Variations in the stimulus salience of cocaine reward influences drug-associated contextual memory.

Drugs of abuse act as reinforcers because they influence learning and memory processes resulting in long-term memory of drug reward. We have previously shown that mice conditioned by fixed daily dose of cocaine (Fix-C) or daily escalating doses of cocaine (Esc-C) resulted in short- and long-term persistence of drug memory, respectively, suggesting different mechanisms in acquisition of cocaine memory. The present study was undertaken to investigate the differential contribution of N-methyl-D-aspartate receptor (NMDAR) subunits in the formation of Fix-C and Esc-C memory in C57BL/6J mice. Training by Esc-C resulted in marked elevation in hippocampal expression of Grin2b mRNA and NR2B protein levels compared with training by Fix-C. The NR2B-containing NMDAR antagonist ifenprodil had similar attenuating effects on acquisition and reconsolidation of Fix-C and Esc-C memory. However, the NMDAR antagonist MK-801 had differential effects: (1) higher doses of MK-801 were required for post-retrieval disruption of reconsolidation of Esc-C memory than Fix-C memory; and (2) pre-retrieval MK-801 inhibited extinction of Fix-C memory but it had no effect on Esc-C memory. In addition, blockade of NMDAR downstream signaling pathways also showed differential regulation of Fix-C and Esc-C memory. Inhibition of neuronal nitric oxide synthase attenuated acquisition and disrupted reconsolidation of Fix-C but not Esc-C memory. In contrast, the mitogen-activating extracellular kinase inhibitor SL327 attenuated reconsolidation of Esc-C but not Fix-C memory. These results suggest that NMDAR downstream signaling molecules associated with consolidation and reconsolidation of cocaine-associated memory may vary upon changes in the salience of cocaine reward during conditioning.

The strength of aversive and appetitive associations and maladaptive behaviors.

Certain maladaptive behaviors are thought to be acquired through classical Pavlovian conditioning. Exaggerated fear response, which can develop through Pavlovian conditioning, is associated with acquired anxiety disorders such as post-traumatic stress disorders (PTSDs). Inflated reward-seeking behavior, which develops through Pavlovian conditioning, underlies some types of addictive behavior (e.g., addiction to drugs, food, and gambling). These maladaptive behaviors are dependent on associative learning and the development of long-term memory (LTM). In animal models, an aversive reinforcer (fear conditioning) encodes an aversive contextual and cued LTM. On the other hand, an appetitive reinforcer results in conditioned place preference (CPP) that encodes an appetitive contextual LTM. The literature on weak and strong associative learning pertaining to the development of aversive and appetitive LTM is relatively scarce; thus, this review is particularly focused on the strength of associative learning. The strength of associative learning is dependent on the valence of the reinforcer and the salience of the conditioned stimulus that ultimately sways the strength of the memory trace. Our studies suggest that labile (weak) aversive and appetitive LTM may share similar signaling pathways, whereas stable (strong) aversive and appetitive LTM is mediated through different pathways. In addition, we provide some evidence suggesting that extinction of aversive fear memory and appetitive drug memory is likely to be mediated through different signaling molecules. We put forward the importance of studies aimed to investigate the molecular mechanisms underlying the development of weak and strong memories (aversive and appetitive), which would ultimately help in the development of targeted pharmacotherapies for the management of maladaptive behaviors that arise from classical Pavlovian conditioning.

Sodium butyrate-induced histone acetylation strengthens the expression of cocaine-associated contextual memory.

