Involvement of the nucleus accumbens and medial prefrontal cortex in the expression of conditioned hyperactivity to a cocaine-associated environment in rats.

This study examined the roles of the nucleus accumbens (NAc), medial prefrontal cortex, basolateral amygdala, and ventral subiculum of the hippocampus in the expression of Pavlovian conditioned hyperactivity responses to cocaine-related stimuli. This was accomplished by pharmacologically inhibiting these regions prior to drug-free tests for conditioned hyperactivity in an environment previously associated with cocaine. The results indicate that conditioned hyperactivity could be disrupted by infusions of the GABA-B agonist, baclofen (0.2 nmol/0.5 microl/side) into the NAc, or completely blocked by infusions of the GABA-A agonist, muscimol (0.1 and 0.2 nmol/0.5 microl/side) into the medial prefrontal cortex. In contrast, conditioned hyperactivity was unaffected by pharmacological inhibition of the basolateral amygdala, the ventral subiculum, or sites dorsal to the NAc or prefrontal cortex. These findings suggest that the NAc and the prefrontal cortex are crucial elements of the neural circuitry underlying the expression of Pavlovian conditioned responses to cocaine-related stimuli.

Expression of fos-related antigens in the nucleus accumbens during opiate withdrawal and their attenuation by a D2 dopamine receptor agonist.

Previous studies from this laboratory indicated that D2 dopamine (DA) receptors within the nucleus accumbens (NAc) are important for regulating somatic signs of opiate withdrawal. The present study measured the expression of Fos-related antigens (FRAs) within the NAc during opiate withdrawal to determine whether decreases in somatic withdrawal signs produced by a D2 receptor agonist are accompanied by related changes in accumbens neuronal activity. In an initial experiment, quantitative analyses of FRA immunoreactivity revealed increases in the number of FRA-positive cells throughout the NAc of opiate dependent animals undergoing naltrexone-precipitated withdrawal relative to dependent or non-dependent animals that did not experience withdrawal. A second experiment showed that somatic signs and FRA expression within the NAc could each be attenuated when the D2 agonist propylnorapomorphine (NPA; 0.1 or 0.3 mg/kg, i.p.) was administered prior to naltrexone-precipitated withdrawal. These findings suggest that D2 regulation of neuronal activity within the NAc may be important for the expression of opiate withdrawal symptoms.

Development of both conditioning and sensitization of the behavioral activating effects of amphetamine is blocked by the non-competitive NMDA receptor antagonist, MK-801.

The non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, has been shown to block the development of sensitization of the behavioral activating effects of amphetamine. Three experiments were designed to determine in rats whether MK-801 had its effects through interference with long-term changes underlying sensitization, per se, or through interference with the development of conditioning of the drug effect to the environment where the drug was given. In experiment 1, conditioning was promoted by explicitly pairing amphetamine (1.5 mg/kg, IP) with the testing environment. In experiment 2, a random-pairing procedure was used to eliminate the possibility of association between the drug and a specific context. Experiment 3 was carried out to assess the duration of the blockade of sensitization by MK-801. The effect of MK-801 (0.25 mg/kg, IP) during amphetamine pre-exposure was studied in tests for conditioning (following saline injections, experiment 1) and in tests for sensitization (following 0.75 mg/kg amphetamine, experiments 1, 2 and 3). It was found in experiment 1 that MK-801 given with amphetamine during the amphetamine pre-exposure phase blocked the development of both conditioning activity and environment-specific sensitization to amphetamine. The results of experiment 2, showing that sensitization to amphetamine was blocked by MK-801 even when conditioning was prevented, suggest that the two effects of MK-801 are independent, and may implicate different sites of action.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of sensitization to the effects of d-amphetamine and apomorphine on sensorimotor gating in rats.

