Andy N. Mead

AMPA‐receptor involvement in c‐fos expression in the medial prefrontal cortex and amygdala dissociates neural substrates of conditioned activity and conditioned reward

Exposure to an environment, previously conditioned to amphetamine (1 mg/kg, i.p.), induced locomotor activity and c‐fos expression (a marker for neuronal activation) in the mouse medial prefrontal cortex (mPFC) and amygdala; acute or repeated amphetamine (1 mg/kg, i.p.) administration induced c‐fos expression additionally in the nucleus accumbens. An α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA)‐receptor antagonist, 2,3‐dihydroxy‐6‐nitro‐7‐sulphamoyl‐benzo(f)quinoxaline (NBQX), blocked expression of conditioned activity, and prevented the increase in c‐fos expression in mPFC, implicating mPFC AMPAergic transmission in the conditioned component of behavioural sensitization to amphetamine. NBQX failed to block the expression of amphetamine‐conditioned place preference, a measure of conditioned reward, or conditioned c‐fos expression in the amygdala, an area implicated in the expression of conditioned place preference. These findings indicate that the conditioned components of behavioural sensitization depend on AMPA‐receptor‐mediated activation in mPFC, but that conditioned reward does not.

AMPA-Receptor GluR1 Subunits are Involved in the Control Over Behavior by Cocaine-Paired Cues

The learning processes underlying the formation of drug–cue associations involve changes in synaptic transmission mediated by AMPA receptors. Here, we examine the consequences of targeted deletion of the gene encoding GluR1 subunits of AMPA receptors (gria1 knockouts (KO)) on cocaine self-administration and on the ability of cocaine-paired cues to affect cocaine-seeking in mice. Cocaine self-administration was unaffected by gria1 deletion, as was the ability of a cocaine-paired cue to reinstate responding following extinction, following either a 3 or a 66 day delay. However, gria1 KOs over-responded during extinction. The KOs were unable initially to learn a new response to access a cue previously conditioned to cocaine (conditioned reinforcement (CR)), although a second test 2 months later revealed that this was a transient deficit. These studies indicate that GluR1-containing AMPA-receptors are not involved in cocaine self-administration, cue-induced reinstatement of cocaine seeking, or incubation of the cocaine seeking response. In order to understand the specificity of the deficits in CR responding, a parallel study was performed with food reward. As with cocaine, there were no effects of gria1 deletion on food self-administration or cue-induced reinstatement, and KOs over-responded during extinction. However, even immediately after instrumental training for food, KO mice demonstrated responding for CR, suggesting that the CR deficit observed with a cocaine cue is specific to drug reward. These data indicate that GluR1-containing AMPA receptors are important in stimulus reward learning, though the method of cue-reward association formation, the reward class, and the behavioral end point are critical variables in determining their involvement.

AMPA-receptors are involved in the expression of amphetamine-induced behavioural sensitisation, but not in the expression of amphetamine-induced conditioned activity in mice.

We investigated the role of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX) in the expression of amphetamine-induced behavioural sensitisation and amphetamine-induced conditioned activity in mice. Repeated weekly administration of amphetamine (0.375 mg/kg) for 7 weeks led to an increased locomotor response when challenged with amphetamine 1 week later. NBQX attenuated this increased response at doses (3 and 30 mg/kg) which had no effect on the acute locomotor response to amphetamine. In a separate experiment, mice given amphetamine (1 mg/kg) in a distinctive environment, showed an increased locomotor response within this environment following a subsequent saline administration. NBQX (5-20 mg/kg) had no effect on the expression of this conditioned response. These results suggest that AMPA receptors are involved in the expression of amphetamine-induced behavioural sensitisation in mice, and that this involvement is limited to either the neurobiological effects of amphetamine or the effects of amphetamine on conditioned associations, rather than drug environment conditioned associations.

