Dopamine activity in the nucleus accumbens modulates blocking in ...

European Journal of Neuroscience, Vol. 24, pp. 3265–3270, 2006

doi:10.1111/j.1460-9568.2006.05195.x

Dopamine activity in the nucleus accumbens modulates blocking in fear conditioning Mihaela D. Iordanova,1,2 R. Frederick Westbrook1 and A. Simon Killcross2 1 2

School of Psychology, The University of New South Wales, Sydney, Australia School of Psychology, Cardiff University, PO Box 901, Park Place, Cardiff CF10 3YG, UK

Keywords: amphetamine, blocking, context, fear, rat

Abstract Associative learning depends on the discrepancy between actual and predicted outcomes. The neurochemical mechanisms involved in regulating this discrepancy in Pavlovian fear conditioning in rats are unknown. We employed the blocking paradigm to show that this learning discrepancy is decreased by heightened activation of dopamine following an accumbal infusion of d-amphetamine, and increased by dopaminegic blockade following an accumbal infusion of cis-(z)-flupenthixol or by combined infusions of the D1 (SCH23390) and D2 (sulpiride) antagonists but not by infusion of either alone.

Introduction The failure of Pavlovian conditioning observed in ‘blocking’ (Kamin, 1969) illustrates the role of the discrepancy between actual and predicted outcomes in modulating associative learning (e.g. Rescorla & Wagner, 1972). In blocking, two groups of rats are trained to fear a compound conditioned stimulus (CS) composed of CSA and CSB by pairing it with a footshock unconditioned stimulus (US). The two groups differ in that one but not the other received CSA–US pretraining. When tested, only the group not pre-trained with CSA shows fear to CSB. The prior CSA–US conditioning is said to have blocked the development of fear to CSB in the normally effective compoundUS conditioning trials. Formal learning theories (e.g. Rescorla & Wagner, 1972; Pearce & Hall, 1980) hold that the formation of a CS–US association is regulated by the discrepancy, or error, between the US actually experienced and that predicted by all available conditioning cues. Hence, rats not pretrained with CSA learn about the relation between CSB and the US in the compound trials because of the large discrepancy between the actual US and that predicted by the available cues. However, rats pre-trained with CSA fail to learn about CSB because the actual US is that predicted by CSA. Hence, pre-training with CSA blocks learning about CSB because the prediction error is small. Investigations into the neural mechanisms of the predictive discrepancy are sparse. Opioid receptors in the nucleus accumbens (Acb) regulate predictive learning as examined in an aversive blocking paradigm in rats (Iordanova et al., 2006). Firing of dopamine (DA) neurones originating from the ventral tegmental area and the substantia nigra in monkeys has also been implicated in coding for the prediction error mechanism in an appetitive blocking procedure (Waelti et al., 2001). Although evidence concerning the role of DA in aversive learning is mixed (Thierry et al., 1976; Abercrombie et al., 1989; Puglisi-Allegra et al., 1991; Horger & Roth, 1996; Young, 2004; but see Ungless et al., 2004), recent studies have reported Correspondence: Dr Mihaela D. Iordanova, 2School of Psychology, as above. E-mail: [email protected] Received 26 October 2005, revised 4 September 2006, accepted 25 September 2006

changes in accumbal DA during the course of fear conditioning. High levels of Acb DA are elicited by an unexpected shock (Sorg & Kalivas, 1991; Young et al., 1993; Young, 2004) and subsequently by the CS that comes to predict the occurrence of shock (Young et al., 1993; Saulskaya & Marsden, 1995; Guarraci & Kapp, 1999; Young, 2004). These findings raise the possibility that accumbal DA may modulate the predictive discrepancy such that changes in DA levels elicited by a pre-trained CSA may regulate the amount learned about CSB during compound conditioning trials in blocking. We examined this possibility in a blocking design by increasing and decreasing accumbal DAergic activity with an infusion of d-amphetamine and cis-(z)-flupenthixol, respectively (Experiment 1a). Subsequently we examined the specificity of the DA receptors involved by antagonizing either D1 or D2 DA receptors with either SCH23390 or sulpiride, respectively (Experiment 1b). Our results indicate that accumbal DA regulates the predictive learning in blocking and does so in a fear conditioning paradigm.

