Introduction ORIGINAL INVESTIGATION. K.J. Stanhope C.T. Dourish

Psychopharmacology (1996) 128 : 293 303 Springer-Verlag 1996 ORIGINAL INVESTIGATION K.J. Stanhope C.T. Dourish Effects of 5-HT 1A receptor agonists, partial agonists and a silent antagonist on the performance of the conditioned emotional response test in the rat Received: 13 March 1996/ Final version: 8 July 1996 Abstract In the present study, the effects of 5-HT 1A receptor ligands with varying degrees of intrinsic activity at the 5-HT 1A receptor were examined in the conditioned emotional response (CER) test and their effects compared to those of the benzodiazepine receptor agonists, diazepam and chlordiazepoxide. Diazepam (3.0 mg/kg) and chlordiazepoxide (3.0 mg/kg), and the 5-HT 1A receptor partial agonists, ipsapirone (10.0 mg/ kg) and gepirone (3.0 mg/ kg), alleviated conditioned suppression of lever pressing. The 5-HT 1A receptor partial agonist, buspirone (0.1 1.0 mg/kg), the 5-HT 1A receptor agonist, 8-OH-DPAT (0.01 0.10 mg/kg), and the 5-HT 1A receptor antagonist, WAY-100635 (0.03 3.0 mg/kg), had no effects on conditioned fear. Neither enhancing the level of food deprivation nor pretreatment with the amnesic agent scopolamine induced anxiolytic-like effects in the present CER test. The anxiolytic-like effects of ipsapirone in this test were completely reversed by WAY-100635. These results indicate that 5-HT 1A agonist, but not antagonist actions, induce an anxiolytic effect in the CER test in rats. Key words Conditioned emotional response (CER) 5-HT 1A receptor agonists and antagonists WAY-100635 Anxiety Fear Introduction The role of 5-HT 1A receptors in the modulation of anxiety is controversial. Although compounds with partial agonist actions at the 5-HT 1A receptor such as the K.J. Stanhope 1 (*) C.T. Dourish Department of Neuropharmacology, Wyeth Research Ltd, Huntercombe Lane South, Taplow, Maidenhead, Bucks SL6 OPH, UK Present address: 1 Cerebrus Ltd., Ascot, Berkshire SL5 7NP, UK pyrimidinylpiperazines buspirone (Riblet et al. 1982), gepirone (Eison et al. 1986) and ipsapirone (Traber and Glaser 1987) have been shown to be anxiolytic in the clinic (Goldberg and Finnerty 1979; Eison et al. 1986; Goa and Ward 1986; Boyer and Feighner 1993), the mechanism of their anxiolytic effects has not been established. It has been suggested that 5-HT 1A receptor partial agonists may exert their actions due to 5-HT 1A receptor agonist or antagonist actions or a combination of both (Dourish et al. 1986; Dourish 1987). The availability of selective 5-HT 1A receptor antagonists such as WAY-100635 (Fletcher et al. 1995; Forster et al. 1995) may help to identify the nature of the anxiolytic action of 5-HT 1A receptor partial agonists. In the present studies, WAY-100635 was used to investigate the role of 5-HT 1A receptors in a preclinical animal model of anxiety, the conditioned emotional response (CER) test. Previous reports of the effects of 5-HT 1A receptor ligands in preclinical animal models of anxiety have been inconsistent. In models involving innate behaviours such as the light/dark box and the elevated plus maze, results have been extremely variable with the demonstration of anxiolytic, anxiogenic or no effects of 5-HT 1A receptor partial agonists (Dourish 1987; Treit 1991; Wilkinson and Dourish 1991). The effects of 5-HT 1A receptor ligands in animal models of anxiety using either conflict procedures (eg., Geller and Seifter 1960; Vogel et al. 1971) or the CER procedure (Estes and Skinner 1941; for a review see Davis 1990) have also proved inconsistent (Barrett and Witkin, 1991; Barrett and Vanover 1993), however, a number of authors have reported that 5-HT 1A receptor partial agonists have anxiolytic effects in such tests (Schefke et al. 1989; Sanger 1990, 1992; Barrett and Witkin, 1991; Charrier et al. 1994). Although Barrett and colleagues (1991, 1993) argue that the most consistent anxiolytic-like effects of 5-HT 1A receptor partial agonists are observed in conflict tests in pigeons, Sanger (1990, 1992), reported robust anxiolytic-like effects with ipsapirone in the CER test in the rat (see also

294 Rittenhouse et al. 1992). Similarly, in another rodent model which employs a CER procedure, the fear potentiated startle paradigm, consistent anxiolytic effects have been reported with buspirone (Davis 1993; see also Mansbach and Geyer 1988). Given that CER procedures appear sensitive to the anxiolytic-like effects of 5-HT 1A receptor partial agonists, a CER procedure was used here to examine the effects of 5-HT 1A receptor ligands with varying degrees of intrinsic activity. As mentioned above, functionally, 5-HT 1A receptor partial agonists could exert their anxiolytic effects due to either agonist (Dourish et al. 1986) or antagonist actions (Dourish 1987). Depending on the assay system that is employed, buspirone, ipsapirone, gepirone, or the aminotetralin 8-hydroxy- 2-(di-n-propylamino)tetralin (8-OH-DPAT) which is generally regarded as a full 5-HT 1A receptor agonist, can display either agonist or antagonist effects (Colino and Halliwell 1987; O Connor et al. 1990; Andrade 1992; Beck et al. 1992). To determine whether agonist or antagonist effects of 5-HT 1A receptor partial agonists induce anxiolytic-like effects, the present series of studies compared the effects of 8-OH-DPAT, ipsapirone, gepirone and buspirone to the effects of the selective and silent 5-HT 1A receptor antagonist, WAY- 100635 (Fletcher et al. 1995; Forster et al. 1995). Before commencing the 5-HT 1A receptor studies we undertook a series of experiments to characterise and validate the CER test, as conducted in our laboratory, as an anxiety model. In the present CER test the conditioned stimulus was occasionally, as opposed to consistently, paired with mild electric footshock. Reducing the predictability of shock has been claimed to change the sensitivity of conflict tests to anxiolytic agents (e.g., Costello et al. 1991). Thus, in order to interpret the effects of the 5-HT 1A receptor ligands, we also examined the effects of two prototypical anxiolytic agents, the benzodiazepine receptor agonists diazepam and chlordiazepoxide. In addition, in order to determine whether false positive effects may be obtained in the present test due to the enhancement of motivation (see Treit 1984), the effects of food deprivation on performance were also examined and compared to the effects of the putative anxiolytic drugs. Finally, to determine whether any cognitive disrupting effects of drugs might induce false positive effects in the present test, the effects of pretreatment with the amnesic agent scopolamine was examined. Materials and methods Subjects Fifty-four male Lister Hooded rats with an average free-feeding weight of 225 g (experiments with chlordiazepoxide, diazepam and scopolamine), 56 male Lister Hooded rats, with a mean free-feeding weight of 264 g (experiment with gepirone) and 56 male Lister Hooded rats with a mean free-feeding weight of 208 g (experiments on food deprivation, buspirone, ipsapirone, WAY-100635, 8-OH- DPAT and WAY-100635 versus ipsapirone) were fed a restricted diet designed to maintain rats at greater than 85% of their initial body weight. Rats were housed in cages of four and fed approximately 55 g of food per cage per day (experiment with chlordiazepoxide, diazepam and scopolamine). In the experiments conducted with the two subsequent groups of rats, the animals were fed approximately 60 g of food per cage per day. A 12:12 light/dark cycle was in effect in the colony room and the rats were trained and tested during the light part of the cycle. Ethics The research described in this manuscript was conducted within the guidelines of the Home Office regulations as outlined in the Animal (Scientific Procedures) Act, 1986, and was approved by an internal ethics committee. Apparatus Eight Colbourn operant chambers with associated Colbourn pellet dispensers and operant levers were used. These chambers were housed in outer sound and light attenuating shells equipped with a ventilation fan which also helped to mask external noise. A single operant lever was positioned on the left side of the front panel of the operant chamber, approximately 2 cm above the grid floor. Colbourn animal test cage grid floor shockers were used to deliver shock (0.4 ma, 0.5 s) to the grid floors (model E-10 10SF). Formula P Noyes food pellets (45 mg) could be delivered to the food magazine centered on the front wall of the chamber approximately 2 cm above the floor. A houselight (4.0 ma, 28 V) was positioned on the front wall of the chamber, above the food magazine, approximately 1.0 cm below the ceiling. The operant chambers were controlled and the number of lever presses recorded by a PDP-11 minicomputer programmed in SKED. The data collected from the PDP-11 were electronically transferred to a VAX computer for statistical analysis using SAS software. Procedure Preliminary training Once the rats had been placed on a restricted feeding regime, magazine training began. The rats received three to four daily 30-min sessions of magazine training during which pellets were delivered on a random time (RT) 30 s schedule. Rats were then given two to four daily sessions of lever press training for food pellets during which each lever press resulted in the delivery of a food pellet (continuous reinforcement or CRF training). These sessions lasted until either 30 min expired or 30 food pellets were delivered. Rats which did not lever press during these initial sessions received additional training. Over the next three or four daily sessions, the session length was increased from 30 to 60 min and the schedule on which the pellets were delivered was changed from a random interval 30 s schedule (RI 30 s) to a RI 90 s schedule. On the RI 90 s schedule pellets could be earned by pressing the lever, on average, once every 90 s. The houselight was off throughout all of the training sessions. Conditioned emotional response training During these sessions, which were usually conducted on 4 days per week, food pellets continued to be available on an RI 90 s schedule as before. However, the rats were now presented with

