Paul Kenneth Hitchcott

Double dissociation of the behavioural effects of R(+) 7–OH–DPAT infusions in the central and basolateral amygdala nuclei upon Pavlovian and instrumental conditioned appetitive behaviours

Abstract Dopaminergic cell bodies located within the ventral mesencephalon innervate the amygdaloid complex, a region critically involved in the attribution of affective significance to environmental stimuli. Recently, we have shown that post-session intra-amygdala administration of a D3 dopamine receptor agonist enhances selectively the acquisition of an appetitive conditioned response. In the present study, we have investigated the potential involvement of the central nucleus and the basolateral nuclei of the amygdala in mediating this effect. Thus, rats were trained to associate an arbitrary stimulus (CS+) with the availability of 10% sucrose reward. Post-session infusions of the D3 receptor-preferring agonist, R(+) 7-OH-DPAT, were made into either the central nucleus or basolateral nuclei. Acquisition of a conditioned approach response was enhanced by R(+) 7-OH-DPAT infusions within the central nucleus, but not within the basolateral nuclei. Drug infusions into either region failed to affect approach behaviour elicited by presentation of a control stimulus (CS−), explicitly unpaired with sucrose reward. The effects of pre-test infusions of R(+) 7-OH-DPAT on the instrumental properties of the stimuli were then determined. Rats were presented with two novel levers, depression of one lever resulted in presentation of the CS+, while presentation of the CS− was contingent upon depression of the other lever. Rates of response upon each lever as well as the ability of the conditioned stimuli subsequently to elicit conditioned approach behaviour were recorded. Data revealed a double dissociation of the effects of R(+) 7-OH-DPAT on the expression of the Pavlovian and instrumental properties of the reward-related stimulus. Thus, within the central nucleus R(+) 7-OH-DPAT dose-dependently attenuated expression of the conditioned approach response, but had no effect upon instrumental responding maintained by the conditioned reward. In contrast, within the basolateral nuclei, R(+) 7-OH-DPAT had no effect upon expression of conditioned approach behaviour, but abolished selectively the ability of the reward-associated stimulus to support the acquisition of a novel instrumental response. Hence, these data indicate that distinct regions of the amygdaloid complex process distinct aspects of conditioned appetitive behaviours.

Enhanced stimulus-reward learning by intra-amygdala administration of a D3 dopamine receptor agonist

Abstract The amygdala is considered to be a critical neural substrate underlying the formation of stimulus-reward associations, and is known to receive substantial innervation from dopaminergic neurons located within the ventral mesencephalon. However, relat- ively little is known about the function of the mesoamygdaloid dopamine projection in stimulus-reward learning. Recently, we have found post-session intra-amygdala microinjections of d-amphetamine to enhance appetitive Pavlovian conditioning as assessed in a discriminative approach task. In the present study, we have examined the effects of dopamine receptor agonists possessing relative selectivity for the D1, D2 and D3 receptor subtypes in order to examine more fully the role of the mesoamygdaloid dopamine projection in stimulus-reward learning. Thus, subjects were trained to associate an initially neutral stimulus (CS+) with 10% sucrose reward (US). A second, control stimulus (CS−) was also presented but never paired with sucrose reward. In order to measure specifically the conditioned response to CS+/CS− presentation, responding during CS and US presentations was measured separately. Immediately following each training session, subjects received bilateral intra-amygdala infusion of 0.1, 1 or 10 nmol/side of SKF-38393, quinpirole or 7-OH-DPAT. Infusions of SKF-38393 or quinpirole were without effect on CS+ approach. However, post-session intra-amygdala infusions of 7-OH-DPAT enhanced selectively CS+ approach in a dose-dependent fashion. No dose of any drug affected CS−approach, US behaviours, or measures of extraneous behaviour. Subsequent acquisition of a novel conditioned instrumental response was also unaffected. Thus, the present data indicate a selective involvement of the D3 dopamine receptor subtype in the modulation of stimulus-reward learning by the mesoamygdaloid dopamine projection.

Enhanced acquisition of discriminative approach following intra-amygdala d-amphetamine.

