Psychopharmacology (1998) 140 : 105–115

© Springer-Verlag 1998

O R I G I NA L I N V E S T I G AT I O N

Justin A. Harris · R. Fred Westbrook

Evidence that GABA transmission mediates context-specific extinction of learned fear

Received : 5 April 1998 / Final version : 29 July 1998

Abstract Six experiments used rats to study the e¤ects of the b-carboline FG 7142 on extinction of fear responses (freezing) to an auditory cue that had signalled footshock. Subcutaneous injection of FG 7142 interfered with the development of extinction without having any detectable e¤ect on the rats’ levels of fear prior to extinction. Injection of FG 7142 also reversed extinction, partially reinstating fear responses that had been extinguished previously. A similar reinstatement of extinguished fear was seen when rats were tested for fear of the cue in a di¤erent chamber. The reinstatement produced by FG 7142 and that caused by context shift were not additive : FG 7142 did not increase extinguished fear if rats were tested in the di¤erent chamber. Finally, FG 7142 had no detectable e¤ect on the latent inhibition of fear produced by repeatedly presenting the cue alone before conditioning with shock, even though this inhibition, like extinction, was a¤ected by a shift in context. The present Þndings indicate that GABA transmission at GABAA receptors is involved in the inhibition of extinguished fear, and that this e¤ect of GABA is regulated by those cues that constitute the extinction context. Key words FG 7142 · b-Carboline · Benzodiazepine inverse agonist · Latent inhibition · Rat

Introduction
Benzodiazepines reduce the expression of fear and “anxiety” responses elicited by innate or learned sources of danger (see File 1988; Green 1991). For
J.A. Harris (*) · R.F. Westbrook School of Psychology, The University of New South Wales, Sydney 2052, Australia e-mail : j.harris@unsw.edu.au, Fax : +612-9385-3641

example, these drugs inhibit the conditioned potentiation of startle and changes in heart rate elicited by a signal for shock (Davis 1979; Pascoe et al. 1983), as well as reducing the freezing, analgesic and passive avoidance responses provoked by a chamber associated with shock or a heated ßoor (Venault et al. 1986; Fanselow and Helmstetter 1988; Harris and Westbrook 1994, 1996). Benzodiazepines act to promote the inhibitory e¤ects of c-amino butyric acid (GABA) at the GABAA receptor complex (Haefely 1990). Thus, the decrease in fear produced by benzodiazepines indicates that the neural mechanisms underlying fear are inhibited by GABA transmission. This conclusion is supported by the observation that GABAA receptors are particularly abundant in the amygdala (McDonald 1985; Thomas et al. 1985), a structure with a wellestablished role in the neural substrate of fear (for recent reviews, see Davis 1992; Maren 1996; Pitkänen et al. 1997). More direct evidence that the amygdaloidbased fear mechanisms are under inhibitory control by GABA comes from demonstrations that microinjection of GABAA receptor agonists (e.g., muscimol or benzodiazepines) into the amygdala reduces learned fear (Helmstetter 1993; Helmstetter and Bellgowan 1994; Harris and Westbrook 1995, 1998; Muller et al. 1997), whereas microinjection of a GABAA receptor antagonist (bicuculline) into the amygdala provokes responses indicative of fear (Sanders and Shekhar 1991). If fear responses are under the inhibitory control of GABA transmission, what is the normal function of that transmission in regulating fear? In the present study, we have addressed this question by investigating the e¤ects of a b-carboline on the expression of fear responses in rats. b-Carbolines are a class of compounds which bind to the benzodiazepine site on the GABAA receptor complex, but in contrast to the benzodiazepines, b-carbolines antagonise the inhibitory e¤ects of GABA (Haefely 1990). Thus, at the behavioural level, one might expect these compounds to

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produce or potentiate fear responses. Indeed, administration of a b-carboline has been reported to provoke analgesia (Fanselow and Kim 1992), to potentiate startle responses (Hijzen and Slangen 1989), and to induce an anxiety-like state in rats (File et al. 1982, 1985; Pellow and File 1986; see Thiébot et al. 1988, for a comprehensive review). Moreover, if GABA transmission underlies normal inhibition of fear, then b-carbolines should have their greatest e¤ect in promoting fear responses under circumstances when fear is inhibited. Accordingly, we examined the e¤ects of the b-carboline FG 7142 on the extinction of fear that results from exposure to a feared cue in the absence of the expected noxious event. The present experiments used freezing as an index of fear to an auditory cue that had signalled footshock in rats (Blanchard and Blanchard 1972). We investigated the e¤ects FG 7142 on this fear response, and in particular, on the loss or extinction of freezing when the cue is repeatedly or continuously presented without shock. Once extinguished, conditioned responses are rapidly reacquired with renewed training, and even show spontaneous recovery after a delay or if the subjects are tested in a di¤erent context (e.g., Pavlov 1927; Bouton 1993). On the basis of these demonstrations, contemporary learning theories hold that extinction is mediated by the acquisition of a separate inhibitory association between the cue and the outcome which opposes the original excitatory association between these events (Rescorla 1979; Wagner 1980; Bouton 1993). Thus, we postulate that the neural basis of this inhibitory association is mediated by inhibitory synapses involving GABAA receptors. To test this hypothesis, in experiment 1, two groups of rats were trained with a single pairing of the auditory cue and shock, while another two groups received exposure to the cue but did not receive shock. The following day, all four groups were injected with FG 7142 or its vehicle and tested for fear when the cue was continuously presented for 8 min.