295 2-min long periods of houselight illumination. The first light presentation occurred approximately 20 min after the start of the session (range 15 25 min) and the second light presentation occurred approximately 20 min after the first light presentation (range 15 25 min). Light presentations were occasionally followed by a 0.5-s 0.4-mA footshock. The session ended 15 min after the second light presentation. In a given session, footshock could follow neither, one, or both of the light presentations. Overall, shock occurred following approximately one in three light presentations. In order to maintain reliable conditioned suppression, at least one light shock pairing occurred in a block of three consecutive sessions. The rate of lever-pressing during each of the light presentations (the during period ) and the rate of lever-pressing in the 2-min interval preceding each light presentation (the pre period ) was recorded. These response rates were used to calculate suppression ratios using the following formula: Response rate during Response rate pre + Response rate during A suppression ratio of 0 indicates that the light has evoked conditioned fear and has completely suppressed lever pressing, whereas a suppression ratio of 0.5 indicates the complete absence of fear: the response rate is unchanged by the light presentation. Once a reliable suppression ratio was observed, occasional test sessions occurred. Test sessions were identical to training sessions except that footshock was never delivered following the first light presentation and treatment with either vehicle or a putative anxiolytic compound occurred before the session began. Testing took place no more frequently than once per week and at least two baseline training sessions occurred before each test session. Prior to test sessions,rats which had suppression ratios of greater than 0.15 during the previous baseline session were excluded from the experiment. The remaining rats were semi-randomly assigned to groups such that the groups were matched for the rate of lever pressing and the level of conditioned suppression displayed during baseline training. Drug treatment in each study was orthogonal to drug treatment which may have been administered to the animals in the previous study. In the food deprivation study the animals were deprived of food for 4, 8, 18 or 23 h prior to testing. Statistical analysis A mixed within-subject (trials) and between-subjects (drug treatment) analysis of variance (ANOVA) was conducted separately on the pre and during light response rates and on the suppression ratios. Significant interactions were followed by one-way analysis of variance on the data collected for each trial. Significant group effects were analysed using post-hoc Student Newman-Keuls tests. In experiments in which significant Treatment Trial interactions were identified, the data for each trial are illustrated on the appropriate figure. Otherwise, the data for each experiment are presented collapsed across the two test trials. Data are graphically presented only in experiments in which significant effects were detected. For all statistical analyses, alpha was set to 0.05. Suppression ratios could not be calculated if no lever presses occurred during the pre light period. Thus, animals which failed to respond during either the first or the second light presentation were excluded from the analysis of suppression ratios. The size of the groups and the number of animals contributing to each analysis are shown in Table 1. Drugs The centrally and peripherally acting muscarinic receptor antagonist scopolamine HBr (0.015, 0.03, and 0.06), was used. A higher dose of scopolamine could not be examined as results from a pilot experiment found that only two out of seven rats treated with 0.1 mg/kg scopolamine HBr were able to lever press. To control for any peripheral effects of scopolamine, a control group treated with a 0.06 mg/ kg dose of the mainly peripherally acting muscarinic Table 1 Number of subjects (n) allotted to groups and included in ANOVA of Pre and Dur response rates (Pre/ Dur) and suppression ratios (Ratio) Experiment Group Pre/ Dur Ratio Experiment Group Pre/ Dur Ratio (n) (n) (n) (n) CDP Vehicle 10 10 Deprivation 4 h 11 11 1.0 mg/kg 9 9 8 h 11 11 3.0 mg/kg 10 10 16 h 14 14 10 mg/kg 9 9 23 h 14 14 Diazepam Vehicle 8 7 Scopolamine Vehicle 10 10 1.0 mg/kg 8 6 0.015 mg/kg 9 9 3.0 mg/kg 8 7 0.03 mg/kg 9 9 10 mg/kg 9 6 0.06 mg/kg 9 9 30 mg/kg 8 0 MeBr 9 9 Ipsapirone Vehicle 9 9 8-OH-DPAT Vehicle 8 7 1.25 mg/kg 10 10 0.01 mg/kg 10 9 2.5 mg/kg 10 10 0.03 mg/kg 8 8 5.0 mg/kg 10 10 0.1 mg/kg 10 4 10.0 mg/kg 9 9 Ipsapirone 10 9 Gepirone Vehicle 6 6 Buspirone Vehicle 11 11 1.0 mg/kg 6 6 0.1 mg/kg 10 10 3.0 mg/kg 5 5 0.3 mg/kg 11 11 Ipsapirone 7 7 1.0 mg/kg 10 5 Ipsapirone 11 10 WAY-100635 Vehicle 11 11 WAY-100635 Veh/ Veh 9 8 0.03 mg/kg 11 11 versus Veh/ ipsapirone 10 10 0.3 mg/kg 11 11 Ipsapirone 0.03/ ipsapirone 10 9 3.0 mg/kg 12 12 0.3/ ipsapirone 10 10 Ipsapirone 11 7 3.0/ ipsapirone 10 8