AMPA receptors and motivation for drug: effect of the selective antagonist NBQX on behavioural sensitization and on self-administration in mice

A series of experiments was carried out in which the potency of the selective α-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA)-receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) (10-100 mg/kg) on locomotor activity was investigated, in mice. NBQX reduced all forms of activity studied, but its potency to do so varied according to the conditions of the experiment. The smallest dose of NBQX significantly reducing spontaneous or cocaineinduced activity was 100 mg/kg. Mice that had been repeatedly treated with 16 mg/kg cocaine once per week, for 7 weeks, showed a sensitized locomotor response to a challenge dose of cocaine (16 mg/kg). NBQX reversed the sensitized response at 30 and 100 mg/kg. The pattern of results obtained leaves open the role that AMPA-receptors may have in the expression of behavioural sensitization. In two further experiments, mice were trained to self-administer cocaine (30 µg per reinforcer) via intravenous catheters, using an operant lever pressing technique. When the amount of cocaine per reinforcer was doubled (to 60 µg) or halved (to 15 µg) the mice adapted lever pressing rates to maintain some constancy of self-dosing (but not at 7.5 µg per reinforcer) and when saline was substituted for cocaine, response rates increased considerably (extinction bursting). NBQX (10 and 30 mg/kg) reduced the self-administration of 30 µg reinforcers of cocaine, but only during the first 30 min of the test session. There was no evidence that NBQX specifically antagonized the reinforcing effect of cocaine, as responding was similarly reduced on both the reinforced and the non-reinforced lever, nor did the response to NBQX mimic behaviour seen following changes in the concentration of the reinforcer. The results of the locomotor experiments and the self-administration experiments are discussed together, in terms of current hypotheses about glutamatergic mechanisms involved in motivation for drug.

Intravenous cocaine induced‐activity and behavioural sensitization in norepinephrine‐, but not dopamine‐transporter knockout mice

Previously, it was reported that both norepinephrine transporter (NET) and dopamine transporter (DAT) knockout (KO) mice were sensitive to the reinforcing effects of cocaine. However, assessing the locomotor‐stimulant effects of cocaine in these subjects has proven difficult due to significant differences in their baseline activity compared to wild‐type controls. The present studies were designed to clarify the role of NET and DAT in the stimulant effects of acute and repeated cocaine utilizing these knockout mice, and thereby assess the role of these substrates in the locomotor stimulant effects of cocaine. Mice were habituated to the test environment for sufficient time to ensure equal baselines at the time of cocaine administration. Mice then received cocaine (3–25 mg/kg) intravenously according to a within‐session cumulative dose–response design. Cocaine dosing was repeated at 48‐h intervals for four sessions to assess behavioural sensitization. NET‐KO mice exhibited a reduced response to acute cocaine administration compared to wild‐type (WT) controls. However, comparable sensitization developed in NET‐KO and WT mice. The DAT‐KO and DAT‐heterozygote (HT) mice displayed no locomotor activation following either acute or repeated cocaine administration. These data suggest a role for the NET in the acute response to cocaine, but no involvement in sensitization to cocaine. In contrast, DAT appears to be necessary for both the acute locomotor response to cocaine and the subsequent development of sensitization. In addition to existing data concerning the reinforcing effects of cocaine in DAT‐KO mice, these data suggest a dissociation between the reinforcing and locomotor stimulant effects of cocaine.

Behavioural effects of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-receptor antagonists and their relevance to substance abuse

This review presents some of the work that has been carried out to investigate the behavioural effects of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-receptor antagonists in animal models of substance abuse. Many of the studies have been conducted in light of current ideas that emphasise the analogous role of glutamatergic mechanisms in synaptic plasticity and long-term behavioural adaptation to drugs. Experiments on behavioural sensitisation indicate that whereas N-methyl-D-aspartate receptors are involved in induction, AMPA-receptors may mediate expression of the established response. In this regard, an important factor may be the degree of drug-environment conditioning. Thus, studies of the effects of AMPA-receptor antagonists on conditioned behaviours are reviewed here. Relatively few studies on the effects of AMPA-receptor antagonists on primary reinforcement from self-administered drugs and the subjective effects of drugs have been carried out, but a profile that contrasts with that of the N-methyl-D-aspartate antagonists appears to be emerging. Studies of withdrawal from opioids suggest that whilst AMPA-receptor antagonists may not be able to prevent tolerance or dependence from developing, they may ameliorate both the physical and emotional consequences of withdrawal. Overall, the AMPA-receptor antagonists may represent a promising new approach for treating the consequences of drug abuse. However, as results are often complicated by the use of the less-selective compounds, it will be important to use better tools in future studies.