Apparatus Rats were trained and tested in four standard conditioning chambers (23 × 21 × 20 cm : depth × width × height). The walls and lid of these chambers were constructed of clear plastic, and the ßoor consisted in stainless steel bars, 2 mm in diameter, spaced 13 mm apart (centre to centre). Unscrambled AC 50-Hz shock from a constant current generator was delivered to the ßoor of each chamber, and the current available to each ßoor was adjusted by reference to an in-line milliamp meter. The chambers were placed in separate compartments of a wooden cabinet, and the door of each compartment was kept open to permit observation of each rat. The auditory cue (a “clicker” consisting in a 75 dB white noise with an interruption rate of 10 Hz) was presented from a loudspeaker, connected to an ampliÞer, mounted on the ceiling of the laboratory directly above the conditioning chambers. During the experiments, the rats were observed and their behaviour recorded using a video camera mounted on the wall of the laboratory opposite the chambers and connected to a video recorder and monitor located in an adjacent room.

Drugs FG 7142 (N-methyl-b-carboline-3-carboxymide) was suspended at a concentration of 10 mg/ml in sterile isotonic saline (0.9% w /v) using 1 drop of Tween 80 per 5 ml saline. FG 7142 or its vehicle (saline plus Tween 80) was administered in a volume of 1.0 ml/kg by subcutaneous injection into the dorsal region of the neck.

Procedure Rats were handled for 2 min a day for at least 3 days and assigned to four weight-matched groups prior to the start of the experiment. On day 1, all four groups of rats (all ns = 8) were placed into the shock chambers, and 2 min later the auditory cue (75dB “clicker”) was presented for 10 s. For rats in two groups, a single 0.8-mA, 1-s shock was delivered through the ßoor of the chamber at the termination of the cue. Shock was not delivered to rats in the other two groups. All rats were left in the chamber for a further 50 s, before being put back in their home cage and returned to the colony room. Between each rat, the chambers were wiped clean with water to remove any odours. On day 2, rats were injected with either FG 7142 or vehicle solution, and 15 min later, were placed into the shock chamber for 10 min. The auditory cue was presented continuously for the Þnal 8 min of that session, but no shock was delivered.

Scoring Each rat was scored for freezing during the full 10 min of the test session. Freezing was scored as the absence of all movement, except those related to breathing (Fanselow 1980). The behaviour of each rat was recorded on videotape, and freezing was rated with a timesampling procedure in which each rat was observed once every 3 s. A percentage score was calculated for the proportion of the total observation period. The test sessions were rated by two observers, one of whom was naive to the rats’ group designations. There was a high degree of agreement between the two observers : the Pearson product-moment correlations between their ratings were above 0.95.

Experiment 1: FG 7142 and freezing
Materials and methods
Subjects Subjects were 32 experimentally naive male Wistar rats (250–350 g), obtained from the colony of SpeciÞc Pathogen Free rats maintained by the Combined Universities Laboratory Animal Service, at Little Bay, Sydney. Rats were housed in groups of eight in plastic boxes (67 × 40 × 22 cm : depth × width × height) kept in a colony room maintained on natural light-dark cycle. Food and water were continuously available. All procedures described here have been approved by the Animal Care and Ethics Committee at The University of New South Wales.

Statistical analyses The data in this and each subsequent experiment were analysed using planned orthogonal contrasts (Hays 1972) testing di¤erences

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Fig. 1 Mean percent of time rats spent freezing during the 10-min test session in experiment 1. The previous day, rats had been placed into a shock chamber, and for two groups (shock) an auditory cue was presented followed by delivery of a footshock, whereas no stimuli were presented for rats in the other two groups (no shock). On test day, rats were injected with either FG 7142 (FG) or its vehicle before being placed into the shock chamber and scored for freezing to the contextual cues for the 2 min. At the end of this “pre-cue” period, the auditory cue was turned on for 8 min while the rats continued to be scored for freezing. –n– Shock FG, –l– shock vehicle, --o-- no shock FG, --¡-- no shock vehicle

between FG 7142 and vehicle-treated rats and trends across minutes of test. SpeciÞc comparisons were tested using post hoc orthogonal contrasts (Rodger 1967). For each analysis, the decision-wise error rate (a) was set at 0.05.

Results The percent of time rats spent freezing on day 2 was broken up into 2-min bins, the Þrst bin (before presentation of the cue) representing freezing to the chamber alone, and the last four bins representing freezing to the chamber and auditory cue. The mean percentages of freezing for all four groups are shown in Fig. 1. The two groups of rats that had not been shocked did not show reliable levels of freezing during the test session, and in this regard, there was no di¤erence between rats injected with FG 7142 and rats given vehicle. Thus, FG 7142 does not induce freezing in rats. For the two groups that were shocked, both groups began freezing immediately on placement in the chamber, before presentation of the auditory cue, and the levels of freezing shown by the two groups were approximately equal during this period. For both groups, the level of freezing immediately increased when the cue was turned on, but this freezing progressively decreased across the 8 min of the cue presentation, as the rats learned that the expected shock would not occur. Importantly, the two groups of rats di¤ered in the rate of this extinction of fear, with the level of freezing decreasing faster in the vehicle-injected rats than in rats injected with FG 7142. These observations were conÞrmed by the planned statistical analysis. This analysis was carried out using only the data