296 antagonist, scopolamine MeBr was included. Scopolamine HBr and MeBr (supplied by Sigma) were administered in saline vehicle in a volume of 1 ml/kg. Diazepam (purchased from Becpharm, 1, 3, 10 or 30 mg/kg) was administered in 0.3% HPMC vehicle and a dosing volume of 1 ml/ kg. Chlordiazepoxide HCl (1.0, 3.0 and 10.0 mg/ kg dissolved in saline) obtained from Sigma was administered in a volume of 1 ml/kg. Buspirone (0.1, 0.3 and 1.0 mg/kg administered in saline), ipsapirone (1.25, 2.5, 5.0 and 10.0 mg/ kg administered in 0.3% HPMC), gepirone (1, 3.0 and 10.0 mg/kg administered in 0.3% HPMC), WAY-100635 (0.03, 0.3, 3.0 mg/ kg administered in saline) and 8-OH-DPAT (0.003, 0.01 0.3 and 0.1 mg/kg administered in saline) were made in the Department of Chemistry, Wyeth Research (UK) Ltd and were administered in a dosing volume of 2 ml/kg. A higher dose of 0.3 mg/kg of 8-OH- DPAT could not be used as rats treated with this dose of 8-OH- DPAT in a pilot study did not lever press. All doses are expressed as base weight and all compounds were administered subcutaneously 30 min prior to testing with the exception of 8-OH-DPAT which was administered with a 5-min pretreatment time. The ph of drugs was adjusted to neutral when necessary using NaOH. In order to facilitate between experiment comparisons and to demonstrate the reliability and sensitivity of the present CER test to anxiolytic-like drug effects, a positive control group given ipsapirone (10.0 mg/ kg, 30-min pretreatment time) was included in the experiments evaluating buspirone, gepirone, 8-OH-DPAT and WAY-100635. Due to the difference in pretreatment time between 8-OH-DPAT and ipsapirone, to ensure that both the 8-OH-DPAT and the ipsapirone treated groups had injections at the same time prior to testing (30 min and 5 min), all animals in the experiment assessing the effects of 8-OH-DPAT received two injections: 0.3% HPMC vehicle and saline vehicle; 0.3% HPMC vehicle and 8-OH-DPAT; or ipsapirone and saline vehicle with the first of the two injections occurring 30 min prior to testing and the second injection occurring 5 min prior to testing. Fig. 1A D Effects of chlordiazepoxide (SC) on mean lever press rates before (open bars) and during (shaded bars) the light presentations (top panels) and on suppression ratios (lower panels). Data for trial 1 are presented in panels A and B and data for trial 2 are presented in panels C and D. Mean ± SEM. *Significantly different relative to the vehicle treated control group Results Chlordiazepoxide Chlordiazepoxide did not significantly affect pre CS response rates (see Fig. 1A, C). Two-way ANOVA on these scores found no significant effect of group nor a significant Group Trial interaction, Fs < 1.0. However, chlordiazepoxide did significantly enhance response rates during the CS presentation. Two-way ANOVA on these scores revealed a significant effect of group, F(3,34) = 4.47,and a significant Group Trial interaction, F(3,34) = 3.97. One-way ANOVA of the during CS scores separately on trial 1 and trial 2 found a significant group difference on both trials, F(3,34) = 4.02 and F(3,34) = 4.86, respectively. Post hoc SNK tests revealed that on trial 1 the vehicle treated group differed significantly from the 10.0 mg/ kg treated group. On trial 2, both the 10 and the 3 mg/kg treated group differed significantly from the vehicle treated group. Two-way ANOVA on the suppression ratios revealed a significant effect of group, F(3,34) = 8.75, but the Group Trial interaction failed to reach statistical significance, F(3,34) = 2.83, P = 0.0532 (see Fig. 1B, D). Post hoc SNK tests revealed that both the 3 and the 10.0 mg/kg treated groups had higher suppression ratios than those observed in the vehicle treated group. Fig. 2A D Effects of diazepam and hours of food deprivation on lever press rates before (open bars) and during (shaded bars) the light presentation (panels A and C, respectively) and on suppression ratios (panels B and D, respectively). Mean ± SEM. * Significantly different relative to the vehicle treated control group (panels A and B) or relative to the 4-h food deprived group (panels C and D) Diazepam Due to the sedative effects of the high dose of diazepam, only three out of eight of the rats treated with 30.0 mg/kg diazepam completed at least one response during both the pre and the dur CS periods on both trials. The data for the group treated with this dose of diazepam were therefore excluded from the statistical analysis of suppression ratios. The results for this experiment are displayed in Fig. 2A, B.