This study assessed whether repeated injections of d-amphetamine or apomorphine could induce sensitization to the disruptive effects of these psychomotor stimulants on sensorimotor gating in rats. In the first experiment, rats were given six pre-exposures to either 2.0 mg/kg d-amphetamine or saline before being tested for the effects of d-amphetamine (0.0, 0.5, 1.0, 2.0 or 4.0 mg/kg, i.p.) on prepulse inhibition of acoustic startle (PPI) and locomotor activity. The tests for PPI confirmed that sensorimotor gating could be disrupted by a high dose of d-amphetamine (4.0 mg/kg). However, comparison of the dose-response curves for the drug and saline pre-exposed groups did not reveal evidence for sensitization to this d-amphetamine effect in drug-pre-exposed rats, despite indications that sensitization had developed to the locomotor stimulant effects of d-amphetamine. A similar pattern of results was obtained in a second experiment that examined the effects of apomorphine (0.0, 0.1, 0.2, 0.4 and 0.8 mg/kg, s.c.) on PPI and locomotion in rats pre-exposed to 2.0 mg/kg of this drug or its vehicle. These findings demonstrate that treatments which induce sensitization to the behavioral activating effects of psychomotor stimulants do not necessarily produce sensitization to the disruptive effects of stimulants on sensorimotor gating. The implications of these results for hypotheses linking sensitization-like processes to the etiology of schizophrenia are discussed.

Differential effects of cholinergic drugs on discriminative cues and self-stimulation produced by electrical stimulation of the ventral tegmental area.

Cholinergic receptors have been shown to modulate a subset of discriminative cues produced by electrical stimulation of the ventral tegmental area (VTA) in rats. The present study identified the specific cholinergic receptor type modulating these electrical brain-stimulation (EBS) cues, and assessed whether these receptors also mediated the rewarding effects of VTA EBS. The EBS cues were enhanced by the acetylcholinesterase inhibitor physostigmine and the muscarinic receptor agonists pilocarpine and RS-86, whereas the nicotinic receptor agonist nicotine had no effect. The enhancing effects of pilocarpine or RS-86 were attenuated by the muscarinic antagonist scopolamine. The EBS cues were not affected when scopolamine was injected alone, although high doses disrupted discriminated responses. Intracranial self-stimulation (ICSS) rates were depressed by physostigmine and pilocarpine and increased by nicotine and scopolamine. These results indicated a facilitatory influence of muscarinic receptors on the EBS cues, and an inhibitory role in VTA ICSS. Nicotinic receptor activation did not affect the EBS cues, but facilitated ICSS. These differential effects of cholinergic receptor activation point to a dissociation of the specific EBS cues measured in this study from the rewarding effects of VTA stimulation.

The retrograde tracer fluoro-gold interferes with the expression of fos-related antigens.

The retrograde tracer, fluoro-gold (FG) has been used in combination with immediate early gene (IEG) immunohistochemistry to identify neural circuits activated by pharmacological, physiological or behavioral manipulations. However, since FG has been shown to be toxic to cell bodies, axons and terminals at the injection site, the question arises as to whether FG alters the detection of IEG products. To examine this question, FG was microiontophoresed unilaterally into the nucleus accumbens (NAc) of rats and Fos-related antigens (FRAs) were examined in both hemispheres 12 days later. Approximately half as many FRA-positive nuclei were observed in the tracer-injected NAc as were found in the contralateral NAc. Similar results were observed in the ventral subiculum of the hippocampus and the basolateral and central amygdaloid nuclei, but not in the lateral septum or lateral habenula. These results suggest that FG microiontophoresed into the NAc interferes with the expression of FRAs at the injection site and also at other ipsilateral limbic sites.

Effects of the competitive N-methyl-D-aspartate receptor antagonist, CPP, on the development and expression of conditioned hyperactivity and sensitization induced by cocaine.