from the two groups of shocked rats; data from the two non-shocked groups were excluded because the scores were at ßoor, thus making their variance artefactually low. Thus, the critical F value with 1,14 df is 4.6. Rats given FG 7142 displayed signiÞcantly more freezing overall during the presentation of the auditory cue than rats injected with vehicle, (F 1,14 = 5.25, P < 0.05). Moreover, there was a signiÞcant di¤erence between the two groups in the linear trend across minutes of test, (F 1,14 = 6.02, P < 0.05), conÞrming that the rate of extinction of freezing in rats injected with FG 7142 was slower than that in rats given vehicle. While the di¤erence in linear trend between the two groups is consistent with the interpretation that FG 7142 has retarded extinction, it is also possible that the di¤erence is an artefact produced by a ceiling e¤ect during the Þrst few minutes of presentation of the auditory cue. According to this interpretation, FG 7142 serves to increase freezing uniformly, but such an e¤ect was not detectable initially because freezing in the vehicle rats was already at ceiling. However, such an interpretation is not supported by the data from the 2 min before presentation of the auditory cue. As is evident in Fig. 1, the level of freezing during this period was well below ceiling, yet there was no apparent di¤erence between the two groups. A post hoc statistical analysis conÞrmed this conclusion. There was a signiÞcant increase in freezing for both groups between the 2 min before onset of the auditory cue and the Þrst 2 min of the cue presentation, (F 3,14 = 34.66; P < 0.001), indicating that both groups were below ceiling during the pre-cue period. Further, there was no di¤erence between the two groups during the pre-cue period (F < 1), nor was there any evidence for a di¤erence between the groups in the degree to which their levels of freezing increased after the onset of the auditory cue (F < 1). In other words, when the level of freezing was relatively low, such as that supported by the chamber alone, then FG 7142 had no detectable e¤ect on freezing. However, if the level of freezing was low due to extinction of conditioned fear, then FG 7142 signiÞcantly increased freezing.

Experiment 2: FG 7142 and the acquisition versus expression of extinction
In experiment 1, the extinction of fear was retarded among rats injected with FG 7142, suggesting that the drug had impaired the acquisition of extinction (i.e., had stopped the rats from learning that the cue no longer signalled shock) and / or had interfered with the expression of that extinction (i.e., had prevented the rats from inhibiting fear responses to the extinguished cue). To examine these alternatives, experiment 2 consisted of three distinct stages : on day 1, the cue was conditioned with shock, on day 2 fear of the cue was

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extinguished, and on day 3, rats were tested for fear of the cue. For extinction training on day 2, the cue was repeatedly presented 6 times for 2 min each in order to increase the carry-over of extinction to test on day 3. Four groups of rats were injected with FG 7142 or vehicle before extinction training on day 2 and / or before test on day 3. Thus, one group received FG 7142 on day 2 and vehicle on day 3, a second group received vehicle on day 2 and FG 7142 on day 3, a third group received FG 7142 on both days, and the fourth group received vehicle on both days. Materials and methods
Subjects and materials Subjects were 40 experimentally naive male Wistar rats (320–400 g), obtained from the same source and maintained in the same manner as described for experiment 1. Rats were trained and tested in the four conditioning chambers with the auditory cue (clicker) described above. FG 7142 was prepared and administered in the manner described above.

Procedure and statistical analyses Rats were handled and allocated to four weight-matched groups (all ns = 10). On day 1, rats were trained with shock signalled by the auditory cue, as in experiment 1. On day 2, two groups of rats were injected with FG 7142 and two groups with vehicle, and 15 min later were placed into the shock chamber for a total of 24 min. Two minutes after being placed into the chamber, the auditory cue was turned on for 2 min. The cue was presented in this way 6 times during the 24 min session, with 2 min interposed between successive presentations. No shock was delivered during this session. Rats were scored for freezing during the 2-min “precue” period at the beginning of the session and during each presentation of the cue. On day 3, rats were again injected with either FG 7142 or its vehicle (of the two groups given FG 7142 on day 2, one was now injected with vehicle and the other with FG 7142, and likewise for the two groups given vehicle on day 2). Fifteen minutes later, each rat was placed into the chamber and, after 2 min, the auditory cue was presented once for 2 min. During this presentation, the rats were scored for freezing.For the data of freezing during the pre-cue period on day 2, a single comparison between rats given FG 7142 and rats given vehicle was made using a Student’s t-test. The remaining data were analysed using planned orthogonal contrasts (Hays 1972) testing for di¤erences between rats given FG 7142 and rats given vehicle on day 2, and between rats given FG 7142 and rats given vehicle on day 3. Because the data were analysed from two separate tests (days 2 and 3), the decision-wise error rate (a) was set at 0.025. Thus, the critical F for the planned contrasts is 5.42.