The conditioned place preference (CPP) paradigm entails Pavlovian conditioning and allows evaluating the acquisition and extinction of drug-associated memory. Epigenetic regulation of chromatin structure by acetylation and deacetylation of histone proteins is critical for formation of long-term memory (LTM). We have recently shown that a single administration of the histone deacetylase (HDAC) inhibitor sodium butyrate (NaB) facilitated extinction of fear-associated memory in mice. Using the CPP paradigm, the present study investigated the effect of NaB on cocaine-associated memory. C57B/6 mice were conditioned by either fixed daily doses of cocaine (5mg/kg×4 and 15mg/kg×4days) or an escalating schedule (3, 6, 12 and 24mg/kg). Acute administration of NaB (1.2g/kg) prior to conditioning by fixed doses of cocaine increased the expression and impaired the extinction of place preference compared to control subjects. Subjects that were conditioned by 15mg/kg×4 cocaine and received a single injection of NaB following the first or the second CPP test showed impaired extinction compared to control mice that received saline instead of NaB. Subjects that were conditioned by escalating schedule of cocaine and subsequently received repeated injections of NaB during daily reexposure to nonreinforced context showed either enhancement or no effect on place preference. Acute administration of NaB (1.2g/kg) to naïve mice resulted in marked increase in acetylation of histone H3 lysine 14 (H3K14) and histone H4 lysine 8 (H4K8) in hippocampus but not amygdala. Results suggest that regardless of the scheduling of either cocaine or NaB administration, NaB-induced histone hyperacetylation in the hippocampus may strengthen cocaine-associated contextual memory.

Histone acetylation rescues contextual fear conditioning in nNOS KO mice and accelerates extinction of cued fear conditioning in wild type mice.

Epigenetic regulation of chromatin structure is an essential molecular mechanism that contributes to the formation of synaptic plasticity and long-term memory (LTM). An important regulatory process of chromatin structure is acetylation and deacetylation of histone proteins. Inhibition of histone deacetylase (HDAC) increases acetylation of histone proteins and facilitate learning and memory. Nitric oxide (NO) signaling pathway has a role in synaptic plasticity, LTM and regulation of histone acetylation. We have previously shown that NO signaling pathway is required for contextual fear conditioning. The present study investigated the effects of systemic administration of the HDAC inhibitor sodium butyrate (NaB) on fear conditioning in neuronal nitric oxide synthase (nNOS) knockout (KO) and wild type (WT) mice. The effect of single administration of NaB on total H3 and H4 histone acetylation in hippocampus and amygdala was also investigated. A single administration of NaB prior to fear conditioning (a) rescued contextual fear conditioning of nNOS KO mice and (b) had long-term (weeks) facilitatory effect on the extinction of cued fear memory of WT mice. The facilitatory effect of NaB on extinction of cued fear memory of WT mice was confirmed in a study whereupon NaB was administered during extinction. Results suggest that (a) the rescue of contextual fear conditioning in nNOS KO mice is associated with NaB-induced increase in H3 histone acetylation and (b) the accelerated extinction of cued fear memory in WT mice is associated with NaB-induced increase in H4 histone acetylation. Hence, a single administration of HDAC inhibitor may rescue NO-dependent cognitive deficits and afford a long-term accelerating effect on extinction of fear memory of WT mice.

Changes in the magnitude of drug-unconditioned stimulus during conditioning modulate cocaine-induced place preference in mice.

Drug reinforcement learning is relevant for the development of addiction. The present study investigated how changes in the magnitude of drug-unconditioned stimulus during associative learning modulate the acquisition and extinction of cocaine-induced conditioned place preference (CPP). B6;129S F2 mice were conditioned by three dosing schedules of cocaine: (1) ascending, (2) fixed and (3) descending daily doses. Following acquisition of CPP, extinction was induced by (1) context re-exposure, (2) reconditioning by saline and (3) reconditioning by descending doses of cocaine. The magnitude of CPP following conditioning by daily ascending doses of cocaine (2, 4, 8 and 16 mg/kg) was significantly higher than that obtained from conditioning by either a fixed daily dose (16 mg/kg × 4 days) or daily descending doses (24, 12, 6 and 3 mg/kg). Extinction following context re-exposure showed persistent CPP in the 'ascending' group compared to the other two groups. However, extinction via reconditioning by saline was equally effective in all groups. Interestingly, reconditioning by descending doses of cocaine (1) extinguished CPP and (2) resulted in partial resistance to the reinstatement of conditioned response by cocaine priming. Results underscore the significance of daily changes in cocaine dosage in the development and extinction of drug-induced conditioned response. Increase and decrease in cocaine dosage strengthens and weakens cocaine-associated memory, respectively. Moreover, extinction by 'tapering down' drug reward may be superior to extinction by saline.

Discrimination between cocaine-associated context and cue in a modified conditioned place preference paradigm: role of the nNOS gene in cue conditioning.