297 Two-way ANOVA on the pre CS scores found a significant effect of Group, F(4,36) = 3.92, and the Group Trial interaction failed to reach statistical significance, F(4,36) = 1.61. Post hoc testing on the mean pre scores revealed that the group treated with 30 mg/kg had a lower response rate than did the vehicle treated group. Two-way ANOVA on the during CS scores, however, revealed a significant group effect, F(4,36) = 3.11, but not a Group Trial interaction, F(4,36) = 1.53. Post hoc tests on the group effect revealed that the group treated with 3.0 mg/kg had a higher during CS response rate than did the vehicle treated group (see Fig. 2A). One rat in both the vehicle and the 3.0 mg/kg treated groups, 2 rats in the 1.0 mg/kg treated group, and three rats in the 10.0 mg/kg treated groups did not respond on both the dur and the pre periods on both trials. Due to the empty data cells, the results for these animals were excluded from the analysis of suppression ratios. Two-way ANOVA on the suppression ratios in the remaining animals revealed a significant group effect, F(3,22) = 7.44, but the Group Trial interaction was not statistically significant, F < 1.0. The post hoc SNK tests revealed that the groups treated with both 10 and 3.0 mg/kg of diazepam had higher suppression ratios than the vehicle treated group (see Fig. 2B). Scopolamine Scopolamine had no significant effects on any of the behavioural measures. Mean pre light response rates (± SEM) for the vehicle, 0.015, 0.03, 0.06 mg/kg scopolamine HBr and the 0.06 mg/kg scopolamine MeBr treated groups were 18.0 (± 3.5), 11.4 (± 1.5), 16.8 (± 3.9), 10.7 (± 2.8) and 16.3 (± 2.0), respectively. The mean during light response rates (± SE mean) were 5.6 (± 3.2), 1.8 (± 0.7), 4.2 (± 2.1), 3.6 (± 1.8) and 2.3 (± 0.7) for the vehicle, 0.015, 0.03 and 0.06 mg/kg treated scopolamine HBr treated groups and the 0.06 mg/ kg scopolamine MeBr treated group, respectively. Finally the suppression ratios (± SEM) were 0.19 (± 0.07), 0.12 (± 0.04), 0.11 (± 0.05), 0.16 (± 0.05) and 0.11 (± 0.02) for the vehicle, 0.015, 0.03, 0.06 mg/kg scopolamine HBr and the 0.06 mg/kg scopolamine MeBr treated groups, respectively. Two-way ANOVA on the group effect failed to find a significant difference, F(4,41) = 1.34, or a significant Group Trial interaction, F < 1. Two-way ANOVA on the during scores and the suppression ratios also failed to reveal any group differences, nor a significant Group Trial interaction, Fs 1.08. Food deprivation Two-way analysis of variance on the pre light scores with drug treatment and trials entered as factors found a significant group effect, F(3,46) = 9.37, but not a significant Trial Dose interaction, F < 1. A post hoc SNK test on the mean pre response rates found that both the 23- and the 16-h food deprived groups responded more than did the 8- and 4-h deprived animals (see Fig. 2C). Although increasing deprivation affected pre light response rates this manipulation did not significantly affect responding during the light. Two-way ANOVA on the during light scores revealed no effect of group, F(3,46) = 2.12, nor a reliable Group Trial interaction, F(3,46) = 1.68. Similarly, changing the deprivation state Fig. 3A D Effects of ipsapirone and 8-OH-DPAT on lever press rates before (open bars) and during (shaded bars) the light presentation (panels A and C, respectively) and on suppression ratios (panels B and D, respectively). Mean ± SEM. * Significantly different relative to the vehicle treated control group

298 did not significantly affect suppression ratios (see Fig. 2D). ANOVA on the suppression ratios did not reveal a reliable group effect, F(3,46) = 2.60, nor a reliable Group Trial interaction, F(3,46) = 1.49. Although the group effect approached statistical significance (P = 0.06), the largest group difference was between the 8- and the 23-h deprived groups with the 4- and 23-h groups having the highest suppression ratios. Ipsapirone Ipsapirone did not significantly suppress pre light response rates (see Fig. 3A). The group effect was not statistically significant, F(4,43) = 1.26, nor was the Group Trial interaction statistically significance, F(4,43) = 1.73. The during light response rates, however, did vary among the drug treatment groups, F(4,43) = 8.32, although the Group Trial interaction did not reach statistical significance, F(4,43) = 1.48. Post hoc testing revealed that the group treated with 10.0 mg/kg ipsapirone responded more during the light presentation than did the vehicle treated group. Analysis of the suppression ratios revealed a significant effect of group, F(4,43) = 18.50, and the Group Trial interaction was not statistically significant, F < 1. Post hoc analysis revealed that the group treated with 10.0 mg/kg ipsapirone had a higher suppression ratio than did the vehicle treated group (see Fig. 3B). 8-OH-DPAT Administration of 8-OH-DPAT did not significantly affect pre light response rates (see Fig. 3C). There was no significant group effect nor a significant Group Trial interaction, Fs(4,41) 1.26. There was a significant drug treatment effect on dur CS scores, F(4,41) = 5.01, and the interaction did not reach statistical significance, F(4,41) = 1.18. The ipsapirone treated group responded more during the CS period than did the vehicle treated group. Analysis of variance on the suppression ratios (see Fig. 3D) revealed a significant group effect, F(4,32) = 4.71, but the Group Trial interaction did not reach statistical significance, F(4,32) = 1.85. Post hoc testing revealed that only the group treated with ipsapirone had a higher suppression ratio than did the vehicle treated group. a significant Drug Trial interaction was observed, F(3,20) = 5.09. A subsequent one-way ANOVA on the group effect on the Pre light scores for the first (see Fig. 4A) and second light presentation (see Fig. 4C) did not reveal the source of this interaction, Fs(3,20) = 2.30 and 0.25, respectively, although the interaction appears to due to the larger trend for a reduction in pre light response rates on trial 1 than on trial 2. There was a statistically significant main effect of drug treatment on during light response rates, F(3,20) = 5.58. The Group Trial interaction did not reach statistical significance, F < 1. The post hoc test of the during light scores indicated that both the 3.0 mg/kg treated group and the ipsapirone treated group had significantly higher during light response rates than did the vehicle treated group. Drug treatment significantly affected the suppression ratios, F(3,20) = 18.18, and the Trial Group interaction did not reach statistical significance, F < 1. The ipsapirone and the gepirone (3.0 mg/ kg) treated groups had significantly higher suppression ratios than did the vehicle treated group (see Fig. 4B, D). Buspirone Buspirone did not induce an anxiolytic-like profile in this test (see Fig. 5). Two-way ANOVA on the pre light rates of lever pressing revealed a significant main effect of drug treatment, F(4,48) = 4.42, and the Drug Trial interaction was also statistically significant, F(4,48) = 3.21. One-way ANOVA on both trial 1 and trial 2 (see Fig. 5A, C) revealed significant group effects, Fs(4,48) = 5.02 and 3.53, respectively. Post hoc tests revealed Gepirone The animals treated with the highest dose of gepirone (10.0 mg/kg) showed complete abolition of lever pressing. Therefore, the results for this group were excluded from the statistical analyses. In the remaining groups, drug treatment did not induce a significant overall suppression in pre light response rates, F(3,20) = 1.06, but Fig. 4A D Effects of gepirone on mean lever press rates before (open bars) and during (shaded bars) the light presentations (top panels) and on suppression ratios (lower panels). Data for trial 1 are presented in panels A and B and data for trial 2 are presented in panels C and D. Mean ± SEM. *Significantly different relative to the vehicle treated control group

299 Fig. 5A D Effects of buspirone (SC) on mean lever press rates before (open bars) and during (shaded bars) the light presentations (top panels) and on suppression ratios (lower panels). Data for trial 1 are presented in panels A and B and data for trial 2 are presented in panels C and D. Mean ± SEM. * Significantly different relative to the vehicle treated control group that on trial 1 both the ipsapirone and the 1.0 mg/kg buspirone treated groups displayed a lower rate of lever pressing than did the vehicle treated animals. On trial 2, only the group treated with 1.0 mg/kg buspirone was suppressed during pre light period relative to the vehicle treated group. Two-way ANOVA on the during light response rates revealed a significant group effect on trial 1, F(4,48) = 4.78, although the Group Trial interaction was not statistically significant, F(4,48) = 1.36. Post hoc testing showed that only the rats in the ipsapirone group responded with a higher rate of lever pressing than the vehicle treated rats during the light presentation. Two-way ANOVA on the suppression ratios revealed a significant effect of group, F(4,42) = 17.50. The Group Trial interaction was also statistically significant, F(4,42) = 3.05. A significant group difference was observed on both trial 1 and 2, Fs(4,42) = 8.70, and 20.05(see Fig. 5B, D, respectively). The suppression ratio in the ipsapirone treated rats was significantly higher than for the vehicle treated rats on trial 1 and trial 2. The animals treated with the highest dose of buspirone also had a suppression ratio that was significantly higher than the vehicle treated group on trial 2. However, as indicated by the pre and dur light response rates, this increase in suppression ratio induced by buspirone is due to a decrease in pre light scores as opposed to an increase in during light response rates and should not be misinterpreted as an anxiolytic-like effect. WAY-100635 At the doses tested, WAY-100635 had no significant effects on the performance of lever pressing in the present test. Two-way ANOVA on pre CS scores did not reveal a group effect, F(4,51) = 1.54; however, the Group Trial interaction did achieve statistical significance, F(4,51) = 3.20. One-way ANOVA on each trial revealed a significant group difference on trial 1, F(4,51) = 2.89, but not on trial 2, F < 1 (see Fig. 6A and C, respectively). On trial 1 the rats treated with ipsapirone had a lower pre CS response rate than did the vehicle treated rats. Two-way ANOVA on the during CS scores revealed a reliable group effect, F(4,51) = 3.49. The Group Trial interaction failed to reach statistical significance, Fig. 6A D Effects of WAY-100635 (SC) on mean lever press rates before (open bars) and during (shaded bars) the light presentations (top panels) and on suppression ratios (lower panels). Data for trial 1 are presented in panels A and B and data for trial 2 are presented in panels C and D. Mean ± SEM. *Significantly different relative to the vehicle treated control group