Fig. 2a,b Mean percent of time rats in experiment 2 spent freezing during the 2-min “pre-cue” period before the Þrst presentation of the auditory cue, and during each of the Þve 2-min presentations of the cue, after being injected with either FG 7142 (FG) or its vehicle (a). Rats were tested again for freezing to the cue the following day (b). On this occasion, half the rats previously given FG were again injected with the drug (FG FG ) and the other half were given vehicle (FG Veh), while half the rats previously given vehicle were injected with FG (Veh FG) or vehicle (Veh Veh). n FG 7142, ¡ vehicle

Results Figure 2 presents the results of experiment 2. During the Þrst 2 min of extinction training, before the initial presentation of the auditory cue, all rats began freezing, and there was no apparent di¤erence in levels of freezing between rats injected with FG 7142 and those given vehicle. During the Þrst presentation of the

cue, the levels of freezing rose sharply, but then fell gradually across successive cue presentations. As in experiment 1, the level of freezing declined more slowly among rats injected with FG 7142 than rats given vehicle. On test the following day, rats that had been injected with FG 7142 before extinction, test, or both, displayed more freezing than rats injected with vehicle before extinction and test. The statistical analysis conÞrmed that, in the 2-min pre-cue period on day 2, there was no statistically reliable di¤erence between rats given FG 7142 and rats given vehicle, (t 1,38 < 1). However, across presentations of the cue during extinction, the overall level of freezing was signiÞcantly higher among rats injected with FG 7142 than rats injected with vehicle, (F 1,38 = 17.0, P < 0.001). Moreover, the two groups di¤ered signiÞcantly in the linear trends across trials (F 1,38 = 20.9, P < 0.001), revealing that rats injected with FG 7142 extinguished more slowly than rats given vehicle.

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Analysis of the data from day 3 identiÞed a signiÞcant main e¤ect for drug administration on the previous extinction day, (F 1,36 = 5.8, P < 0.025). That is, the two groups that had been injected with FG 7142 before extinction on day 2 showed more freezing at test on day 3 than the two groups extinguished with vehicle. Moreover, among the two groups given vehicle before extinction on day 2, rats injected with FG 7142 before test on day 3 (Group Veh FG) froze signiÞcantly more than rats injected again with vehicle before test (Group Veh Veh), (F 1,36 = 7.1, P < 0.025). In contrast, among the two groups extinguished under FG 7142 on day 2, there was no di¤erence between rats injected again with the drug (FG FG) and those given vehicle (FG Veh) (F < 1). Thus, the acquisition and expression of extinction of fear was reduced if rats were injected with FG 7142 before extinction training, before test, or before both extinction and test.

The cue was presented in this way three times during the 12-min session, with 2 min interposed between successive presentations. No shock was delivered during this session. This procedure was repeated on day 3. On day 4, rats were injected with one of the three solutions of FG 7142 or with vehicle. Thus, di¤erent groups received 0, 2.5, 5.0 and 10.0 mg / kg of FG 7142. Fifteen minutes later, each rat was placed into the chamber and, after 2 min, the auditory cue was presented once for 2 min. During this presentation, the rats were scored for freezing.The data were analysed using planned orthogonal contrasts (Hays 1972) testing linear, quadratic and cubic trends across drug doses. Post hoc orthogonal contrasts (Rodger 1967) were also tested. With the decision-wise error rate (a) set at 0.05, the critical F for the planned and post hoc contrasts is 4.20 and 5.90, respectively.

Results The mean percent of time spent freezing on test for rats in experiment 3 is presented in Fig. 3. The three groups injected with FG 7142 froze more than the group given vehicle. However, this e¤ect of FG 7142 varied as a function of the dose : rats given the low dose of FG 7142 (2.5 mg / kg) showed only a slight increase in freezing relative to control rats given vehicle, whereas rats given the middle and high doses (5.0 and 10.0 mg / kg, respectively) showed much greater levels of freezing. The planned statistical analysis identiÞed a signiÞcant linear trend for freezing as a function of drug dose (F 1,28 = 6.16, P < 0.05), but there were no signiÞcant quadratic or linear trends, (Fs < 1). The post hoc analysis conÞrmed that the two groups injected with 5.0 and 10.0 mg / kg FG 7142 froze signiÞcantly more than two groups given 0 and 2.5 mg / kg FG 7142 (F 3,28 = 6.08, P < 0.05). There were no signiÞcant di¤erences between rats given 5.0 and rats given 10.0 mg / kg FG 7142 (F < 1), nor between rats given 0 and rats given 2.5 mg / kg of the drug (F < 1).

Experiment 3: dose-response function for FG 7142 and extinguished fear
Experiment 2 revealed that FG 7142 not only interferes with the acquisition of extinction, but that the drug also reverses the expression of that extinction, reinstating extinguished freezing. Experiment 3 examined the dose-response function for this reinstatement of extinguished fear by FG 7142. Four groups of rats were trained with the auditory cue and shock on day 1, their fear of the cue was extinguished on days 2 and 3, then they were tested on day 4 after being injected with 0, 2.5, 5.0 or 10.0 mg / kg FG 7142. The extinction training was conducted over 2 days, so that the levels of performance on test would be very low, thereby allowing us to investigate the generality of the e¤ects of FG 7142 on extinguished fear. Materials and methods
Subjects and materials Subjects were 32 experimentally naive male Wistar rats (300–380 g), obtained from the same source, and maintained in the same manner as described for experiment 1. Rats were trained and tested in the four conditioning chambers with the auditory cue (clicker) described above. Three separate solutions of FG 7142 were prepared : the concentration of FG 7142 being 2.5, 5.0 or 10.0 mg / ml in vehicle (saline plus 1 drop of Tween 80 per 5 ml). FG 7142 or vehicle was injected in the manner described above.

Procedure and statistical analyses Rats were handled and allocated to four weight-matched groups (all ns = 8). On day 1, rats were trained with shock signalled by the auditory cue, as in experiment 1. On day 2, all four groups were placed into the shock chamber for 12 min. Two minutes after being placed into the chamber, the auditory cue was turned on for 2 min.