The conditioned place preference (CPP) paradigm entails appetitive learning and is utilized to investigate the motivational effects of drug and natural reward in rodents. However, a typical CPP design does not allow dissociation between cue- and context-dependent appetitive learning. In humans, context and cues that had been associated with drug reward can elicit conditioned response and drug craving. Therefore, we investigated (a) methods by which to discriminate between cue- and context-dependent appetitive learning, and (b) the role of the neuronal nitric oxide synthase (nNOS) gene in appetitive learning. Wild-type (WT) and nNOS knockout (KO) mice were trained by cocaine (20 mg/kg) in a discrete context paired with a light cue (a compound context-cue stimulus). In test 1, approach behaviour to either the training context or to the cue in a novel context was determined. WT mice showed robust preference for both cocaine-associated context and cue. nNOS KO mice acquired approach behaviour for the cocaine-associated context but not cue. This finding suggests that the nNOS gene is required for cue-dependent appetitive learning. On the following day (test 2), mice were tested for approach behaviour to the compound context-cue stimulus. Context but not cue exposure in test 1 reduced approach behaviour to the compound context-cue stimulus in test 2, suggesting that repeated context but not cue exposures diminished the conditioned response. Hence, this modified CPP paradigm is useful for the investigation of approach behaviour for both drug-associated context and cue, and allows further investigation of mechanisms underlying cue- and context-dependent appetitive learning.

Impairments in fear conditioning in mice lacking the nNOS gene.

The fear conditioning paradigm is used to investigate the roles of various genes, neurotransmitters, and substrates in the formation of fear learning related to contextual and auditory cues. In the brain, nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) functions as a retrograde neuronal messenger that facilitates synaptic plasticity, including the late phase of long-term potentiation (LTP) and formation of long-term memory (LTM). Evidence has implicated NO signaling in synaptic plasticity and LTM formation following fear conditioning, yet little is known about the role of the nNOS gene in fear learning. Using knockout (KO) mice with targeted mutation of the nNOS gene and their wild-type (WT) counterparts, the role of NO signaling in fear conditioning was investigated. Plasma levels of the stress hormone corticosterone were measured to determine the relationship between physiological and behavioral response to fear conditioning. Contextual fear learning was severely impaired in male and female nNOS KO mice compared with WT counterparts; cued fear learning was slightly impaired in nNOS KO mice. Sex-dependent differences in both contextual and cued fear learning were not observed in either genotype. Deficits in contextual fear learning in nNOS KO mice were partially overcome by multiple trainings. A relationship between increase in plasma corticosterone levels following footshock administration and the magnitude of contextual, but not cued freezing was also observed. Results suggest that the nNOS gene contributes more to optimal contextual fear learning than to cued fear learning, and therefore, inhibition of the nNOS enzyme may ameliorate context-dependent fear response.

Development and persistence of long-lasting behavioral sensitization to cocaine in female mice: role of the nNOS gene.

Our recent studies have shown that the neuronal nitric oxide synthase (nNOS) gene is required for the development and persistence of psychomotor sensitization to cocaine in adult but not adolescent male mice (Balda, M.A., Anderson, K.L., Itzhak, Y., 2008. Differential role of the nNOS gene in the development of behavioral sensitization to cocaine in adolescent and adult B6;129S mice. Psychopharmacology (Berlin) 200, 509-519.). The aim of the present study was to investigate the contribution of the nNOS gene to cocaine-induced behavioral sensitization in adolescent and adult female mice. Adolescent and adult wild type (WT) and nNOS knockout (KO) mice received saline or cocaine (20 mg/kg) for 5 days and then were challenged with cocaine (20 mg/kg) after a drug-free period of either 10, 30, or 90 days. Context-dependent sensitization was determined by measuring saline-induced locomotor activity in the previously cocaine-paired environment. Results show that adolescent females of both genotypes, like their adult counterparts, developed long-lasting behavioral sensitization to cocaine (a three-month period), suggesting high vulnerability of females to cocaine regardless of age. An effect of genotype was observed in the initiation of sensitization, e.g., delayed onset in the absence of the nNOS gene. The only age-dependent difference observed was that adult, but not adolescent mice developed context-dependent sensitization. The present study suggests that long-term expression of cocaine-induced behavioral sensitization in females (adolescent and adult) is nNOS-independent, unlike our previous findings in adult males.