This study used novel behavioral measures to examine the effects of the competitive N-methyl-D-aspartate (NMDA) receptor antagonist, CPP, on the development and expression of conditioned hyperactivity and sensitization produced with cocaine. The first experiment confirmed that horizontal locomotor activity measured in the central zone of an activity enclosure could be increased by 10.0 mg/kg cocaine. This increased activity showed sensitization after repeated cocaine injections, and it could be conditioned to the test environment. Subsequent experiments demonstrated that CPP (0.2 and 0.4 nmol, i.c.v.) could block the development, but not the expression, of conditioned hyperactivity and sensitization in the central zone. These findings confirm that NMDA receptors are critically involved in the development of conditioned hyperactivity and sensitization, but indicate that such receptors may not be necessary for the expression of these neurobehavioral adaptations.

D-amphetamine-like stimulus properties are produced by morphine injections into the ventral tegmental area but not into the nucleus accumbens.

We investigated whether injections of morphine into the ventral tegmental area (VTA) or the nucleus accumbens (NAS) could produce amphetamine-like stimulus properties and locomotor stimulant effects. Rats were trained to discriminate 1.0 mg/kg D-amphetamine from saline using both VI-30 and FR-16 reinforcement schedules and they were then tested following bilateral injections of morphine sulfate (2.5, 5.0 and 10.0 micrograms/side) either into the VTA or the NAS. Intra-VTA injections of morphine produced significant increases in amphetamine-lever responding that were comparable to increases observed following intra-NAS D-amphetamine (2.5, 5.0 and 10.0 micrograms/side). Such increases were not observed, however, following intra-NAS injections of morphine. Tests for the effects of intracerebral morphine and D-amphetamine on locomotor activity produced a similar pattern of results. Locomotor activity was increased following intra-VTA morphine and intra-NAS D-amphetamine injections, but not after intra-NAS morphine injections. Together, these findings indicate that activation of opioid receptors within the VTA, but not the NAS, can produce a behavioral state which mimics to some degree the state produced by systemic and intra-NAS injections of D-amphetamine.

Amphetamine-like stimulus properties produced by electrical stimulation of reward sites in the ventral tegmental area.

The present study examined whether the stimulus properties of D-amphetamine could be mimicked by electrical stimulation of the ventral tegmental area (VTA). Rats trained to discriminate 1.0 mg/kg D-amphetamine from saline were given generalization tests with a range of D-amphetamine doses administered either alone or in combination with VTA stimulation. The results suggested that the VTA stimulation could enhance the cueing effects of D-amphetamine, as levels of responding on the D-amphetamine-appropriate lever during stimulation trials were increased relative to tests without stimulation. Individual differences were observed in the amount of drug-lever responding elicited by the VTA stimulation during drug-free substitution tests, and the different levels of drug-lever responding correlated positively with the response rates obtained from these rats during subsequent intracranial self-stimulation tests. These finding suggest that VTA stimulation can have D-amphetamine-like stimulus properties and such stimulus properties may be related to the rewarding effects of the brain stimulation.

Origin of noradrenergic afferents to the shell subregion of the nucleus accumbens: anterograde and retrograde tract-tracing studies in the rat.

The nucleus accumbens (NAcc) can be subdivided into 'core' and 'shell' based on anatomical connections and histochemical markers. Previous studies have demonstrated dopamine-beta-hydroxylase immunoreactive (DBH-ir) fibers in the NAcc shell, but the source of these noradrenergic (NE) afferents has not been determined. Therefore, we have investigated in detail the anatomy of NE afferents to this subregion. Dual immunohistochemistry for DBH and substance P demonstrated numerous DBH-ir fibers in the caudal NAcc shell. Neurons projecting to the NAcc were identified with Fluoro-Gold (FG) or cholera toxin B (CTb) retrograde tracing and tyrosine hydroxylase (TH) immunohistochemistry. Single- and double-labeled neurons were observed in the A2 and A1 NE cell groups following FG injections into the caudal NAcc shell. Numerous FG and CTb single-labeled neurons were found in the rostral locus coeruleus (LC), subcoeruleus and pericoerulear dendritic region, with an occasional double-labeled neuron in the LC. Few labeled neurons were seen in the brainstem after FG injections into the NAcc core, consistent with the lack of DBH-ir in this subterritory. To confirm these results, injections of Phaseolus vulgaris leucoagglutinin or biotinylated dextran amine were made into the LC or nucleus tractus solitarius (NTS). Virtually no labeled fibers were observed in the NAcc following injections into central LC. However, fibers were observed in the NAcc shell after injections in the NTS. These results indicate that the primary source(s) of NE afferents to the NAcc shell is the A2 region of the NTS, with lesser contributions from A1 and LC.