Fig. 3 Mean percent of time rats spent freezing during the 2-min presentation of an auditory cue in experiment 3. On day 1, the cue was paired with shock, then on days 2 and 3, the rats’ fear of the cue was extinguished in the conditioning chamber. On day 4, rats were injected with FG 7142, at a dose of 2.5, 5.0 or 10.0 mg / kg, or with vehicle (0 mg / kg) before being tested for fear of the cue in the conditioning chamber

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Experiments 4 and 5: FG 7142 and context-specific extinction
Across a variety of paradigms, the extinction of Pavlovian conditioned responses has been shown to be relatively speciÞc to the environmental context in which extinction took place (Bouton 1993). For example, if rats are Þrst trained to fear a cue and then that fear is extinguished, the rats will display renewed fear if the cue is presented in a di¤erent context (Bouton and Bolles 1979; Bouton and King 1983). Accordingly, experiments 4 and 5 investigate whether the e¤ects of FG 7142 interact with the regulation of extinction by environmental contexts by comparing the e¤ects of FG 7142 when rats are tested in the chamber where their fear had been extinguished against the e¤ects of the drug when rats are tested in a di¤erent chamber. In experiment 4, four groups of rats were conditioned with an auditory cue and footshock, and on the following 2 days, their fear of the cue was extinguished in the conditioning chamber. Finally, rats were injected with either FG 7142 or its vehicle and tested for fear of the cue either in the conditioning chamber (groups Same) or in a novel chamber (groups Di¤erent). Experiment 5 used three distinctive chambers in order to counterbalance fully between the extinction context and the di¤erent context. Thus, four groups were conditioned with the cue and shock in the shock chamber, then their fear of the cue was extinguished in a second chamber while they received comparable exposures to a third chamber. The rats were later tested in either the extinction context (groups Same) or in the third context (groups Di¤erent) after injection of FG 7142 or its vehicle. Materials and methods
Subjects and materials Subjects in experiment 4 were 40 experimentally naive male Wistar rats (320–400 g). Subjects in experiment 5 were 40 experimentally naive female Wistar rats (250–300 g). Rats were obtained from the same source, and maintained in the same manner as described for experiment 1. Rats were trained and tested in the four conditioning chambers with the auditory cue (clicker) described above. Two sets of alternative chambers were also used. One set consisted in four chambers (30 × 23 × 35 cm; depth × width × height). The front walls of these chambers were made of clear plastic, the hinged lids were made of wood, and the side and rear walls were constructed of steel and painted with 2-cm wide black-and-white stripes. The stripes were horizontal in two chambers and vertical in the other two. The ßoors of these chambers consisted in stainless steel rods, 2 mm in diameter, spaced 13 mm apart (centre to centre). The third set consisted in four chambers (24 × 24 × 19 cm : depth × width × height). The front and rear walls of these chambers, as well as the hinged lid, were constructed of clear plastic, and the end walls were made of stainless steel. The ßoor of these chambers consisted in stainless steel rods, 10 mm in diameter, spaced 22.5 mm apart (centre to centre). FG 7142 was prepared and administered in the manner described for experiment 1.

Procedure and statistical analyses Rats were handled and allocated to four weight-matched groups (all ns = 10). On day 1, rats were trained with the auditory cue and shock, as in experiment 1. On days 2 and 3, all groups received extinction training with the cue as described in experiment 3. In experiment 4, this extinction training took place in the shock chamber. In experiment 5, the cue was extinguished in a second context, and the rats received additional matched exposures to a third context (but without the cue). The chambers that constituted these two contexts were counterbalanced. Rats were tested on day 4 for fear of the auditory cue. Rats were injected with either 10 mg / kg FG 7142 or its vehicle, and 20 min later were placed in a chamber where, after 2 min, the auditory cue was turned on for 2 min. During this time, each rat was scored for freezing. In experiment 4, this test was conducted either in the shock chamber (where the rats had also been extinguished : groups Same) or in a novel chamber (the chambers with black and white striped walls : groups Di¤erent). In experiment 5, the test was conducted either in the chambers where the cue had been extinguished (groups Same) or in the third context (groups Di¤erent). The data were analysed using planned orthogonal contrasts (Hays 1972) testing for the di¤erence between the two groups tested in the extinction chamber (groups Same) and the two groups tested in the alternative chamber (groups Di¤erent), and for pairwise di¤erences between rats injected with FG 7142 and rats given vehicle. Post hoc orthogonal contrasts (Rodger 1967) were also tested. With the decision-wise error rate (a) set at 0.05, the critical F for the planned and post hoc contrasts is 4.11, and 5.78, respectively.

Results Experiment 4 The results of experiment 4 are presented in Fig. 4. The level of freezing in all rats was reduced as a consequence of extinction training. However, freezing was lowest in rats injected with vehicle and tested in the shock chamber. Relative to these rats, freezing was elevated in rats given FG 7142 and tested in the same chamber, as well as in both groups of rats tested in the

Fig. 4 Mean percent of time rats spent freezing during the 2-min presentation of an auditory cue in experiment 4. Four groups of rats had received paired exposures to the cue and footshock, and their fear of the cue was then extinguished in the conditioning chamber. On test day, rats were injected with either FG 7142 (n) or its vehicle ( ) before being placed into either the conditioning chamber (Same) or a novel chamber (Di¤erent) where the auditory cue was presented for 2 min while the rats were scored for freezing