Differential role of the nNOS gene in the development of behavioral sensitization to cocaine in adolescent and adult B6;129S mice.

RATIONALE: Previous studies have suggested the involvement of neuronal nitric oxide synthase (nNOS) in the development of sensitization to psychostimulants. Ontogeny-dependent differences in the response to psychostimulants have been reported. OBJECTIVE: The objectives were to investigate (a) the short- and long-term consequences of adolescent and adult cocaine exposure on behavioral sensitization and (b) the role of the nNOS gene in behavioral sensitization in adolescent and adult mice. MATERIALS AND METHODS: Adolescent and adult wild type (WT) and nNOS knockout (KO) mice received saline or cocaine (20 mg/kg) for 5 days and then were challenged with cocaine (20 mg/kg) after a drug-free period of 10 or 30 days. Locomotor activity was recorded by infrared beam interruptions. nNOS immunoreactive (ir) neurons in the dorsal and ventral striatum were quantified 24 h after repeated administration of cocaine to adolescent and adult WT mice. RESULTS: Repeated administration of cocaine to either WT or nNOS KO mice during adolescence resulted in locomotor sensitization, which persisted into adulthood. WT but not KO adult mice developed long-term sensitization to cocaine. Repeated cocaine administration resulted in a 96% increase in the expression of nNOS-ir neurons in the dorsal striatum of adult but not adolescent WT mice. CONCLUSIONS: The nNOS gene is essential for the induction of behavioral sensitization to cocaine in adulthood but not in adolescence. The increased expression of nNOS-ir neurons in the dorsal striatum may underlie the induction of behavioral sensitization in adulthood. Thus, the NO-signaling pathway has an ontogeny-dependent role in the neuroplasticity underlying cocaine behavioral sensitization.

Ethanol-induced behavioral sensitization in adolescent and adult mice: role of the nNOS gene.

BACKGROUND: In the brain, nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) has a role in synaptic plasticity. Recent evidence suggests the role of NO in a variety of effects produced by alcohol in the central nervous system. The current study investigated the role of the nNOS gene in the development of behavioral sensitization to ethanol in adolescent and adult mice. METHODS: Adolescent and adult wild type (WT; B6;129SF2) and nNOS knockout (KO; B6;129S4-Nos1) mice of both sexes received saline or ethanol (1.5 g/kg; intraperitoneally) for 5 consecutive days, and locomotor activity was recorded daily. The locomotor response to challenge ethanol and saline injections was investigated at various time points following withdrawal from ethanol. RESULTS: Adolescent WT but not nNOS KO mice developed a long-lasting sensitized response to ethanol as well as context-dependent hyperlocomotion (in response to saline) from adolescence through adulthood; sex-dependent differences were not observed. Compared to adolescent WT mice, adult WT males developed a short-term sensitized response to ethanol and context-dependent hyperlocomotion; adult WT females showed only short-term context-dependent hyperlocomotion. Adult nNOS KO males (like their adolescent counterparts) did not develop behavioral sensitization; no significant differences between adult nNOS KO and WT females were observed. Blood ethanol concentrations did not show genotype- or sex-dependent differences. CONCLUSIONS: (1) The nNOS gene is required for the development of behavioral sensitization to ethanol in adolescent male and female mice. (2) Adolescent exposure to ethanol results in long-lasting behavioral sensitization through adulthood, while adult exposure to ethanol results in a shorter behavioral sensitization. (3) Sex-dependent differences are observed when ethanol exposure begins in adulthood but not in adolescence. (4) Ethanol-induced behavioral sensitization in adulthood is nNOS-dependent in males but not in females. Taken together, results suggest genotype-, ontogeny-, and sex-dependent differences in the development of behavioral sensitization to ethanol.

Impairment in consolidation of learned place preference following dopaminergic neurotoxicity in mice is ameliorated by N-acetylcysteine but not D1 and D2 dopamine receptor agonists.