Materials and methods Subjects The subjects used in Experiment 1a were 61 male Lister Hooded rats (Harlan, UK) and those used in Experiment 1b were 63 male Australian Albino Wistar rats (Gore Hill Research Laboratories, Sydney, New South Wales, Australia). All rats weighed between 250 and 380 g. In Experiment 1 rats were housed in pairs whereas in Experiment 2 rats were housed in groups of eight in a temperaturecontrolled (21 ± 2 C) colony room maintained on a 12-h light ⁄ dark cycle. Food and water were available ad libitum throughout the experiment. All procedures were conducted during the light phase, between 08:00 and 18:00 h. Animal husbandry and experimental procedures were conducted in accordance with the Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985), the UK Animals (Scientific Procedures) Act 1986, and the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (publication DHHS NIH 86-23).

ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

3266 M. D. Iordanova et al. Surgery Prior to behavioural testing, rats were anaesthetized with isoflurane (Experiment 1a) or injected i.p. with 1.3 ml/kg of the anaesthetic ketamine (Ketapex; Apex Laboratories, Sydney, Australia) at a concentration of 100 mg/ml, and with 0.3 ml/kg of the muscle relaxant xylazine (Rompun; Bayer, Sydney, Australia) at a concentration of 20 mg/ml (Experiment 1b). Rats were then implanted with 26-gauge guide cannulae (Plastics One, Roanoke, VA, USA) into the right hemisphere of the brain (as reported previously by Iordanova et al., 2006). The tip of the guide cannula was aimed at the Acb by positioning it 7.0 mm below bregma through a hole drilled 1.4 mm anterior to and 1.8 mm lateral to bregma. Guide cannulae were fixed in position with dental cement and anchored by Super Glue (Selleys, Sydney, Australia). Dummy cannulae were kept in the guide at all times except during microinjections when 33-gauge microinjection cannulae were inserted into the guide cannulae and connected to a 25-lL glass syringe operated by an infusion pump (Harvard Apparatus, South Natick, MA, USA). Microinjection cannulae projected a further 1 mm ventral to the tip of guide cannulae. Rats were allowed 7 days to recover from surgery, during which time they were handled and weighed daily.

Behavioural procedures During Stage I (i.e. Day 1) of the experiments, rats were transported to the laboratory and placed in conditioning chambers (Experiment 1a, Campden Instruments Ltd, UK, 24.5 cm wide · 20.5 cm high · 22cm deep; Experiment 1b, laboratory made, 20 cm high · 23 cm long · 21 cm wide). At 3 min following placement into the conditioning chambers rats in blocking (Block) groups received three footshocks spaced 3 min apart, establishing strong context–shock associations. Rats remained in the chambers for 60 s following the last shock presentation. The shock duration for both experiments was 1.0 s and the shock intensity was 0.4 mA for Experiment 1a and 0.8 mA for Experiment 1b (adjusted appropriately for the different shock generators used in each experiment). The background noise in the room was 69 dB. Rats in the control conditions were handled during Stage I. The shock was identical for Stage I and Stage II training. During Stage II (i.e. Day 2) at 20 min prior to conditioning, rats in Experiment 1a received 1-lL infusions into the Acb (0.5 lL ⁄ min plus 1 min of diffusion) of either artificial cerebrospinal fluid (ACSF), 10 lg ⁄ lL d-amphetamine sulphate (Sigma-Aldrich, UK) or 10 lg ⁄ lL cis-(z)flupenthixol (Sigma-Aldrich). d-Amphetamine and cis-(z)-flupenthixol were dissolved in sterile ACSF. Rats in Experiment 1b were infused with either saline or with 2.5, 5 and 7.5 lg ⁄ lL of SCH23390 or sulpiride (Sigma, Sydney, Australia) or a combination of 2.5 lg ⁄ lL SCH23390 and 2.5 lg ⁄ lL sulpiride. SCH23390 and sulpiride were dissolved in sterile non-pyrogenic saline (0.9% w ⁄ v; Astra, Sydney, Australia). Following infusions all rats were placed in the conditioning chambers where they received two pairings of a CS consisting of a train of clicks (10 s, 75–81-dB 10-Hz clicker; rise time < 10 ls, decay time 250 ls) and footshock, 220 s apart, at 5 min following placement in the chambers. Rats remained in the chambers for 60 s following the second clicker–shock pairing. To ensure equal exposure to all neuroactive agents, 6 h later, rats infused with a drug received an infusion of ACSF (Experiment 1a) or saline (Experiment 1b) and those infused with ACSF ⁄ saline received a drug infusion. On Days 3 and 4 rats were tested for fear to the conditioning context or the clicker CS for 10 min, although only the first minute of the test was used for the statistical analysis. The order of testing (context and CS) was counterbalanced. In Experiment 1a novel chambers, which were