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novel chamber. These observations were conÞrmed by the statistical analysis, which revealed a signiÞcant di¤erence between rats tested in the shock chamber (groups Same) and rats tested in the novel chamber (groups Di¤erent), (F 1,36 = 5.23, P < 0.05). Further, there was a signiÞcant di¤erence between the group given FG 7142 and the group given vehicle for those rats tested in the shock chamber (groups Same) (F 1,36 = 9.91, P < 0.01), but not between the two groups tested in the novel chamber (groups Di¤erent), (F 1,36 = 1.72, P > 0.1). Post hoc analysis showed that, for the two groups injected with vehicle, the group tested in the Same context froze signiÞcantly less than the group tested in the Di¤erent context, (F 3,36 = 6.43, P < 0.05), while there was no such di¤erence between the two groups injected with FG 7142 (F < 1). Thus, the level of extinguished fear was increased either by testing rats in a di¤erent chamber or by injecting rats with FG 7142 before test, but these two e¤ects were not additive in that the drug did not reliably increase fear in rats already tested in the di¤erent chamber. Experiment 5 The results of experiment 5 are presented in Fig. 5. While freezing in all rats was reduced by the extinction training, freezing was lowest among rats injected with vehicle and tested in the same chamber where their fear was extinguished. Relative to these rats, freezing was greater among rats given FG 7142 and tested in the extinction chamber, as well as in both groups of rats tested in the di¤erent chamber, thus replicating the results of experiment 4. The statistical analyses conÞrmed these observations. While the di¤erence between the two groups tested in the extinction con-

text (groups Same) and the two groups tested in the alternative context (groups Di¤erent) did not reach statistical signiÞcance, (F 1,36 = 2.7, P > 0.1), there was a signiÞcant di¤erence between the group given FG 7142 and the group given vehicle for those rats tested in the extinction context (groups Same), (F 1,36 = 4.81, P < 0.05). Further, there was no di¤erence between rats given FG 7142 and those given vehicle when both groups were tested in the di¤erent context, (F < 1). Post hoc analysis additionally revealed that rats injected with vehicle and tested in the di¤erent context froze more than rats injected with vehicle and tested in the extinction context, (F 3,36 = 7.58, P < 0.05), whereas there was no di¤erence between the two groups of rats injected with FG 7142, (F < 1). Thus, as in experiment 4, the level of extinguished fear was increased either by testing rats in a di¤erent chamber or by injecting rats with FG 7142 before test, but these two e¤ects were not additive in that the drug had no e¤ect in rats tested in the di¤erent chamber.

Experiment 6: FG 7142 and latent inhibition
This experiment investigates the e¤ects of FG 7142 on latent inhibition of fear, in order to determine whether the drug acts speciÞcally on extinction of fear or more generally in other instances where fear is inhibited. Latent inhibition refers to the decrement in conditioned responding produced by extensively preexposing rats to the cue in advance of conditioning (Lubow and Moore 1959). Thus, the procedure for inducing latent inhibition mirrors that for extinction : in the case of extinction, fear is inhibited by repeatedly presenting the cue alone after rats have been conditioned with the cue and shock, whereas with latent inhibition, the inhibition of fear is caused by repeated presentation of the cue alone before conditioning with shock. Moreover, like extinction, latent inhibition is context-speciÞc. That is, rats will show little fear of a cue if presented in the chamber where the cue was preexposed, but will show recovery of fear if the cue is presented elsewhere (e.g., Westbrook et al. 1997). Thus, experiment 6 examined the e¤ects of FG 7142 when rats were tested in the chamber where the cue had been preexposed and compared that with the e¤ects of the drug in rats tested in a di¤erent chamber. Four groups of rats (Preexposed) were exposed to the cue in one context while receiving equivalent exposures to a second context but without presentations of the cue. Two further groups (Not preexposed) were given matched exposures to the two chambers but the cue was not presented in either. All rats were then conditioned with the cue and shock in a third context, and subsequently tested for fear of the cue either in the Þrst context (groups Same) or in the second context (groups Di¤erent) after injection of FG 7142 or its vehicle.

Fig. 5 Mean percent of time rats spent freezing during the 2-min presentation of an auditory cue in experiment 5. Four groups of rats had received paired exposures to the cue and footshock, and their fear of the cue was then extinguished in one of two alternative chambers. On test day, rats were injected with either FG 7142 (n) or its vehicle ( ) before being placed into either the extinction chamber (Same) or the other alternative chamber (Di¤erent) where the auditory cue was presented for 2 min while the rats were scored for freezing

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Materials and methods
Subjects and materials Subjects were 48 experimentally naive male Wistar rats (300–420 g) obtained from the same source, and maintained in the same manner as described for experiment 1. Rats were trained and tested in the four conditioning chambers with the auditory cue (clicker) described for experiment 1. The two additional sets of chambers described for experiment 5 were also used. FG 7142 was prepared and administered in the manner described for experiment 1.