Some of the major concerns related to methamphetamine (METH) abuse are the neuronal damage inflicted at dopamine (DA) nerve terminals and the cognitive deficits observed in human METH abusers. We have shown that a high dose of METH selectively depleted dopaminergic markers in striatum, frontal cortex and amygdala of Swiss Webster mice, and impaired learned place preference. In this study, we investigated whether deficits in consolidation of place learning, as a consequence of METH neurotoxicity, underlie the underperformance of cocaine conditioned place preference (CPP). Administration of METH (5 mg/kg x 3) to Swiss Webster mice decreased striatal tyrosine hydroxylase (TH) immunoreactive neurons and significantly increased glial fibrillary acidic protein (GFAP) expression, confirming the neurotoxic potential of METH in mice. This treatment significantly attenuated the establishment of cocaine (15 mg/kg) CPP compared to control. To investigate whether manipulation of the consolidation phase improves learned place preference, mice were trained by cocaine and received daily post-training injections of DA receptor agonists or N-acetylcysteine (NAC). As memory consolidation occurs shortly after training, drugs were administered either immediately or 2 h post-training. Immediate post-training administration of the D1 DA receptor agonist SKF38393 (5, 10, and 20 mg/kg) or the D2 DA receptor agonist quinpirole (0.25, 0.5, and 1.0 mg/kg) did not improve the establishment of CPP following METH neurotoxicity. However, immediate but not delayed NAC administration (50 and 100 mg/kg) enhanced cocaine CPP following METH neurotoxicity and had no effect on control CPP. The levels of the reduced form of glutathione (GSH) in striatum, amygdala, hippocampus and frontal cortex were significantly lower in METH-treated mice compared to control during the period of CPP training. Acute and repeated administration of NAC to METH-treated mice restored the decreased brain GSH but had no effect on controls. Results suggest that METH-induced dopaminergic neurotoxicity is associated with impairment of consolidation of learned place preference, and that this impairment is improved by immediate post-training administration of the glutathione precursor NAC and not by D1 or D2 DA receptor agonists. Restoration of brain glutathione levels immediately post-training may facilitate the consolidation process.

Role of nitrergic system in behavioral and neurotoxic effects of amphetamine analogs.

Several amphetamine analogs are potent psychostimulants and major drugs of abuse. In animal models, the psychomotor and reinforcing effects of amphetamine, methamphetamine (METH), 3,4-methylenedioxymethamphetamine (MDMA; Ecstasy), and methylphenidate (MPD; Ritalin) are thought to be dependent on increased extracellular levels of dopamine (DA) in mesocorticolimbic and mesostriatal pathways. However, amphetamine analogs that increase primarily serotonergic transmission, such as p-chloroamphetamine (PCA) and fenfluramine (FEN), have no potential for abuse. High doses of METH, MDMA, PCA, and FEN produce depletions of dopaminergic and serotonergic nerve terminal markers and are considered as potential neurotoxicants. The first part of this review briefly summarizes the behavioral and neurotoxic effects of amphetamines that have a different spectrum of activity on dopaminergic and serotonergic systems. The second part discusses evidence supporting involvement of the nitrergic system in dopamine-mediated effects of amphetamines. The nitrergic system in this context corresponds to nitric oxide (NO) produced from neuronal nitric oxide synthase (nNOS) that has roles in nonsynaptic interneuronal communication and excitotoxic neuronal injury. Increasing evidence now suggests cross talk between dopamine, glutamate, and NO. Results from our laboratory indicate that dopamine-dependent psychomotor, reinforcing, and neurotoxic effects of amphetamines are diminished by pharmacological blockade of nNOS or deletion of the nNOS gene. These findings, and evidence supporting the role of NO in synaptic plasticity and neurotoxic insults, suggest that NO functions as a neuronal messenger and a neurotoxicant subsequent to exposure to amphetamine-like psychostimulants.

Long-term memory of cocaine-associated context: disruption and reinstatement.

Long-term memory of cocaine-associated context was established by conditioned place preference learning. After 1 week, exposure to context in the absence of cocaine (memory retrieval) was paired with one of the following treatments: saline, scopolamine (muscarinic acetylcholine receptor antagonist), dizocilpine (MK-801; noncompetitive N-methyl-D-aspartate antagonist) or D-cycloserine (partial N-methyl-D-aspartate agonist). In subsequent conditioned place preference tests, place preference was suppressed in the drug-treated groups but not saline-treated groups. Results suggest that the amnesic agents, scopolamine and MK-801, disrupted reconsolidation of cocaine-associated contextual memory. In contrast, the mnemonic agent D-cycloserine might have facilitated extinction learning during context exposure in the absence of cocaine. Challenge administration of cocaine reinstated place preference in all groups except the MK-801 group, suggesting that suppression of conditioned response may or may not suppress memory evoked by drug-context reexposure.

