Effects of concomitant methylphenidate and ethanol administration on working and reference memory in rats.

Recent studies have suggested that college students are heavily engaged in non-medical use of stimulant drugs prescribed to treat attention deficit hyperactivity disorder. This age group is also at high risk for alcohol use. Despite their potential co-abuse, little work has examined how these drugs interact to affect cognitive abilities. In fact, these drugs have opposing effects on working memory, which brings into question how they may interact to affect this particular behavior. The purpose of this research was to examine the concomitant effects of methylphenidate (MPH) and ethanol (EtOH) on working and reference memory. Rats were first trained on the radial arm maze task to establish a baseline performance rate measured as average number of reference and working memory errors. Performance was then assessed after injections of saline, MPH alone, EtOH alone, and MPH+EtOH combined. While both doses of MPH caused nonsignificant improvements in working memory, when combined with EtOH, there was an overall impairment in working and reference memory compared to other conditions. EtOH alone also decreased memory. These data indicate increased impairment of memory function with combined MPH and EtOH use. By understanding how the combination of methylphenidate and alcohol affects memory, we can better assess the risks of taking both substances simultaneously.

Nicotine Enhances Footshock- and Lithium Chloride-Conditioned Place Avoidance in Male Rats.

INTRODUCTION: Numerous studies have shown that nicotine (NIC) can enhance the reinforcing effects of non-NIC stimuli through a nonassociative mechanism. To date, it is unclear whether NIC reinforcement enhancement serves to increase behaviors motivated by rewarding stimuli only, or whether NIC potentiates behavior motivated by all stimuli, regardless of valence. METHODS: The current study used a place conditioning procedure to examine whether acute NIC injection modulates avoidance of an environment previously associated with an aversive stimulus. Separate groups of rats underwent place conditioning using either lithium chloride (125mg/kg/ml, i.p.) or footshock (0.75 mA) as the aversive stimulus. Other rats served as nonconditioned controls. The magnitude of place avoidance was assessed after acute NIC (0.1 or 0.4mg/kg/ml, s.c.) or saline. RESULTS: Rats avoided chambers previously paired with either lithium chloride or footshock, and conditioned place avoidance was significantly enhanced by NIC pre-treatment. CONCLUSIONS: These results demonstrate that the ability of NIC to enhance motivated behavior extends to behaviors elicited by aversive stimuli, evidence that NIC affects behavior motivated by a broader range of stimuli than previously appreciated. IMPLICATIONS: The current study examined whether the reinforcement enhancement properties of NIC apply to aversive stimuli by testing NIC enhancement of conditioned place avoidance in rats. The results demonstrate that NIC enhances the motivational impact of these distinct aversive stimuli, providing novel evidence that NIC affects behavior motivated by a broader range of stimuli than has previously been demonstrated.

Effects of MAO inhibition and a combination of minor alkaloids, ß-carbolines, and acetaldehyde on nicotine self-administration in adult male rats.

INTRODUCTION: Although nicotine is the primary reinforcing constituent in cigarettes, there is evidence that other constituents in cigarette smoke may interact with nicotine to reinforce smoking behavior. METHODS: The present experiments investigated whether a novel combination of these cigarette smoke constituents would increase nicotine self-administration in adult male rats. The constituents included five minor alkaloids (anabasine, nornicotine, cotinine, myosmine, and anatabine), two ß-carbolines (harman and norharman), and acetaldehyde. All doses were indexed to be proportional to concentrations in cigarette smoke given a standard dose of nicotine used in rodent self-administration, or ten times higher than this standard. To model MAO inhibition seen in chronic smokers, some groups received separate injections of tranylcypromine prior to each self-administration session. RESULTS: Tranylcypromine increased low-dose nicotine self-administration independent of other smoke constituents, which had no effect on self-administration behavior. The effect of tranylcypromine was confirmed across a large range of reinforcement schedules. The effect of tranylcypromine on low-dose nicotine self-administration was observed regardless of whether the injection was delivered 1-h or 23-h prior to the self-administration session, consistent with the interpretation that MAO inhibition was responsible for the increase in self-administration, instead of acute off-target effects. CONCLUSIONS: These data suggest that this cocktail of constituents does not significantly alter the primary reinforcing effects of nicotine, but constituents that inhibit MAO may increase the primary reinforcing effects of nicotine, especially at low doses.

Behavioral mechanisms underlying nicotine reinforcement.

Cigarette smoking is the leading cause of preventable deaths worldwide, and nicotine, the primary psychoactive constituent in tobacco, drives sustained use. The behavioral actions of nicotine are complex and extend well beyond the actions of the drug as a primary reinforcer. Stimuli that are consistently paired with nicotine can, through associative learning, take on reinforcing properties as conditioned stimuli. These conditioned stimuli can then impact the rate and probability of behavior and even function as conditioning reinforcers that maintain behavior in the absence of nicotine. Nicotine can also act as a conditioned stimulus (CS), predicting the delivery of other reinforcers, which may allow nicotine to acquire value as a conditioned reinforcer. These associative effects, establishing non-nicotine stimuli as conditioned stimuli with discriminative stimulus and conditioned reinforcing properties as well as establishing nicotine as a CS, are predicted by basic conditioning principles. However, nicotine can also act non-associatively. Nicotine directly enhances the reinforcing efficacy of other reinforcing stimuli in the environment, an effect that does not require a temporal or predictive relationship between nicotine and either the stimulus or the behavior. Hence, the reinforcing actions of nicotine stem both from the primary reinforcing actions of the drug (and the subsequent associative learning effects) as well as the reinforcement enhancement action of nicotine which is non-associative in nature. Gaining a better understanding of how nicotine impacts behavior will allow for maximally effective tobacco control efforts aimed at reducing the harm associated with tobacco use by reducing and/or treating its addictiveness.