Procedure and statistical analyses On each of days 1–4, six groups of rats were placed into both sets of alternative chambers for 20 min. For four groups of rats (groups Preexposed) the auditory cue was presented 5 times, each time for 2 min, and each presentation was separated by 2 min, during exposures to one of these contexts. The cue was not presented during exposures to the other context. The speciÞc chambers that served as the cue and no-cue contexts were counterbalanced. For the other two groups of rats (groups Not preexposed), the auditory cue was never presented in either context on these occasions. The daily exposures to the two contexts occurred 6 h apart, and the timing (morning versus afternoon) of the context exposures alternated between days (i.e., on 2 days, rats were exposed to one set of chambers during the morning and to the other set during the afternoon, and on the other 2 days the order of exposures was reversed). On day 5, all rats were trained with the cue and shock in the shock chambers, as in experiment 1. On day 6, rats were injected with either FG 7142 or its vehicle, and 15 min later were tested for fear of the auditory cue in one of the two alternative chambers. For Preexposed rats, two groups were tested in the chamber where the cue had been preexposed (groups Same) and two groups were tested in the other alternative chamber (groups Di¤erent). Rats from the two Not preexposed groups were tested in either chamber in a counterbalanced fashion. The data were analysed using planned orthogonal contrasts (Hays 1972) testing for di¤erences between 1) Preexposed and Not preexposed rats, 2) Preexposed rats tested in the Same context and Preexposed rats tested in the Di¤erent context, and 3) rats given FG 7142 and rats given vehicle. With the decision-wise error rate (a) set at 0.05, the critical F is 4.07. Fig. 6 Mean percent of time rats spent freezing during the 2-min presentation of an auditory cue in experiment 6. Four groups of rats (Preexposed) were exposed to the cue across 4 days in one of two alternative chambers, while another two groups (Not preexposed) were exposed to the chambers without the cue. All six groups then received a paired exposure to the cue and footshock in a third chamber. On test the following day, rats were injected with either FG 7142 (n) or its vehicle ( ) and placed into one of the two alternative chambers where the auditory cue was presented for 2 min while the rats were scored for freezing. For Preexposed rats, this test took place either in the chamber where the cue had been preexposed (Same) or the other alternative chamber (Di¤erent). For the Not preexposed groups, half the rats were tested in each of the two chambers

vehicle (all Fs < 1). Therefore, preexposure to the auditory cue had reduced conditioned fear of that cue, and this latent inhibition of fear was linked to the environmental context in which the cue had been preexposed. However, injection with FG 7142 had no detectable e¤ect on the expression of this latent inhibition.

General discussion
The present experiments have shown that administration of a b-carboline interferes with the extinction of conditioned fear in rats. Rats acquired fear of an auditory cue that had signalled the delivery of footshock. In experiment 1, when tested for fear of the cue the following day, rats displayed high levels of fear (measured as freezing) during the Þrst minutes of cue presentation, but their fear gradually extinguished as they learned that the cue no longer signalled shock. However, injection of the b-carboline FG 7142 signiÞcantly retarded the rate at which fear was extinguished : rats injected with FG 7142 continued to display higher levels of freezing across the course of the test session than rats injected with vehicle solution. This di¤erence in level of freezing between rats given FG 7142 and vehicle was only observed during the later portion of the test, once the level of fear had begun to extinguish. In particular, no di¤erence between rats treated with FG 7142 and rats given vehicle was seen during the Þrst 2 min of test, before presentation of the

Results The results of experiment 6 are presented in Fig. 6. The levels of freezing among the four Preexposed groups were much lower than those for the two groups not preexposed to the cue. Further, among the Preexposed rats, the two Same groups tested in the context where the cue had been preexposed displayed less freezing than the two Di¤erent groups tested in the other context. Finally, there was no detectable e¤ect of FG 7142 on freezing among any of the groups. These observations were conÞrmed by the statistical analysis. The two groups of rats not preexposed to the auditory cue froze more than the four Preexposed groups, (F 1,42 = 70.44, P < 0.001). Further, the two Preexposed groups tested in the Same context where the cue had been preexposed froze less than the two Preexposed groups tested in the Di¤erent context, (F 1,42 = 6.43, P < 0.05). However, there were no pairwise di¤erences between rats injected with FG 7142 and rats injected with

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cue, even though the level of freezing supported by fear of the chamber in this period was well below ceiling. Moreover, FG 7142 did not elicit freezing in rats that had not been shocked, indicating that the b-carboline does not induce fear (at least as measured by freezing). Thus, it appears that the b-carboline acted selectively to impair extinction of fear rather than to produce a general increase in fear and freezing. Experiment 2 conÞrmed that administration of FG 7142 does not a¤ect freezing before the development of extinction, but retards the rate at which freezing extinguishes when rats are repeatedly presented with the auditory cue in the absence of shock. This experiment additionally demonstrated that FG 7142 interferes with both the acquisition of extinction as well as with the expression of that extinction. Rats given extinction training under the inßuence of FG 7142 showed little evidence of that extinction when tested the following day in the absence of the drug. Conversely, rats that had been extinguished without FG 7142 showed a reinstatement of fear when tested under the inßuence of FG 7142. This reinstatement of extinguished fear was shown to be dose-dependent (experiment 3). The e¤ects of FG 7142 in blocking the acquisition and reversing the expression of extinction cannot be attributed to a drug-induced generalisation decrement because rats that had been extinguished under FG 7142 and tested on the following day under the inßuence of the drug also showed increased levels of freezing. The present experiments have conÞrmed that extinction of conditioned responding is tied to the environmental context in which extinction training took place. That is, if rats are trained to fear a cue (which signals shock) and then their fear of the cue is extinguished in a speciÞc chamber, the rats show little fear when the cue is presented in that chamber but show renewed fear if the cue is presented in a di¤erent chamber. Importantly, experiments 4 and 5 revealed that the ability of FG 7142 to reverse extinction is contingent upon rats being tested in the extinction context. SpeciÞcally, injection of FG 7142 reinstated freezing if rats were tested in the chamber where their fear had been extinguished, but not if rats were tested in a di¤erent chamber. The apparent selectivity of FG 7142 for reversing extinction in rats tested in the extinction context was clearly not due to a ceiling e¤ect which prevents the detection of an e¤ect when rats were tested with the drug in the di¤erent context. The levels of freezing among rats tested in the di¤erent context were still low, and well within the range of levels at which FG 7142 can produce an increment of freezing (see experiments 1 and 2). Rather, the present results indicate that FG 7142 selectively reversed the component of extinction that was linked to the environmental context where extinction had occurred. Finally, experiment 6 failed to provide any evidence that FG 7142 inßuenced the expression of latently