Adolescent and adult responsiveness to the incentive value of cocaine reward in mice: role of neuronal nitric oxide synthase (nNOS) gene.

A major concern in adolescent psychostimulant abuse is the long-term consequence of this practice, because early drug exposure may cause long-term adaptations, which render the organism more susceptible to drug abuse later in life. The incentive value of drug and natural reward in rodents is commonly assessed by the conditioned place preference (CPP) paradigm, which involves Pavlovian learning. The aims of the present study were to investigate: a) the acquisition, expression, maintenance and reinstatement of cocaine CPP from periadolescence (PD24-45) through adulthood (PD70); b) potential sexual dimorphism in adolescence and adulthood in response to cocaine-induced CPP; and c) the role of the neuronal nitric oxide synthase (nNOS) gene in long-term neural plasticity underlying responsiveness to cocaine and cocaine-associated cues. Adolescent wild type (WT) mice acquired significant cocaine (20 mg/kg) CPP that was maintained from PD24 through PD43. Upon extinction, CPP was reinstated in adulthood (PD70) following a priming injection of cocaine (5 mg/kg). In contrast, cocaine CPP acquired between PD26 and PD31 in adolescent nNOS knockout (KO) mice, was neither maintained nor reinstated by cocaine. There was no sexual dimorphism in adolescent WT and KO mice. Genotype differences and sexual dimorphism were observed in adult mice. Cocaine CPP in adult WT males (PD89-94) was maintained for 4 weeks post training, and subsequently reinstated by cocaine priming; the magnitude of CPP in adult WT males was lower than in female counterparts. CPP in adult KO males (PD88-93) was neither maintained nor reinstated by cocaine priming; in contrast, CPP in adult KO females was not significantly different from adult WT females. Results suggest that the nNOS gene is essential during adolescence of both sexes for the development of long-term neural plasticity underlying responsiveness to the incentive value of cocaine reward. Sexual dimorphism in response to cocaine CPP emerges in adulthood; nNOS contribution to long-term plasticity is therefore sexually dimorphic and age-dependent in female but not in male subjects.

Methamphetamine-induced selective dopaminergic neurotoxicity is accompanied by an increase in striatal nitrate in the mouse.

Exposure to high doses of methamphetamine (METH), a major drug of abuse, may cause neuronal damage. Previous studies have implicated the role of peroxynitrite, produced by nitric oxide (NO) and reactive oxygen species, in dopaminergic neurotoxicity produced by METH in mice. The present article was undertaken to investigate if a neurotoxic regimen of METH is associated with changes in tissue levels of nitrate and nitrite, which are the stable products of NO. Administration of METH (5 mg/kg x 3) to Swiss Webster mice resulted in marked depletion of dopamine (DA) and DA transporter (DAT) binding sites but no change in 5-hydroxytryptamine (5-HT) and 5-HT transporter (5-HTT) binding sites in the striatum, amygdala, frontal cortex, and hippocampus, suggesting that METH causes selective neurotoxicity to DA nerve terminals. The concentration of nitrate in the striatum was increased by about two-fold after METH administration; however, no changes in nitrate concentration were detected in other brain regions that endured dopaminergic neurotoxicity. These findings suggest that (a) a neurotoxic regimen of METH produces selective increase in NO in the striatum, which may generate toxic species such as peroxynitrite, and (b) toxins other than NO-related derivatives may mediate dopaminergic neurotoxicity in the amygdala and frontal cortex.

Differential effects of amphetamines-induced neurotoxicity on appetitive and aversive Pavlovian conditioning in mice.