Abstract This study examined the role of the mesoamygdaloid dopamine projection in stimulus-reward learning. Bilateral post-session intra-amygdala microinjections of d-amphetamine were carried out in rats during training in a discriminative approach task known to be sensitive to experimental manipulations of the amygdala. The experiment consisted of two phases: discriminative approach training, and a subsequent assessment of instrumental conditioned reward efficacy. During discriminative approach training, subjects were trained to associate a neutral stimulus with 10% w/v sucrose reward. Each trial consisted of a 1-s light stimulus followed by a 5-s presentation of the sucrose reward. Approach behaviour into the recess housing sucrose reward was measured during each trial. Inappropriate approach behaviour (approach outside of the trial periods) was punished by delaying the next trial. Intra-amygdala d-amphetamine (10 μg/side) enhanced the rate of acquisition of discriminative approach behaviour. This effect was most evident early during training (sessions 2–4) and by the tenth session both groups had reached similar asymptotic performance. Horizontal and vertical activity increased slightly across sessions, but there was no indication of a differential effect of d-amphetamine. Thus, intra-amygdala microinjections of d-amphetamine enhanced selectively the acquisition of the stimulus-reward association. During a subsequent test of instrumental conditioned reward, presentation of the conditioned light stimulus was made contingent upon performance of a novel lever-pressing response (probability 0.5). Responding on a second, control lever was without programmed consequences. Sucrose reward was not available at any point, and subjects were tested drug-free. In both groups the conditioned stimulus was found to possess significant conditioned rewarding efficacy. Extraneous behaviour was increased in the d-amphetamine group but the rewarding properties of the conditioned stimulus were unaltered. These findings demonstrate that the mesoamygdaloid dopamine projection modulates the acquisition of a stimulus-reward association, but is apparently without subsequent effect on the rewarding efficacy of a conditioned stimulus.

Repeated d-amphetamine enhances stimulated mesoamygdaloid dopamine transmission

Abstract The mesoaccumbens dopamine pathway exhibits an enhanced dopaminergic response to a challenge injection of d-amphetamine or cocaine after repeated intermittent exposure to that drug. Much research has focused on the potential role of this sensitised response in the enhanced propensity of drug-associated stimuli to elicit relapse. However, the amygdala is acknowledged to play a critical role in stimulus-reward learning, and recent work suggests that the mesoamygdaloid dopamine pathway exerts a significant influence upon amygdala function. In the present study, rats were administered d-amphetamine (1 mg/kg, IP) or vehicle once per day, for 14 days. After 11 untreated days, a locomotor assay showed that prior repeated administration of d-amphetamine led to a markedly enhanced locomotor response to 0.5 mg/kg d-amphetamine. There was no effect of d-amphetamine pretreatment upon the response to a novel environment, or to injection with vehicle. Following a total of 14 days in the home cage, subjects were implanted with microdialysis probes within the amygdala, and for comparison also within the nucleus accumbens. Baseline and d-amphetamine-stimulated (0.5 mg/kg) levels of extracellular dopamine were assessed for each brain region. Results showed that baseline levels of dopamine were very similar in sensitised and control animals. By contrast, prior treatment with d-amphetamine enhanced dopamine overflow in response to a challenge with d-amphetamine both in the nucleus accumbens and amygdala. These results indicate that changes in the pattern of dopamine transmission both in the nucleus accumbens, and the amygdala, accompany the behavioural sensitisation observed after repeated exposure to d-amphetamine. Hence, an enhanced propensity of drug-associated stimuli to elicit relapse may not depend solely upon changes relating to the mesoaccumbens dopamine projection.

Facilitation of appetitive Pavlovian conditioning by d-amphetamine in the shell, but not the core, of the nucleus accumbens

The effects of postsession d-amphetamine within subregions of the ventral and dorsal striatum on appetitive Pavlovian learning were assessed. Rats acquired a conditioned approach response on presentation of a stimulus predictive of 10% sucrose solution (unconditioned stimulus [US]), but not during equally frequent presentations of a stimulus uncorrelated with the US. In Experiment 1, postsession d-amphetamine infusions enhanced acquisition of conditioned responding, with no effect on control measures. In Experiment 2, rats received postsession d-amphetamine in the accumbens shell or core. Shell infusions facilitated conditioning; core infusions did not. In Experiment 3, dorsomedial striatal infusions of d-amphetamine also were ineffective. In sum, dopaminergic activation within the shell, but not the core, of the nucleus accumbens facilitates the acquisition of a Pavlovian association.