inhibited fear. In this experiment, rats were exposed to the auditory cue before conditioning with the cue and shock. In accordance with previous demonstrations, preexposure to the cue greatly reduced the level of fear rats showed to the cue after it had been paired with shock. Further, rats tested in the chamber where the cue had been preexposed showed less fear of the cue than rats tested in a second chamber. Thus, like the extinction of fear, the latent inhibition of fear was relatively speciÞc to the environmental context in which preexposure took place. However, in contrast to its e¤ects on extinguished fear, FG 7142 had no e¤ect on the expression of latently inhibited fear. Thus, the present Þndings identify a basic di¤erence between the processes underlying extinction and those mediating latent inhibition of fear, such that GABA is critical for the former but not the latter.The failure to detect evidence for an involvement of GABA in latent inhibition is inconsistent with a proposed model of the neural mechanisms underlying latent inhibition. SpeciÞcally, Gray (1995) has suggested that GABAergic projections from the nucleus accumbens to the ventral pallidum contribute to the processes by which the accumbens mediates latent inhibition. In support of this assertion, administration of a benzodiazepine agonist (chlordiazepoxide) has been shown to disrupt the acquisition of latent inhibition (Feldon and Weiner 1989). However, most investigations into the neural bases of latent inhibition, including those upon which Gray’s proposal is based (see Gray et al. 1997), have focused on the neurochemical processes involved either in the acquisition of latent inhibition (i.e., during preexposure) or in the impact of latent inhibition during conditioning. Thus, Gray’s model does not describe the role of speciÞc neurotransmitters in the inhibition of conditioned responding during test, as has been investigated here. The failure to observe a loss of latent inhibition among rats given FG 7142 in experiment 6 has important implications for the interpretation of the results of experiments 4 and 5. In those experiments, it might have been possible to argue that the interoceptive cues produced by FG 7142 caused a shift in internal state in the rat, and that this shift in state reduced extinction in much the same way as a shift in the environmental context had done. However, in experiment 6, FG 7142 did not reverse latent inhibition even though latent inhibition, like extinction, is sensitive to a shift in context. Thus, it would seem that FG 7142 does not induce an alteration in internal state such as might a¤ect expression of extinction and latent inhibition. In summary, we have shown that systemic injection of the b-carboline FG 7142 retards the rate at which fear is extinguished in rats. This e¤ect of FG 7142 was related to both an impairment in the acquisition of extinction and in the expression of that extinction. The e¤ect of FG 7142 was relatively speciÞc to extinguished

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fear because the drug did not 1) elicit freezing in rats not exposed to a frightening cue, 2) increase freezing prior to the development of extinction, and 3) increase freezing in rats whose fear of the cue was latently inhibited by preexposure to the cue. Finally, FG 7142 was found to reverse extinction only when rats were tested in the extinction context, indicating that the drug only reverses context-speciÞc extinction. We interpret these Þndings as indicating that extinction of fear involves the acquisition of a context-gated inhibitory association between the conditioned stimulus and noxious unconditioned stimulus, and this inhibitory association is mediated by GABA binding to GABAA receptors. There is no direct evidence from the current Þndings to indicate whether the same GABAA receptors that underlie the acquisition of extinction also mediate the expression of that extinction. However, the fact that administration of FG 7142 before both extinction training and test did not a¤ect extinction any more than administration of the drug before either extinction training or test alone (experiment 2) suggests that the drug was acting on a common mechanism in each case. In other words, this Þnding provides indirect evidence that the GABAergic synapses which mediate the acquisition of extinction also contribute to the expression of extinction. Further, we speculate that this process is mediated speciÞcally by GABA transmission within the basolateral amygdala, as suggested by evidence that GABA transmission within that nucleus inhibits both the acquisition and expression of conditioned fear (e.g., Helmstetter and Bellgowan 1994; Muller et al. 1997; Harris and Westbrook 1998). Finally, we should note that treatment with FG 7142 reduced, but did not eliminate, extinction. This failure to eliminate extinction may reßect the nature of the drug’s pharmacological action, namely that FG 7142 reduces the inhibitory e¤ects of GABA without actually blocking GABA receptors. Alternatively, this observation may indicate that GABA transmission represents only one of several independent mechanisms that mediate extinction. For example, in addition to the view that extinction is mediated by inhibition of the memory of the unconditioned stimulus, Robbins (1990) has proposed that extinction involves a decrement in attentional processing of the conditioned stimulus. Decrements in attentional processing are thought to be mediated by cholinergic inputs to the hippocampus (Han et al. 1995; Baxter et al. 1997). Thus, manipulations which interfere with hippocampal acetylcholine might be expected to reduce both latent inhibition and that component of extinction not a¤ected by FG 7142.
Acknowledgements The authors wish to thank R. Richardson, P. Carrive and P. Lovibond for comments on an earlier version of the manuscript. The research was funded by grants from the Australian Research Council.

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