The abuse of substituted amphetamines such as methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA/Ecstasy) can result in neurotoxicity, manifested as the depletion of dopamine (DA) and 5-hydroxytriptamine (5-HT; serotonin) axon terminal markers in humans and animal models. Human METH and MDMA users exhibit impairments in memory and executive functions, which may be a direct consequence of the neurotoxic potential of amphetamines. The objective of this study was to investigate the influence of amphetamines-induced neurotoxicity on Pavlovian learning. Using mouse models of selective DA neurotoxicity (METH; 5 mg/kg x 3), selective 5-HT neurotoxicity (fenfluramine /FEN; 25 mg/kg x 4) and dual DA and 5-HT neurotoxicity (MDMA; 15 mg/kg x 4), appetitive and aversive conditioning were investigated. Dopaminergic neurotoxicity significantly impaired METH and cocaine conditioned place preference (CPP), but had no effect on LiCl-induced conditioned place aversion (CPA). In contrast, serotonergic neurotoxicity significantly enhanced CPP, and had no effect on CPA. Dual dopaminergic/serotonergic neurotoxicity had no apparent effect on CPP; however, CPA was significantly attenuated. Postmortem analysis revealed that significantly diminished levels of DA and 5-HT markers persisted in the striatum, frontal cortex, hippocampus, and amygdala. These findings suggest that amphetamines-induced dopaminergic and serotonergic neurotoxicity exert opposing influences on the affective state produced by subsequent drug reward, while dual dopaminergic/serotonergic neurotoxicity impairs associative learning of aversive conditioning. Furthermore, results revealed that amphetamines-induced DA and 5-HT neurotoxicity modulates appetitive Pavlovian conditioning similar to other DA and 5-HT neurotoxins. Modulation of Pavlovian conditioning by amphetamines-induced neurotoxicity may be relevant to compulsive drug-seeking behavior in METH and MDMA abusers.

Cocaine-induced conditioned place preference in mice: induction, extinction and reinstatement by related psychostimulants.

Reinstatement of drug-seeking behavior in animals is relevant to drug relapse in humans. In the present study, we employed the conditioned place preference (CPP) paradigm to investigate the extinction and reinstatement of the place-conditioned response, a model that is consistent with drug-seeking behavior. Cocaine-induced CPP was rendered in Swiss Webster mice and then extinguished after repeated saline injections (8 days) in both the previously cocaine-paired compartment and the saline-paired compartment. Following the extinction phase, the reinstatement of CPP was investigated. Cocaine-experienced mice were challenged with one of the following psychostimulants, cocaine (15 mg/kg), methamphetamine (METH; 0.5 mg/kg), methylphenidate (MPD; 20 mg/kg) and phencyclidine (PCP; 5 mg/kg). The priming injection of cocaine, METH and MPD, unlike PCP, induced a marked preference for the previously cocaine-paired compartment. This finding suggests that all three psychostimulants reinstated the CPP response, and METH and MPD substituted for the reinforcing cue of cocaine. A challenge injection of cocaine administered two and four weeks after the reinstatement of CPP indicated that CPP was maintained up to two weeks. The finding that METH and MPD but not PCP reinstated and supported cocaine-induced CPP suggests that the CPP paradigm may be a useful tool for drug discrimination studies and the reinstatement of drug-seeking behavior.

Long-lasting behavioral sensitization to psychostimulants following p-chloroamphetamine-induced neurotoxicity in mice.

Amphetamine analogs such as p-chloroamphetamine (PCA) cause serotonergic and dopaminergic neurotoxicity. The behavioral consequences and the responsiveness to psychostimulants following the neurotoxic insult are unclear. The present study was undertaken to investigate the outcome of neurotoxic and non-neurotoxic PCA pre-treatments on the sensitivity of Swiss Webster mice to the psychomotor stimulating effects of PCA, 3,4-methylenedioxymethamphetamine (MDMA) and cocaine. PCA (15 mg/kg x 2; i.p.) caused 37-70% depletion of dopaminergic and serotonergic markers in mouse brain. Saline and PCA (15 mg/kg x 2) mice were challenged on days 5, 12, 40 and 74 with one of the following drugs: PCA (5 mg/kg), MDMA (10 mg/kg) and cocaine (20 mg/kg). The PCA pre-exposed mice showed marked locomotor sensitization from days 5-74 to all three drugs tested. The time course of the sensitized response coincided with the time course of the neurotoxic insult as determined by reduced densities of striatal dopamine transporter and frontal cortex serotonin transporter binding sites. A single injection of PCA (5 mg/kg) caused neither neurotoxicity nor sensitization to the locomotor stimulating effects of PCA, MDMA and cocaine. Repeated administration of a low non-neurotoxic dose of PCA (5 mg/kg/day; 6 days) caused a transient locomotor sensitization to PCA that dissipated after one month. Results of the present study suggest that PCA-induced serotonergic and dopaminergic neurotoxicity coincides with long-lasting locomotor sensitization to psychostimulants. These findings may be relevant to the psychopathology of amphetamines-induced neurotoxicity.