identical to the conditioning chambers with the alteration of having added checked walls and a blue cardboard floor, were used for the clicker test. Rats remained in a novel context for 2 min before the clicker was switched on. In Experiment 1b the novel chambers were plastic tubs (16 cm high · 40 cm long · 26 cm wide). In addition, the odour used to wipe the chambers between rats was switched from 1% acetic acid (used during conditioning) to 1% vanilla. The room lights were also switched off during clicker testing. These changes were made in order to eliminate contextual effects on freezing performance to the clicker during test. Freezing to the novel context prior to clicker delivery was below 10%.

Scoring and statistics During the test the behaviour of all rats in each experiment was recorded on videotape, and freezing was rated with a time-sampling procedure in which each rat was observed every 2 s and scored as either freezing or moving. Freezing was defined as the absence of all movements, except for those related to breathing (Fanselow, 1980). A percentage score was calculated for the proportion of the total observation period that each rat spent freezing. Freezing was rated by two observers, one of whom was unaware of the subjects’ group designations. There was a high degree of agreement between the two observers (the Pearson product-moment correlation between their ratings was above 0.90). The data were analysed by anova testing sets of planned contrasts. The familywise error rate was controlled for each family of contrasts tested (where k is the number of contrasts in each family) using the Bonferroni inequality procedure (Stevens, 1986). Significance was set at the 0.05 level.

Histology At the end of each experiment, the rats were given an overdose of sodium pentobarbital (0.5 mL) and their brains were removed. The unfixed brains were sectioned coronally at 40 mm through the Acb. Every fourth section was collected on a slide, stained with cresyl violet and coverslipped with Permount. The sections were examined under a microscope and cannula placements were verified to the boundaries defined by Paxinos & Watson (1998). Figures 1 and 2 show the location of microinjection tips, predominantly within the core subregion of the Acb, for each of the experimental groups in Experiments 1a and 1b, respectively. The data from three rats were excluded from the statistical analysis as subsequent examination of histology revealed that the cannula tip was more than 0.5 mm outside the Acb. Another rat was excluded from the analysis as its cannula cap became displaced prior to test.

Results Figure 3A shows the mean ± SEM levels of freezing for the first minute of the clicker test in Experiment 1a. Inspection of Fig. 3A indicates that prior conditioning of the context with shock reduced or blocked the level of conditioned fear acquired by the clicker following clicker–shock pairings in the conditioning context. Freezing in the Block ACSF group (n ¼ 18) was lower relative to the Control groups (n ¼ 22; F1,61 ¼ 15.5; k ¼ 5; P < 0.05). Accumbal microinjections of d-amphetamine prior to compound conditioning in Stage II augmented this blocking of clicker conditioning by the context. The Block dAmph group (n ¼ 15) exhibited lower levels of freezing to the clicker relative to the Block ACSF group (F1,61 ¼ 11.9; P < 0.05). Accumbal infusions of cis-(z)-flupenthixol prevented the blocking

ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 24, 3265–3270

Accumbal dopamine and blocking 3267

Fig. 1. Area of placements of cannulae tips into the nucleus accumbens relative to bregma point for Experiment 1a. ACSF, artificial cerebrospinal fluid; cis(z)Flu, cis-(z)-flupenthixol; dAmph, d-amphetamine.

