[4, 52] = 82.48), and an interaction between days and group (F[72, 936] = 11.42; p ⬍ 0.001). As indicated in Fig. 4, the groups given CDP or the any of the doses of TP13 responded slower then the vehicle group on extinction days 6 to 15. The effect of
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Fig. 4. Log-transformed overall group mean IRTs in Experiment 3 for the last four days of acquisition and 15 extinction days. Values plotted are log-transformed means of the 30 IRTs (measured in centiseconds) that occurred in each daily session (2.0 log units = 10 s). Vehicle/CDP group received CDP from extinction session 11. CDP dose was 15mg/kg, doses of TP13 are indicated on the graph.
TP13 was dose-related: the 10 mg/kg TP13 group responded slower than the 1 mg/kg TP13 group on extinction days 8, 9, and 11 to 15, and slower than the 3 mg/kg TP13 group on extinction days 8 and 11 to 15. The 3 mg/kg TP13 group responded slower than the 1 mg/kg TP13 group on extinction days 8 and 11 to 15. There was also a dose-related difference in the effect of TP13 from that of CDP: the CDP group showed no differences from the 1 mg/kg TP13 group, but responded faster than the 3 mg/kg TP13 group on extinction days 8 and 11 to 15, and faster than the 10 mg/kg TP13 group on extinction days 6 to 9 and 11 to 15. As shown in Fig. 4, the group given vehicle followed by CDP showed no differences from the vehicle group until extinction days 11 to 15 on which CDP was administered. On all those days, the group given vehicle followed by CDP responded slower than the vehicle group. There was no significant difference in mean IRT between the group given vehicle followed by CDP and the group given CDP on extinction days 11 to 14. All trials (100%) were completed by all groups on acquisition days and the first six extinction days. Over the last three extinction days (13 to 15), the saline group completed 100% of trials, the CDP group completed an
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average of 66.0% trials, the lowed by CDP completed an and the three groups given (1mg/kg), 26.9% (3 mg/kg), trials respectively.
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group given vehicle folaverage of 91.9 % trials, TP13 completed 58.1% and 4.0% (10 mg/kg) of
3.4. Locomotor effects of CDP and buspirone A one-way ANOVA on the total time in Experiment 4 spent mobile during the 30 minute locomotor test period showed significant variation between the drug groups (F[4,39] = 7.27) (see Fig. 5). Post-hoc analysis showed that CDP-treated mice (10 mg/kg or 15 mg/kg) did not show a significant decrease in activity (p ⬎ 0.05). Indeed, the 10 mg/kg CDP group showed a trend towards increased activity, although this did not reach statistical significance. Both groups treated with buspirone showed a decrease in activity, although only 4 mg/kg buspirone produced a significant decrease (p ⬍ 0.05). Analysis of the number of beam broken during the test yielded similar results (F[4,39] = 6.08) and posthoc analysis indicated that only 4 mg/kg buspirone significantly altered activity (p ⬍ 0.05; data not shown). L838,417 is not sedating in mice and nor is TP13 (McKernan et al., 2000).
4. Discussion The present series of studies provides evidence that GABAergic anxiolytic compounds selectively facilitate extinction of previously food-reinforced operant behaviour in at least one of the in-bred strains (C57Bl/6) of mice typically used as background in genetic manipulation studies. Using C57Bl/6 mice in the present series of experiments, we found that buspirone at a higher dose (4 mg/kg) had an effect that could be interpreted as seda-
Fig. 5. Total locomotor activity in 30 minute session of mice treated with CDP (10 or 15 mg/kg), buspirone (3 or 4 mg/kg) or vehicle (0.9% saline i.p.). Mice treated with 4 mg/kg buspirone showed a significant decrease in locomotor activity, whereas as all other groups did not significantly differ from vehicle. ∗p ⬍ 0.05 compared with vehicle.
tive or due to locomotor impairment, while at a lower dose (3 mg/kg) effects on extinction were similar to those found with the GABAergic compounds. Parallel findings were obtained with the same doses in an activity test, with the higher does of buspirone producing a marked reduction in activity. This suggests that the FR5 operant procedure may also be selectively affected by buspirone (although we have obtained lack of effect with a range of other 5-HTergic compounds, see McCabe, 2002), and this may occur because buspirone indirectly increases GABA by acting through neuronal networks (Siemiatkowski et al., 2000; Soderpalm et al., 1997). In these experiments, FR5 training in mice results in sustained responding during extinction. In vehicletreated mice responding slowed across 15 extinction sessions, with mean IRTs typically increasing by a factor greater than 10 fold. However, close to 100% of trials were still being completed during the 15th extinction session. Compared to this baseline, we found that CDP significantly slowed responding in late, but not early, extinction sessions. We obtained separate evidence (Experiment 4) that at the dose used in the extinction experiments, CDP did not reduce activity, suggesting that the effects on extinction were not due to sedation. Furthermore, subtype-selective compounds, which are devoid of sedative-like effects in animals (McKernan et al., 2000), similarly slowed responding in the late stages of extinction. The possibility that the facilitation of extinction by CDP was due to accumulation of the drug is not supported by the finding in Experiment 3. In this experiment, mice were switched from vehicle to CDP from extinction session 11 onwards and an immediate increase in the mean IRT was observed. This suggests that CDP interacts with a time-dependent extinction process. One hypothesis regarding the effects of anxiolytic-like drugs in extinction of appetitively-motivated behaviours is that they act to reduce frustrative non-reward (Gray, 1977; see Gray & McNaughton, 2000 for further discussion). The present results do not support this interpretation of the data. It might be expected that frustration would be highest at the onset of extinction and would decrease with subsequent sessions. Thus the effects of an acutely acting anxiolytic drug, such as CDP, would be expected to act immediately to slow responding from the onset of extinction by reducing frustration (Gray, 1977). In the current study, as in Williams et al. (1990), a clearly different pattern is observed. One theory of extinction is that ‘new learning’ is required to change to behaviour (Myers and Davis, 2002). In addition, it has recently been suggested that the changes in behaviour observed in extinction are timeand GABA-dependent. In recent studies, reducing GABAergic tone produced no difference in the amount of conditioned freezing observed initially in extinction, but then slowed extinction of conditioned freezing over