300 F(4,51) = 1.89. Post hoc testing on the group effect revealed that the ipsapirone group had a higher during CS response rate than did the vehicle treated group. WAY-100635 did not affect suppression ratios (see Fig. 6B, D). A significant main effect of group, F(4,47) = 16.9, but not a significant Group Trial interaction, F < 1, was observed when a two-way ANOVA was conducted on the suppression ratios. Post hoc testing revealed that only the group treated with ipsapirone had significantly higher suppression ratio than did the vehicle treated group. Two-way ANOVA on the suppression ratios revealed a significant effect of group, F(4,40) = 18.47, and the Group Trial interaction failed to reach statistical significance, F(4,40) = 2.52. Post hoc testing on the group effect revealed that the group treated with ipsapirone alone had a suppression ratio significantly higher than the ipsapirone +0.03 mg/ kg WAY-100635 group which in turn had a suppression ratio significantly higher than all the other treatment groups (see Fig. 7B). WAY-100635 versus ipsapirone The anxiolytic-like effects of ipsapirone (10.0 mg/ kg) were dose dependently reversed by WAY-100635. Twoway ANOVA on the pre CS scores revealed no significant group effect, F(4,44) = 1.75, nor a significant Group Trial interaction, F(4,44) = 1.69. Two-way ANOVA on the during CS scores did reveal a significant group effect, F(4,44) = 10.47, but the Group Trial interaction failed to reach statistical significance, F(4,44) = 2.15. Post hoc testing revealed that the group treated with ipsapirone alone or ipsapirone +0.03 mg/ kg WAY-100635 differed significantly from all the other treatment groups. Thus, 0.3 and 3.0 mg/ kg WAY- 100635 completely reversed the ipsapirone induced increase in dur CS responding (see Fig. 7A). Fig. 7A,B Effects of WAY-100635 and ipsapirone (SC) on lever press rates before (open bars) and during (shaded bars) the light presentation (panel A) and on suppression ratios (panel B). Mean ± SEM. * Significantly different relative to the vehicle/ vehicle treated control group. + Significantly different relative to the ipsapirone/ vehicle treated group Discussion The benzodiazepine receptor agonists, chlordiazepoxide (3.0 mg/kg) and diazepam (3.0 mg/kg), and the 5-HT 1A receptor partial agonists, ipsapirone (10.0 mg/kg) and gepirone (3.0 mg/kg), induced anxiolytic-like effects in the CER test. However, within the range of doses tested, 8-OH-DPAT, buspirone and WAY-100635 failed to induce an anxiolytic-like effect in this test. At the highest doses, 8-OH-DPAT and buspirone significantly suppressed responding for food pellets, presumably due to induction of the behavioural 5-HT syndrome (Tricklebank 1987) although the suppression of lever pressing induced by buspirone may also be due to its D 2 receptor antagonist effects (see Ryan et al. 1993). Clearly, the anxiolytic-like actions of drugs which disrupt lever pressing are difficult to assess in this model. By contrast, WAY-100635 had no effect on any of the behavioural measures in the CER test. The failure to observe a behavioural effect of WAY- 100635 cannot be due to an insufficient dose: WAY- 100635 was administered at doses which are know to be sufficient to block the 5-HT behavioural syndrome, hypothermia, hyperphagia and the disruption of lever pressing for food induced by 8-OH-DPAT (Fletcher et al. 1995). Moreover, WAY-100635 was found to block the anxiolytic action of ipsapirone. It therefore appears that a silent 5-HT 1A receptor antagonist, in contrast to the partial agonists, ipsapirone and gepirone, is inactive in the CER test. The present failure to observe an anxiolytic-like effect with WAY-100635 contrasts with previous reports of anxiolytic-like actions of the 5-HT 1A receptor antagonists WAY-100135, WAY-100635 and (S)-UH-301 in the light/dark box in mice, and elevated maze test in rats and mice (Moreau et al. 1992; Bill and Fletcher 1994a,b; Rodgers and Cole 1994). In contrast to the data obtained in these ethological models of anxiety, Charrier et al. (1994) failed to observe an anxiolyticlike action of WAY-100135 in the safety signal withdrawal conflict procedure in the rat. Perhaps the most straightforward explanation for the discrepancy between the results from these various studies is due to differences in the models used. Positive effects were obtained with 5-HT 1A receptor antagonists in

301 ethological models measuring innate or unlearned fear whereas negative effects were observed in conditioning models employing learned fear. It has previously been suggested that the type of model used to measure responses to 5-HT 1A receptor ligands appears critical to the determination of their anxiolytic effects. Treit et al. (1993), for example, reported that equivalent doses of 8-OH-DPAT induced an anxiogenic effect in the rat elevated plus maze test and an anxiolytic effect in the rat shock-probe burying test in which a probe is associated with electric shock. Although we have consistently found anxiolytic-like effects with ipsapirone in the CER test, unpublished work conducted by Savraj Grewal in our laboratory has found that acute or chronic treatment with ipsapirone produces either anxiogenic or sedative effects in the rat elevated maze test. The clinical implications of the positive effects obtained in the ethological tests and the negative results obtained with 5-HT 1A receptor antagonists in learned anxiety tests is unknown. It is clear, however, that determination of the effects of 5-HT 1A receptor antagonists in the clinic will have important implications for the predictive validity of both ethological and conditioned fear models of anxiety in animals. Interestingly, Charrier et al. (1994) reported that the 5-HT 1A receptor antagonist WAY-100135 failed to block the anxiolytic action of buspirone in the safety signal withdrawal model, questioning the pharmacological specificity of the anxiolytic effects of buspirone in their test. However, the 5-HT 1A receptor antagonist, WAY-100635, which is 100-fold selective relative to binding at other major neurotransmitter receptors (see Forster et al. 1995), completely and dose-dependently reversed the anxiolytic-like effects of ipsapirone in the present test. This is consistent with previous results from studies using non-selective 5-HT 1A receptor antagonists (Chojnacka-Wojcik and Przegalinski 1991) and provides compelling evidence that the anxiolytic-like actions of ipsapirone are due to its agonist effects at the 5-HT 1A receptor. Given that both ipsapirone and gepirone induced an