effect. The Block Flu group (n ¼ 12) exhibited higher levels of freezing relative to the Block ACSF group (F1,61 ¼ 10.8; P < 0.05). No differences in freezing levels were detected between the control groups (Fs < 1). The differences in levels of freezing to the clicker seen among the block groups were not accompanied by differences in freezing to the context (maximum F1,61 ¼ 2.4; P > 0.05; data not shown). Similarly, no differences were obtained among the control groups in freezing to the context (Fs < 1; P > 0.05). Figure 3B shows the mean ± SEM levels of freezing for the first minute of clicker in Experiment 1b. Inspection of Fig. 3B indicates no effect of drug vs. saline infusions on freezing to the clicker (F1,63 ¼ 3.2, k ¼ 5; P > 0.05). Prior conditioning of the context with shock reduced or blocked the level of conditioned fear acquired by the clicker following clicker–shock pairings in the conditioning context. The Block Saline group (n ¼ 10) exhibited lower levels of freezing to the clicker than the Control Saline group (n ¼ 7; F1,63 ¼ 8.9; P < 0.05). Accumbal microinjections of SCH23390 or sulpiride at any of the three doses prior to compound conditioning in Stage II failed to prevent blocking of clicker conditioning by the context. The SCH23390 (n ¼ 8, 8 and 7 at doses of 2.5, 5.0 and 7.5 lg ⁄ mL, respectively) and Sulpiride (n ¼ 8 for each of the three doses) groups exhibited equivalent levels of freezing to the clicker relative to the Block Saline group (Fs < 1). However, combined SCH23390

(2.5 lg ⁄ mL) and sulpiride (2.5 lg ⁄ mL) infusions into the Acb prior to Stage II training prevented the blocking of clicker conditioning by the context. The SCH23390 + Sulpiride group (n ¼ 8) exhibited higher levels of freezing relative to the Block Saline group (F1,63 ¼ 7.6; P < 0.05). Again, as in Experiment 1a, the effect of combined infusion of SCH23390 + sulpiride on clicker conditioning was not accompanied by a similar effect on context freezing. Levels of freezing to the context did not differ between the Sulpiride + SCH23390 and Block Saline groups (F < 1; data not shown). These results show once again that prior conditioning to the context in the Block Saline group retarded conditioning to the clicker in Stage II. Further, this reduction was prevented by the combined antagonism of D1 and D2 receptors but not either receptor type alone, and was specific in regulating learning about the blocked (clicker) and not the blocking (context) stimulus.

Discussion Rats that had already learned to fear a context developed less fear of a CS paired with shock in that context than rats not pre-trained to fear that context. The prior training with the shocked context blocked the development of fear in the subsequent CS–shock pairings. Infusion of


3268 M. D. Iordanova et al.

Fig. 2. Area of placements of cannulae tips into the nucleus accumbens relative to bregma point for Experiment 1b.

the DA agonist d-amphetamine into the Acb in advance of the CS– shock pairings increased the ability of the pre-trained context to block fear conditioning to the context, i.e. these infusions produced even less

fear conditioning to the CS. In contrast, infusion of the DA antagonist cis-(z)-flupenthixol into the Acb decreased the ability of the pretrained context to block fear conditioning to the CS in its pairings with


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Fig. 3. (A) Mean and SEM levels of conditioned freezing to the clicker on test in Experiment 1a. Freezing in the Block Artificial Cerebrospinal Fluid (ACSF) group was lower relative to the Control groups. Infusion of d-amphetamine (dAmph) reduced whereas an infusion of cis-(z)-flupenthixol [cis-(z)-Flu] increased freezing relative to the Block ACSF group. No differences in freezing levels were detected between the control groups. (B) Mean and SEM levels of conditioned freezing to the clicker on test in Experiment 1b. Freezing in the Block Saline group was lower relative to Control Saline. Rats infused with either SCH23390 or sulpiride in the three doses (2.5, 5.0 and 7.5 lg ⁄ mL) exhibited equivalent levels of freezing to Block Saline groups. Infusion of SCH23390 (2.5 lg ⁄ mL) combined with sulpiride (2.5 lg ⁄ mL) increased freezing to the clicker relative to the Block Saline group.

the US; these infusions increased fear conditioning to the CS. This effect of DA antagonism depended on the combined action of D1 and D2 DA receptors, as infusion of either D1 or D2 antagonists in three different doses failed to modulate the ability of the pre-trained context to block fear conditioning to the CS. The selectivity of SCH23390 and sulpiride for DA suggests that the effects reported here are unlikely to be due to changes in norepinephrine, which has been reported to increase following administration of amphetamine (Segal & Kuczenski, 1997). The increase (i.e. reduction in blocking) and decrease (i.e. increase in blocking) in fear conditioning to the CS produced by DA receptor blockade and heightened DAergic activation, respectively, cannot be attributed to state-dependent learning because the effects on CS conditioning were not mirrored in context conditioning. Similarly, control groups exhibited no differences in either context or CS conditioning regardless of drug treatment. Hence, blockade of DAergic receptors in the Acb cannot have increased clicker conditioning (i.e. prevented blocking) by reproducing some component of the shock US (e.g. more painful or otherwise aversive) or