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several trials (Harris and Westbrook, 1998; Marsicano et al., 2002; Shumyatsky et al., 2002). Thus one possible role for the inhibitory effects of GABA during extinction is that it facilitates the inhibition of a behaviour that is no longer required by the new contingencies (Myers and Davis, 2002). However, as learning about new contingencies is experience- or time-dependent, there is a delay in the ability of GABA to influence inappropriate behaviour. Once this has occurred GABA may facilitate the inhibition of the inappropriate behaviour. Our data are consistent with this hypothesis, as CDP administered throughout extinction does not have an immediate effect on the mean IRT, but CDP treatment from extinction day 11 onwards does. Clearly further studies are required to demonstrate that a GABA-independent learning process is occurring in the initial extinction sessions. The precise brain region(s) responsible for GABAmediated potentiation of extinction is not known. However, previous work with this model in rats has shown the septo-hippocampal system to be important in facilitating extinction (Williams et al., 1990). The amygdala is also known to be important in appetitively-motivated behaviour (Baxter and Murray, 2002; Balleine et al., 2003) as well and in extinction processes of fear conditioned responses (for review see Myers and Davis, 2002). Both the amygdala and hippocampus show a high density of GABAergic neurones. Detailed mapping studies using both in situ hybridisation (Wisden et al., 1992) and receptor subunit-specific antibodies (Pirker et al., 2000; Sperk et al., 1997) have shown a highly heterogeneous distribution of these subunits. Cell bodies and dendritic processes express a1 subunits throughout the amygdala, whereas a3-expressing cell bodies are largely confined to the central nucleus (Pirker et al., 2000). Other amygdalar nuclei only show diffuse staining for a2 and a3, and very little a5 (Pirker et al., 2000). In contrast, the hippocampus expresses mostly a1, a2 and a5 subunits, with very little a3 expression (Pirker et al., 2000; Sperk et al., 1997). A further layer of complexity is that GABA receptor subtypes are differently expressed on different cell types within any one brain region. Elegant studies in the hippocampus have shown that different populations of GABAergic interneurones express discrete combinations of GABA receptors and form synapses in a spatially-controlled manner with glutamatergic projection neurones (Fritschy and Brunig, 2003). Although the highly complex expression patterns of GABA receptor subtypes makes it difficult to determine exactly which brain regions or neuronal populations are responsible for extinction processes, the differing pharmacology of the two novel GABAergic drugs does give us some clues as to which subtypes are involved. Whilst both drugs show equivalent effects in the rat plus maze and fear-potentiated startle anxiolytic models (McKernan et al., 2000; personal communication), L838,417 was less efficacious than CDP or TP13 in this
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model. Both TP13 and L-838,417 share similar efficacy at the a2 and a3 subtypes of the GABAA receptor and it is this efficacy at the amygdala a2/a3 and/or the hippocampal a2 subunits that is likely to underlie the majority of extinction-facilitating effects seen here. Interestingly, TP13 had a greater extinction-facilitating effect than L-838,417 or CDP (note that more-or-less complete extinction occurred with the group given the highest dose of TP13). The reason for this perhaps relates to its differing efficacy profile at a1 and a5 subtypes. In contrast to L-838,417 and CDP, TP13 is a partial agonist at the a1 subtype. This subtype has been shown to be responsible for the sedative, anti-convulsant and amnesic effects of BZs (Crestani et al., 2000; McKernan et al., 2000; Rudolph et al., 1999). The locomotor activity data shows clearly that a direct sedative action is not mediating the facilitation of extinction seen here. However, there may be other functional consequences of efficacy at a1 that do play a role in facilitating extinction and further investigation will be required to clarify these. Genetically removing or reducing expression of the a5 subtype is known to improve performance in hippocampal-dependent learning and memory tasks (Collinson et al., 2002; Crestani et al., 2002), which may have some impact in this procedure. Certainly CDP and L-838,417 have higher a5 efficacy than TP13, and TP13 produces a greater facilitatory effect than either of these drugs, which would be consistent with this hypothesis. A final point to note is that both novel drugs are partial agonists at those receptor subtypes, indicating that full BZ agonism is not necessary to produce extinction-facilitating effects. In summary, the data presented here indicate that operant procedures first developed for rats can be transferred for use in mice. This is of particular importance in recent years with the explosion in genetic techniques for creating mice with specific mutations. Detailed behavioural analysis of such mice has been somewhat hampered by the lack of operant procedures, where many parameters of these models can be tightly controlled. The present findings show that both non-selective BZs and novel subtype-selective compounds facilitate the extinction of prior learning in a mouse operant model. This has been extensively demonstrated with aversive conditioning (Myers and Davis, 2002) and is demonstrated here for the first time following foodreinforced operant conditioning.
Acknowledgements We are grateful to Maree Holmes, Helen Sharkey, Sarah Strange and Gillian O’Meara for their assistance in running experiments.
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