The mGluR5 antagonist MTEP dissociates the acquisition of predictive and incentive motivational properties of reward-paired stimuli in mice

An environmental stimulus paired with reward (a conditioned stimulus; CS) can acquire predictive properties that signal reward availability and may also acquire incentive motivational properties that enable the CS to influence appetitive behaviors. The neural mechanisms involved in the acquisition and expression of these CS properties are not fully understood. The metabotropic glutamate receptor, mGluR5, contributes to synaptic plasticity underlying learning and memory processes. We examined the role of mGluR5 in the acquisition and expression of learning that enables a CS to predict reward (goal-tracking) and acquire incentive properties (conditioned reinforcement). Mice were injected with vehicle or the mGluR5 antagonist, MTEP (3 or 10 mg/kg), before each Pavlovian conditioning session in which a stimulus (CS+) was paired with food delivery. Subsequently, in the absence of the primary food reward, we determined whether the CS+ could reinforce a novel instrumental response (conditioned reinforcement) and direct behavior toward the place of reward delivery (goal-tracking). MTEP did not affect performance during the conditioning phase, or the ability of the CS+ to elicit a goal-tracking response. In contrast, 10 mg/kg MTEP given before each conditioning session prevented the subsequent expression of conditioned reinforcement. This dose of MTEP did not affect conditioned reinforcement when administered before the test, in mice that had received vehicle before conditioning sessions. Thus, mGluR5 has a critical role in the acquisition of incentive properties by a CS, but is not required for the expression of incentive learning, or for the CS to acquire predictive properties that signal reward availability.

AMPA receptor GluR2, but not GluR1, subunit deletion impairs emotional response conditioning in mice.

Deletions of gria1 or gria2 genes encoding alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic-acid-receptor subunits differ in their effects on appetitive conditioning. The authors investigated whether similar differences would occur in an aversive conditioning test. The ability of a discrete stimulus paired with footshock to subsequently inhibit food-maintained operant responding (conditioned emotional response) was examined in mice with deletions of gria1 or gria2 genes. Whereas gria1 knockout (KO) mice performed normally compared with wild-type (WT) controls, gria2 KO mice displayed no reduction in response rates when the shock-paired stimulus was presented. Nevertheless, gria2 KOs displayed evidence of freezing in a footshock-paired context, indicating that aversive learning could occur. In addition, gria1 KO mice showed some evidence of increased anxiety, and gria2 KOs showed reduced anxiety, in the elevated plus-maze.

Studying the neurobiology of stimulant and alcohol abuse and dependence in genetically manipulated mice.

The ability to manipulate the genetic makeup of organisms by specific targeting of selected genes has provided a novel means of investigating the neurobiological mechanisms underlying drug abuse and dependence. However, as with other techniques, there are a number of potential pitfalls in the use of genetically manipulated animals (usually mice) in behavioural experiments. This review discusses the techniques involved in creating genetically manipulated mice, and points to opportunities and insights into addictive processes provided by the new science, while illustrating some of the potential problems encountered in interpretation of data obtained from such animals. The use of the mouse as an experimental animal also raises some specific problems which limit the usefulness of the technique at present. Examples taken from research into alcohol and psychostimulant abuse and dependence are used to illustrate the usefulness of genetically manipulated animals in addiction research, the problems of interpretation which sometimes arise, and how techniques are being developed to overcome present limitations to this exciting area of research.

Previous experience of withdrawal from chronic diazepam ameliorates the aversiveness of precipitated withdrawal and reduces withdrawal‐induced c‐fos expression in nucleus accumbens

Flumazenil (20 mg/kg, i.p.)‐precipitated withdrawal from chronic treatment with diazepam (DZP, 15 mg/kg, s.c. in sesame oil for 21 days) resulted in a decreased seizure threshold to the convulsant, pentylenetetrazole (PTZ), infused into the tail vein; withdrawal from 21‐day chronic diazepam treatment, interspersed with two periods of drug withdrawal, resulted in a greater decrease in convulsant threshold. A separate experiment showed that consumption of a sucrose solution immediately prior to precipitated withdrawal resulted in a decreased subsequent consumption of the sucrose solution; no such evidence of a conditioned taste aversion (CTA) was seen in mice given prior experience of withdrawal. Thus, prior experience of withdrawal enhanced the effects of a subsequent precipitated withdrawal in increasing seizure sensitivity, but weakened the ability of this withdrawal to serve as an aversive unconditioned stimulus (US). The weakening of the aversive properties of precipitated withdrawal may reflect habituation to the withdrawal stimulus, and was accompanied by a loss of the ability of withdrawal to induce c‐fos expression in the shell of the nucleus accumbens, an area sensitive to both novel, and stressful, as well as rewarding stimuli.