Amygdala and hippocampus control dissociable aspects of drug-associated conditioned rewards

Abstract Limbic innervation of the nucleus accumbens via the ventral subiculum/hippocampus and basolateral area of the amygdala has been shown to determine dissociable aspects of behaviour controlled by stimuli associated with natural rewards. However, the respective contributions of the ventral subiculum and amygdala to behaviour governed by drug-associated stimuli remain to be determined. Experiments consisted of two phases: drug-stimulus training, and subsequent stimulus-only testing. Initial training sessions were of two alternating forms. During drug sessions, responding upon one lever resulted in an infusion of 1 μg d-amphetamine into the nucleus accumbens, whilst during saline sessions d-amphetamine was replaced with saline. Each infusion (drug or saline) was preceded with either a light, or tone. Responding upon a control lever had no programmed consequences. Following training, the levers were retracted, and instead two novel vertical bars were extended from the chamber ceiling. Movement of one bar produced the drug-associated stimulus, whilst the alternative bar produced the saline-associated stimulus. Infusions of the AMPA receptor antagonist CNQX into the ventral subiculum or basolateral area of the amygdala (0, 0.2, 2.0 nmol) were made immediately before the start of each session. Intra-basolateral area of the amygdala CNQX reduced responding upon the drug-associated stimulus bar, but at the same time increased responding upon the saline-associated stimulus bar. By contrast, intra-ventral subiculum CNQX reduced drug-associated stimulus responding selectively. Neither manipulation affected levels of activity within the operant chamber extraneous to the bar-pushing response. Hence, the basolateral area of the amygdala appeared to have determined the degree of discriminative control exerted over behaviour by the drug-associated stimulus, whilst the ventral subiculum is suggested to have determined the efficacy of the conditioned reward.

Immunohistochemical assessment of mesotelencephalic dopamine activity during the acquisition and expression of Pavlovian versus instrumental behaviours

Dopaminergic activity during Pavlovian or instrumental learning in key target regions of the mesotelencephalic dopamine system was investigated immunohistochemically using antibodies raised against glutaraldehyde-conjugated dopamine. Experiment 1 examined dopamine immunoreactivity during acquisition of a Pavlovian conditioned-approach response. Observations were taken at three stages of learning: initial, intermediate and asymptotic; each with a conditioned stimulus+ (CS+) group for whom visual or auditory stimuli immediately preceded an unconditioned stimulus (sucrose), and a conditioned stimulus- (CS-) group for whom stimuli and the unconditioned stimulus were unpaired. Animals learned to approach the alcove during CS+ presentations, whilst approach behaviour of the CS- group remained low. In general, target regions exhibiting a dopaminergic reaction responded maximally during the intermediate stage of acquisition, and were less responsive initially, and not responsive at all at asymptote. Specifically, the pattern of dopaminergic response was: shell more than core of the nucleus accumbens; prefrontal cortex, central and basolateral nuclei of the amygdala also significantly responsive. Mediodorsal and laterodorsal striatal regions were reactive only very early in training. Experiment 2 examined dopaminergic reaction following acquisition of a novel conditioned instrumental response. The conditioned response+ (CR+) group responded at a much higher rate on the lever for which unconditioned stimulus-associated stimuli were presented, than on the control lever. The conditioned response- (CR-) group responded at a low rate on both levers. In contrast with experiment 1, the most responsive regions were the core of the nucleus accumbens, medial prefrontal cortex and basolateral area of the amygdala. Thus, the acquisition, but not expression of Pavlovian associations activated dopamine within several key target regions of the mesotelencephalic dopamine system, and preferentially within the shell rather than core of the nucleus accumbens. By contrast, acquisition of a novel instrumental response preferentially activated the core of the nucleus accumbens, and basolateral area of the amygdala. These data carry significant implications for the potential role of these regions in learning and memory.

Effects of intra-amygdala R(+) 7-OH-DPAT on intra-accumbens d-amphetamine-associated learning. I. Pavlovian conditioning

Abstract We have previously obtained evidence that the mesoamygdaloid dopamine projection modulates the acquisition of a conditioned response (CR) elicited by presentation of a conditioned stimulus (CS) predicting the availability of a natural (sucrose) reward. This property was found to be dependent upon D3, but not D1 or D2, dopamine receptor activation. The aim of the present study was to determine whether the mesoamygdaloid dopamine projection is similarly involved in the acquisition of a drug-associated CR. Thus, two groups of rats with guide cannulae aimed at the nucleus accumbens and amygdala were trained using a Pavlovian conditioning procedure in which an initially neutral CS was paired with a computer-controlled, bilateral intra-accumbens infusion of d-amphetamine (the unconditioned stimulus; US). Conditioning sessions were conducted in standard operant chambers, with each session consisting of a single CS-US trial. For one group of rats, CS presentation was positively correlated with the drug US (Paired group), while for the second group CS and US presentations were negatively correlated (Unpaired group). During training, locomotor activity was recorded and was utilised as the measure both of the unconditioned (UR) and conditioned response (CR). A within-subjects design was utilised to investigate the effect of post-session bilateral intra-amygdala administration of R(+) 7-OH-DPAT on the development of the drug-associated CR. Hence, both Paired and Unpaired groups were exposed to two different CSs which were presented on alternate sessions. Post-session bilateral intra-amygdala administration of R(+) 7-OH-DPAT (10 nmol) followed sessions in which one CS was presented, while intra-amygdala vehicle followed sessions in which the alternate CS was presented. The development of a CR occurred only in the presence of a CS that had been positively correlated with presentation of the drug US. Post-session, intra-amygdala administration of R(+) 7-OH-DPAT enhanced the acquisition of this CR. However, R(+) 7-OH-DPAT was without effect upon the unconditioned response to intra-accumbens d-amphetamine. Our previous data indicate a comparable effect of R(+) 7-OH-DPAT on conditioning to a CS associated with a non-drug, natural reward. Therefore, taken together, these findings suggest that D3 dopamine receptors within the amygdala modulate specifically the acquisition of Pavlovian conditioned responses, regardless of whether drug or natural rewards constitute the US.