anxiolytic-like effect, it is curious that 8-OH-DPAT and buspirone were inactive in the present test. As mentioned above, the failure to observe an anxiolytic-like effect with buspirone could be due to its D 2 receptor antagonist properties which may disrupt lever pressing and thereby mask any anxiolytic-like effect due to its 5-HT 1A receptor agonist actions. However, the disruption of lever pressing induced by 8-OH-DPAT is almost certainly due to the induction of the 5-HT behavioural syndrome, an effect which is also mediated by an agonist action at the 5-HT 1A receptor (Tricklebank 1987). It is unclear why ipsapirone and gepirone, but not 8- OH-DPAT, appear able to alleviate conditioned suppression of responding at doses lower than those which induce the 5-HT behavioural syndrome and thereby impair the animal s ability to perform the test. Our results are, however, similar to those obtained by Sanger who observed robust anxiolytic-like effects with ipsapirone, but no evidence of an anxiolytic-like effect of 8-OH-DPAT in a CER test (Sanger 1990) and a punishment procedure (Sanger 1992). The CER test as conducted here has certain advantages over other CER tests in which the conditioned stimulus is consistently paired with shock and over conflict tests such as the Geller-Seifter (see Treit 1984), as it allows for the anxiolytic activity of drugs to be examined in the absence of any potential confounding analgesic effects. This is because on drug test days the behavioural data are collected before the animal is exposed to the aversive footshock. However, another potential confound in most shock-induced response suppression tests that are used to screen for anxiolytic drugs is the effect of drugs on appetite or satiety mechanisms. As pointed out by Treit (1984), conflict tests, by definition, involve crossed motivations (e.g., the attainment of food or water and the avoidance of shock (p. 209). Therefore, compounds such as benzodiazepines and 5-HT 1A receptor partial agonists which are know to increase feeding in rodents (e.g. Dourish et al., 1985a, b; Cooper, 1989), may induce false positives in a conflict or a conditioned suppression model by enhancing the animal motivation to lever press for food (e.g., Leslie, 1977). Due to ceiling effects in baseline response rates, enhanced motivation could lead to an increase in the rate of responding during the signal for shock with no effect on response rates when the signal is off. This pattern of results could be misinterpreted as an anxiolytic effect. It seems unlikely that enhanced motivation to respond for food can account for the anxiolytic-like effects of ipsapirone observed in the present studies. Ipsapirone (10.0 mg/kg) did not increase pre light rates of lever pressing in any of the present experiments including those experiments in which the baseline response rates were clearly submaximal (e.g., see Figs. 3C and 7A). By contrast, enhancing the level of food deprivation from 4 to 23 h resulted in a significant increase in pre, but not during light rates of lever pressing. Thus, the ipspairone induced increase in lever pressing during the light signal for shock in the present paradigm appears to be independent of any effects of the drug on motivation to respond for food. It is also possible that memory disrupting drugs may induce false positive anxiolytic-like effects in the CER test. Both benzodiazepine and 5-HT 1A receptor agonists have been claimed to have cognitive disrupting effects (e.g., Hunter and Roberts 1987; Winter and Petti 1987; Preston et al. 1989) and could induce the forgetting of conditioned fear. This explanation seems unlikely given that the CER procedure involves simple associative learning that is well established prior to the commencement of drug testing (see Hoffman et al. 1963; Hendersen 1985). Moreover, the amnesic agent scopolamine has not been found to produce a false positive anxiolytic-like effects in a CER test (e.g.,

302 Miczek 1973). However, the unpredictable nature of shock in the present CER test could make it more sensitive to the amnesic effects of drugs than traditional CER tests such as that conducted by Miczek (1973). To determine whether an amnesic agent would produce an anxiolytic-like effect in the present studies, we examined the effects of the muscarinic receptor antagonist, scopolamine HBr, which has well established cognitive disrupting effects (Weingartner 1985; e.g., Preston et al. 1988). When administered at doses up to 0.06 mg/kg, scopolamine HBr had no effects on conditioned suppression of responding. However, in cognitive experiments conducted in our laboratory doses as low as 0.025 mg/kg scopolamine has been shown to disrupt the performance of working memory in a delayed matching to position task (see Stanhope et al. 1995). By contrast, unpublished results from the delayed matching-to-position task conducted in our laboratory have failed to find any memory impairing effects of ipsapirone when administered up to a dose of 10 mg/kg (for a similar result see Cole et al. 1994). Given that scopolamine appears to have a more pronounced cognitive disruptive effect than ipsapirone, yet ipsapirone, but not scopolamine induced an anxiolyticlike effect in the present studies, it seems unlikely that this anxiolytic-like effect is due to the forgetting of conditioned fear. In conclusion, the present studies show that 5-HT 1A receptor partial agonists but not a full 5-HT 1A receptor agonist or a silent 5-HT 1A receptor antagonist have anxiolytic effects in the present CER model. These anxiolytic-like effects are not secondary to changes in the sensitivity of the animals to the aversive stimulus, changes in motivation to respond for food, or the induction of amnesia resulting in the forgetting of conditioned fear. 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