Enhanced norepinephrine release in prefrontal cortex with burst stimulation of the locus coeruleus.

The present study was designed to determine the relationship between the discharge of noradrenergic locus coeruleus (LC) neurons and norepinephrine release in the medial prefrontal cortex, a target of LC projections. The LC was electrically stimulated at varying frequencies and patterns for 20 min and extracellular norepinephrine levels were measured in the medial prefrontal cortex of halothane-anesthetized rats using in vivo microdialysis. Electrical stimulation of the LC at 3-10 Hz with an evenly spaced pattern of pulses (tonic stimulation) increased cortical norepinephrine levels in a frequency-dependent manner, with 5- and 10-Hz stimulation increasing norepinephrine levels by 49 +/- 3% and 66 +/- 20%, respectively. The LC was also stimulated with bursts of pulses designed to deliver physiologically relevant phasic stimulation using the same number of stimuli in a 20-min period as delivered by tonic stimulation at 3 Hz. Results revealed that norepinephrine levels were significantly higher with phasic stimulation compared to tonic stimulation. The present findings indicate that both frequency and pattern of LC discharge are determinants of norepinephrine terminal release. Additionally, bursts of LC activity, similar to those that occur in behaving animals, may be more effective in increasing terminal norepinephrine release on a per spike basis than tonic increases in activity.

The development of behavioral sensitization to apomorphine is blocked by MK-801.

The effects of the non-competitive NMDA receptor antagonist, MK-801 (dizocilpine), on the development of sensitization to the mixed dopamine receptor agonist, apomorphine were examined. Rats pretreated with MK-801 (0.25 mg/kg) prior to 2.0 mg/kg apomorphine failed to show a progressive sensitization to the activating effect of apomorphine across six preexposure sessions, nor did they show enhanced activity relative to control rats in response to 0.5 mg/kg apomorphine, given alone, during a subsequent test for sensitization. These results support the view that MK-801 interferes with long-term neural changes underlying sensitization to stimulant drugs.

Influence of some drugs of abuse on the discriminative stimulus properties of amphetamine.

The present study assessed the utility of a drug interaction paradigm for measuring the effects of various psychoactive drugs on the stimulus properties of d-amphetamine. In the first experiment, rats trained to discriminate 1.0mg/kg d-amphetamine from saline were tested for generalization to a range of d-amphetamine doses (0.25, 0.50 and 1.0mg/kg) injected alone or with cocaine, apomorphine or haloperidol. When given alone d-amphetamine yielded an orderly dose-response function and this was altered by the test drugs in a manner consistent with the dopamine agonist or antagonist actions of each compound. In the second experiment, the cueing effects of d-amphetamine were potentiated by nicotine and attenuated by morphine and midazolam. Ethanol enhanced the cueing effects of the lower d-amphetamine doses but produced no drug-lever responding when given alone. These findings confirm the results of previous studies which have assessed the effects of these classes of drugs on the stimulus properties of amphetamine. However, the results are inconsistent with the hypothesis that all drugs of abuse possess psychomotor stimulant properties.

Rats' short-term memory for brief electrical stimulation of the ventral tegmental area of the brain.