Relevance of MDMA ("ecstasy")-induced neurotoxicity to long-lasting psychomotor stimulation in mice.

RATIONALE: Although many studies have focused on the mechanisms underlying MDMA-induced neurotoxicity, little is known about the subsequent long-term response to psychostimulants following exposure to a neurotoxic dose of MDMA. OBJECTIVES: We investigated the effect of pre-exposure to neurotoxic and non-neurotoxic doses of MDMA on the response of mice to the psychomotor stimulating effects of MDMA and cocaine. METHODS: To investigate MDMA-induced neurotoxicity, male Swiss Webster mice were subjected to three regimens of MDMA: i) 40 mg/kg x 2, ii) 30 mg/kg x 2, and iii) 15 mg/kg x 2 for 2 days. On day 5 following the last exposure to MDMA, the levels of dopaminergic and serotonergic markers were determined. For the behavioral experiments, mice received either a single injection of 10 mg/kg MDMA [MDMA(L)] or one of the following doses of MDMA: 30 mg/kg x 2 or 15 mg/kg x 2 for 2 days [MDMA (H)]. A third group received saline as a control. On day 5 after the last pretreatment injection, the first MDMA (10 mg/kg) challenge was given, and on day 12, cocaine (20 mg/kg) was administered. Subsequently, mice were re-challenged with MDMA on days 35, 50 and 80, after which locomotor activity was monitored by infrared beam-interrupts. On day 83, mice were killed to detect the levels of dopaminergic and serotonergic markers. RESULTS: MDMA-induced mortality and depletion of dopaminergic and serotonergic markers were dose-dependent. MDMA (H) mice endured a sensitized response to MDMA challenge from days 5 through 80, e.g. a persistent 3-fold increase in locomotor activity compared to the response of mice that were not pretreated with a neurotoxic dose of MDMA. The depletion of DAT and 5-HTT binding sites was sustained throughout this time period (64-68% of control). The MDMA (L) mice showed a sensitized response to MDMA only on day 5. Both MDMA (L) and MDMA (H) mice were sensitized to the cocaine challenge. CONCLUSIONS: The induction of sensitization to the locomotor stimulating effects of MDMA and cocaine was independent of MDMA-induced neurotoxicity. However, the long-lasting maintenance of the sensitized response to MDMA may be related to the enduring neurotoxicity caused by MDMA.

Behavioral consequences of methamphetamine-induced neurotoxicity in mice: relevance to the psychopathology of methamphetamine addiction.

Methamphetamine (METH) is a major drug of abuse in the United States. A high dose of METH given to mice and rats causes long-lasting depletion of tyrosine hydroxylase activity, dopamine (DA), and DA-transporter (DAT) binding sites in the striatum. In human METH-abusers, a marked decrease of the DAT in the caudate putamen was observed. Despite intensive investigations of the mechanism associated with METH-induced neurotoxicity, the behavioral consequences of this phenomenon are not clear. We used the mouse model of METH-induced neurotoxicity to investigate the response of the animals to the psychomotor-stimulating effect of METH and the rewarding effect of the drug. Mice pre-exposed to a neurotoxic dose of METH developed a marked sensitization to the psychomotor-stimulating effect of METH, which lasted for more than two months. The rewarding effect of METH was determined by the conditioned place preference (CPP) paradigm. Mice pre-exposed to the neurotoxic dose of METH showed reduced sensitivity to the rewarding effect of METH compared with control animals. While CPP was maintained for three months in the control group, the conditioned response in the METH pre-exposed animals lasted only a few days. These findings indicate that METH neurotoxicity is associated with opposing and long-lasting behavioral outcomes: (a) sensitization to the psychomotor-stimulating effect of the drug and (b) desensitization to the rewarding properties of the drug. These consequences may be relevant to the psychopathology of METH abuse. Sensitization is pertinent to compulsive drug-seeking behavior that is accompanied by desensitization to the rewarding effect of METH.