Blockade of sensitisation-induced facilitation of appetitive conditioning by post-session intra-amygdala nafadotride

Prior D-amphetamine experience has been reported to enhance appetitive Pavlovian conditioning. The present study assessed the involvement of the mesoamygdaloid dopamine projection in this effect. Bilateral post-session intra-amygdala infusions of the D3 dopamine receptor antagonist, L-nafadotride, or vehicle were given during acquisition of a Pavlovian association in sensitised and unsensitised rats. During these sessions, subjects received presentations of a stimulus (CS(+)) paired with 10% sucrose availability. A second stimulus (CS(-)) was also presented but never paired with sucrose. Sensitised animals infused post-session with vehicle acquired a Pavlovian conditioned approach response during CS(+) presentations more rapidly than controls, as we have shown previously. However, post-session intra-amygdala L-nafadotride selectively retarded conditioned responding to the CS(+) in both groups of animals, abolishing the difference between sensitised and unsensitised rats. These results, therefore, extend the evidence for the involvement of the mesoamygdaloid dopamine projection in Pavlovian conditioning, and the facilitation of associative learning following sensitisation.

Effects of intra-amygdala R(+) 7-OH-DPAT on intra-accumbens d-amphetamine-associated learning II. Instrumental conditioning

Abstract Rats were trained to associate an initially neutral conditioned stimulus (CS) with a response-independent, intra-accumbens infusion of d-amphetamine (the unconditioned stimulus; US). Elsewhere, we have reported that as a result of this training, presentations of the CS alone elicited a conditioned response consisting of increased locomotor activity and that acquisition of this conditioned response was enhanced by post-session, intra-amygdala infusion of the dopamine D3 receptor preferring agonist, R(+) 7-OH-DPAT. Here, in this same group of animals, we have examined the conditioned rewarding properties of the drug-associated CS by determining its ability to support the acquisition of a novel instrumental response in the absence of drug reward. Thus, rats were presented with two novel levers. Presentation of the drug-associated CS was made contingent upon depression of one of the levers (CR lever), while responding upon the other lever (NCR lever) had no programmed consequences. Preferential responding upon the lever delivering the drug-associated CS was observed despite a 6-week interval between CS-US training and the conditioned reward test. Intra-accumbens administration of d-amphetamine (0–20 μg) increased the control over behaviour exerted by the CS, increasing CR, but not NCR lever responding. In contrast, rats that received three pairings of an intra-accumbens infusion of d-amphetamine in combination with intra-amygdala infusion of R(+) 7-OH-DPAT, 3 weeks prior to testing, displayed similar rates of response upon both levers and were insensitive to the potentiation of responding for conditioned reward following intra-accumbens d-amphetamine. However, intra-accumbens d-amphetamine stimulated locomotor activity in a similar, dose-related manner in both groups. In this way, rats that had received intra-accumbens infusion of d-amphetamine in combination with intra-amygdala infusion of R(+) 7-OH-DPAT appeared exactly like control group rats, for which the CS had been paired with intra-accumbens d-amphetamine on a negative basis only. A locomotor activity test indicated that one behavioural consequence of intra-amygdala administration of R(+) 7-OH-DPAT was the reduction of the unconditioned locomotor response resulting from intra-accumbens administration of d-amphetamine. Hence, the present data demonstrate that the conditioned rewarding properties of a drug-associated CS are specific to the CS-US association and are relatively insensitive to decay over time. However, the rewarding properties of a drug-associated CS were selectively abolished following activation of amygdala D3 receptors during presentation of the drug reward. Potential explanations for this effect are discussed, including the possibility that intra-amygdala R(+) 7-OH-DPAT reduced the incentive value of the US.