Short-term memory for brief electrical stimulation of rats' ventral tegmentum was assessed in a delayed conditional discrimination procedure in which trials began with a train of either low- or high-intensity stimulations. After a delay (i.e., retention interval) ranging from 0.01 to 4 sec, a lever was inserted, accompanied by a dim or a bright light. Lever-pressing was rewarded with food on low intensity-dim light and high intensity-bright light trials, but not on low-bright or high-dim trials. Discrimination ratios, based on relative responding on rewarded trials, indicated that subjects were able to remember the electrical stimulations, but that there was sometimes substantial forgetting after the 4-sec retention interval. The implications of these results for an understanding of the stimulus properties of electrical brain stimulation were discussed.

Dissociation of dopaminergic and non-dopaminergic substrates for cues produced by electrical stimulation of the ventral tegmental area.

The present study provides evidence for the existence of multiple substrates for cues produced by electrical stimulation of the ventral tegmental area in rats. Two different procedures were employed to assess the effects of amphetamine and haloperidol on the discrimination of high and low intensity cues produced by electrical brain stimulation (EBS). When the procedure involved frequent presentation of brief trials, amphetamine and haloperidol had no effect on the discrimination of EBS. In contrast, when the trials were less frequent and extended in duration, amphetamine enhanced the perceived intensity of the cues whereas haloperidol had the opposite effect. These results indicate that the use of different discrimination procedures may result in the measurement of separate dopaminergic and non-dopaminergic substrates for cue properties of EBS in the ventral tegmental area.

Differential effects of physostigmine on cues produced by electrical stimulation of the ventral tegmental area using two discrimination procedures.

Two procedures were employed to assess the effects of physostigmine on the discrimination of cues produced by either high or low intensity electrical brain stimulation (EBS) of the ventral tegmental area in rats. When the procedure involved frequent presentation of brief trials, physostigmine enhanced the perceived intensities of the cues, causing the rats to respond to low intensities as though they had higher values. In contrast, physostigmine had no effects on the discrimination when the trials were less frequent and extended in duration. These results confirm the existence of multiple substrates for cues produced by stimulation of the ventral tegmental area in rats and implicate cholinergic neurons as substrates for the non-dopaminergic cues identified in the companion paper.

Expression of Fos-related antigens in the nucleus accumbens and associated regions following exposure to a cocaine-paired environment.

This study examined whether conditioned hyperactivity measured in a cocaine-paired environment was associated with increased expression of Fos-related antigens (FRA) within the nucleus accumbens (NAc) and associated forebrain regions of rats. Three groups of rats were given repeated injections of either cocaine in the test environment and saline in the colony room (group Paired), saline in the test environment and cocaine in the colony room (group Unpaired), or saline in both environments (group Control). All rats were subsequently given a drug-free test for conditioned hyperactivity in the test environment, and their brains were removed so that FRA immunohistochemistry could be conducted. Rats in the Paired group showed conditioned hyperactivity during the conditioning test, and this behavioural response was associated with increased FRA expression within the caudal NAc, the medial prefrontal cortex and the lateral septum relative to the Unpaired and Control groups. Paired rats also showed increased FRA expression within the orbital prefrontal cortex, the claustrum, the caudal amygdala (basolateral and central regions), the paraventricular thalamic nucleus, the subiculum of the hippocampus, and the lateral habenula relative to the Control group. However, the FRA levels in these latter sites were not significantly increased relative to those of Unpaired rats, indicating that genomic responses in these regions were not entirely context dependent. The correspondence between conditioned hyperactivity and enhanced FRA expression within the caudal NAc, the medial prefrontal cortex and lateral septum suggests that these regions may participate in the expression of conditioned responses to cocaine-related stimuli.

MK-801 increases locomotor activity without elevating extracellular dopamine levels in the nucleus accumbens.