vice versa in the case of increased DAergic activity. Further, any such increase in the effectiveness of the shock US by antagonism of DA in the Acb would have been accompanied by a general increase in fear conditioning, yet no differences were observed in context fear in the Block groups or among the Control groups in either contextor clicker-induced fear. Similarly, increased DAergic activity could not have reduced the effectiveness of the shock US as, again, there were no differences in context conditioning among the Block groups or in context and clicker conditioning among the Control groups. Thus, the series of experiments reported here supports two conclusions. Firstly, DAergic activity within the Acb modulates the effect of the discrepancy between actual and predicted outcomes on learning and behaviour. Secondly, combined D1 and D2 DA receptor activity in the Acb, and not either alone, is critical for this modulation and the lack of effect following either D1 or D2 DA receptor blockade alone is not a consequence of dose-dependent effects. Although the effects of DAergic modulation on the predictive relationship between stimuli obtained here are reminiscent of the


3270 M. D. Iordanova et al. electrophysiological data presented by Waelti et al. (2001), three caveats prevent us from drawing parallels between the two findings. Firstly, infusions of DAergic agonists and antagonists into the Acb may modulate both transient or phasic DA release caused by DA neurone firing as well as sustained, tonic DA (Grace, 1991), thus making it difficult to infer how such agents influence specific DAergic signals emanating from the ventral tegmental area. Secondly, it is unclear how and if the Acb provides feedback to the ventral tegmental area with regard to the learning discrepancy. Finally, no evidence exists implicating the ventral tegmental area in predictive learning in fear conditioning. However, the results reported here do provide clear evidence that accumbal DA modulates predictive learning in an aversive blocking paradigm. Given this role of accumbal DA receptors in regulating predictive learning (such that heightened DAergic activity lowers and blockade of DA augments the impact of the predictive discrepancy in learning a behaviour), it might be expected that variations in CS conditioning of the sort seen in the blocking groups should also have been present in the control groups, i.e. increases in levels of DAergic activation in the CS–US pairings should have produced less fear to the CS (and the context), whereas decreases in these levels should have produced more fear to the CS (and context). However, neither an increase nor a decrease in fear of the CS (or context) was detected in these variations in DA levels among the control groups. The failure to detect any differences in baseline conditioning could have been due to the fact that the parameters used did not allow any such differences to be observed. Alternatively, it is also possible that the manipulation of levels of DA activation only modulate the effect of the predictive discrepancy on learning and behaviour when there is already a high level of DA activation in the Acb, as is likely to have been the case in the Block but not the Control groups, i.e. the pretrained context in the Block groups may have provoked increased DA activation in the Acb in advance of the CS–shock pairings. Thus, the impact of the drugs would have been to increase or reduce this level of DA, thereby regulating the effectiveness of the pre-trained context as a predictor of the US. In the absence of a strong predictor, changes in accumbal DA levels would have limited impact on learning in the Control groups (especially with few conditioning trials). This latter interpretation has possible implications for the mechanism underlying predictive learning mediated by accumbal DA. Specifically, the effect of accumbal DA activation on predictive learning in Block but not in Control groups suggests that accumbal DA modulates the ability of good predictors to regulate learning about other, poorer predictors (and, in the absence of any predictor, has no effect). This is captured in CS-processing theories of predictive learning, which hold that the establishment of associative relations depends upon the ability of a CS to gain access to attentional processing. CSs compete for this processing, such that good predictors (here, the context) of biologically significant events capture attention and are processed at the expense of poor predictors (here, the clicker) of the same events (Mackintosh, 1975). Our results show that in fear learning accumbal DA modulates stimulus competition such that heightened DAergic activation in the Acb facilitates the reduction in processing of poor predictors in the presence of good predictors. In contrast, blockade of accumbal DA prevents the reduction in the processing of these poor predictors.

Acknowledgements This research was supported by an Australian Research Council grant (A10007151) to R.F.W. and a Wellcome Trust Biomedical Research Collaboration grant (063095 ⁄ Z ⁄ 00 ⁄ Z) to A.S.K. and R.F.W. We thank Gavan McNally for his helpful discussions.

Abbreviations Acb, nucleus accumbens; ACSF, artificial cerebrospinal fluid; CS, conditioned stimulus; DA, dopamine; US, unconditioned stimulus.

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