In vivo microdialysis was used in freely moving rats to determine whether the locomotor stimulant effects of dizocilpine maleate (MK-801) were related to increased dopamine (DA) release within the nucleus accumbens (N. Acc.). Each experiment began with a baseline period of at least 2 h (starting 15-20 h after insertion of concentric, removable dialysis probes), during with activity records and dialysate samples were collected every 20 min. Rats in the first experiment then were injected with MK-801 (0.125, 0.25, or 0.50 mg/kg, i.p.) or saline, and activity and extracellular levels of DA, dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were measured for a further 160 min post-injection. In a second experiment, rats were given 1.5 mg/kg d-amphetamine sulphate 40 min after receiving the same doses of MK-801, and testing was continued for 120 min. Rats in a third experiment were given low, autoreceptor-preferring doses of apomorphine hydrochloride (25 or 50 micrograms/kg, s.c.) or its vehicle 40 min after injection of 0.25 mg/kg MK-801 and then monitored for 120 min. MK-801 produced strong and consistent increases in locomotor activity that were augmented by amphetamine and greatly reduced by the low doses of apomorphine. MK-801 did not increase extracellular DA levels within the N. Acc. when given alone, and it failed to influence the changes in extracellular DA produced by d-amphetamine and apomorphine. MK-801 did produce consistent, dose-related increases in DOPAC and HVA that were probably not related to transmitter release. These results indicate that the increases in locomotor activity seen following MK-801 do not arise from a drug-induced increase in DA levels within the N. Acc.

Behavioral activation induced by D(2)-like receptor stimulation during opiate withdrawal.

Withdrawal is a potent motivator of drug-seeking behavior in human opiate addicts. Paradoxically, opiate withdrawal reduces dopamine release and suppresses behavioral responding in several animal models of addiction. These findings pose critical questions about how a withdrawal state that depresses dopaminergic and behavioral functioning contributes to drug seeking. This study addressed this issue by investigating factors that increase behavioral activity during opiate withdrawal. Initial experiments revealed that the D(2)-like agonists propylnorapomorphine HCl (NPA; 0.05-0.4 mg/kg, i. p.) and quinpirole (0.2-0.4 mg/kg, s.c.) each produced strong locomotor activating effects during opiate withdrawal that were not apparent in the absence of withdrawal. Concurrent stereotypy ratings indicated that these effects of NPA and quinpirole during withdrawal were not an indirect consequence of changes in the stereotypy-inducing effects of these drugs. Subsequent experiments showed that locomotion was not increased when opiate withdrawal was induced in the presence of the D(1)-like agonist SKF 38393 (1.0-8.0 mg/kg, i.p.), that the locomotor activation produced by NPA during withdrawal could be attenuated by the D(2)-like antagonist eticlopride (0.1-0.2 mg/kg, i.p.), and that locomotor activating effects of NPA could be observed when withdrawal was induced by extracting the implanted morphine pellets, but not when the NPA was given after naltrexone antagonism of acute morphine treatment in nondependent rats. These findings indicate that opiate withdrawal regulates the behavioral impact of D(2)-like receptor stimulation so that locomotion is markedly increased when these receptors are stimulated during periods of withdrawal. This potentiation may be important for facilitating behavioral responses during periods of opiate detoxification.

Noradrenaline in the ventral forebrain is critical for opiate withdrawal-induced aversion.

Cessation of drug use in chronic opiate abusers produces a severe withdrawal syndrome that is highly aversive, and avoidance of withdrawal or associated stimuli is a major factor contributing to opiate abuse. Increased noradrenaline in the brain has long been implicated in opiate withdrawal, but it has not been clear which noradrenergic systems are involved. Here we show that microinjection of beta-noradrenergic-receptor antagonists, or of an alpha2-receptor agonist, into the bed nucleus of the stria terminalis (BNST) in rats markedly attenuates opiate-withdrawal-induced conditioned place aversion. Immunohistochemical studies revealed that numerous BNST-projecting cells in the A1 and A2 noradrenergic cell groups of the caudal medulla were activated during withdrawal. Lesion of these ascending medullary projections also greatly reduced opiate-withdrawal-induced place aversion, whereas lesion of locus coeruleus noradrenergic projections had no effect on opiate-withdrawal behaviour. We conclude that noradrenergic inputs to the BNST from the caudal medulla are critically involved in the aversiveness of opiate withdrawal.