Low-dose nicotine self-administration is reduced in adult male rats naïve to high doses of nicotine: implications for nicotine product standards.

Product standards that greatly reduce the content of nicotine within cigarettes may result in improved public health. The study presented here used an animal model to investigate whether individuals who start smoking after implementation of regulation may be affected differently from current smokers who form the basis of most clinical studies. One group of adult male rats (n = 14/group) acquired nicotine self-administration at a high nicotine dose (60 µg/kg/infusion) before experiencing a reduction to one to three low doses of nicotine (3.75, 7.5, or 15 µg/kg/infusion) or vehicle. Their self-administration behavior at the low doses was compared with a group of adult male rats given the opportunity to acquire nicotine self-administration at one of the same low doses or vehicle (n = 7-14/group). Second, the self-administration behavior of the acquisition group of rats was compared with their own self-administration behavior after experience self-administering a high dose of nicotine. A cocktail of non-nicotine cigarette smoke constituents was included in the vehicle for all rats across all phases of the study. Rats with a history of self-administering a high dose of nicotine had a higher rate of self-administration across the low doses than rats with no history. In addition, the number of earned infusions increased after rats experienced self-administration of a higher dose of nicotine. These data show that low-dose nicotine self-administration is higher after a dose reduction than during acquisition. If a nicotine reduction policy were implemented, then this policy may be especially effective at reducing acquisition of smoking.

Nicotine enhances the expression of a sucrose or cocaine conditioned place preference in adult male rats.

Nicotine has been shown to enhance the motivational properties of non-nicotine stimuli. This reinforcement-enhancing property of nicotine has the potential to promote the use of other illicit substances as well as maladaptive patterns of food intake. Therefore, the current study aimed to examine whether nicotine enhances preference for contexts paired with cocaine or sucrose utilizing a place conditioning procedure. Separate groups of adult male rats were administered sucrose or cocaine in one of two compartments of a standard CPP chamber on four consecutive days. Preference was then assessed following no injection, a single subcutaneous (s.c.) injection of nicotine, and a s.c. saline injection. The animals preferred the chamber paired with either sucrose or cocaine, as evident from an increased time spent in the paired chamber compared to baseline. Nicotine further increased the time spent in the sucrose- or cocaine-paired chamber, consistent with a reinforcement-enhancement effect. Previous results demonstrate an interaction between nicotine and intake of other drugs or food. The present findings provide an additional mechanism that may underlie these effects and which may have implications for drug dependence and obesity.

Gradual and immediate nicotine reduction result in similar low-dose nicotine self-administration.

INTRODUCTION: Food and Drug Administration-mandated product standards that drastically reduce nicotine content in cigarettes aim to decrease smoking and thus improve health outcomes for millions of U.S. smokers. Researchers have suggested that nicotine reduction should be implemented gradually, but a gradual nicotine reduction may shift the minimum level of nicotine required to reinforce behavior or may result in different levels of compensatory smoking behavior. METHOD: Rats were given the opportunity to acquire nicotine self-administration at 60 µg/kg/infusion nicotine with a cocktail of other tobacco constituents included as the vehicle. Rats were subsequently assigned to one of six immediate dose reductions (30, 15, 7.5, 3.75, 1.875, or 0.0 µg/kg/infusion) for 10 sessions (n = 9-15). Rats in the 30 µg/kg/infusion reduction group continued to have their nicotine dose reduced by half after at least 10 sessions at each dose until reaching 1.875 µg/kg/infusion (i.e., gradual reduction). RESULTS: For both methods of reduction, reduction to 3.75 µg/kg/infusion resulted in significant decreases in behavior. Reduction to doses above 3.75 µg/kg/infusion resulted in only limited compensation. The largest compensation was temporary. There was no compensation following reduction to 3.75 µg/kg/infusion or below. CONCLUSION: This study suggests that reduction to the same nicotine dose will result in similar reductions in behavior for both gradual and immediate reductions, and both methods result in similar compensation. Future studies using humans should investigate differences in other outcomes such as withdrawal and craving.

The effects of varied extinction procedures on contingent cue-induced reinstatement in Sprague-Dawley rats.

RATIONALE: Cue exposure therapy, which attempts to limit relapse by reducing reactivity to cocaine-paired cues through repeated exposures, has had limited success. OBJECTIVES: The current experiments examined cocaine cue-induced anxiogenesis and investigated whether a model of cue exposure therapy would reduce reinstatement of cocaine seeking in rats with a history of cocaine self-administration. METHODS: Male rats experienced daily intravenous cocaine self-administration. Rats then experienced exposure to either the self-administration context or the context plus noncontingent presentations of cocaine-paired cues. Immediately following exposure, anxiety-like behavior was measured using elevated plus maze and defensive burying tests. In a second group of rats, self-administration was followed by 7 days of exposure to the context, context + noncontingent cue exposure, lever extinction, or cue + lever extinction. All animals then underwent two contingent cue-induced reinstatement tests separated by 7 days of lever extinction. RESULTS: Exposure to noncontingent cocaine-paired cues in the self-administration context increased anxiety-like behavior on the defensive burying test. Animals that experienced lever + cue extinction displayed the least cocaine seeking on the first reinstatement test, and lever extinction reduced cocaine seeking below context exposure or context + noncontingent cue exposure. All animals had similar levels of cocaine seeking on the second reinstatement test. CONCLUSION: Noncontingent cue exposure causes anxiety, and noncontingent cue and context exposure are less effective at reducing contingent cue-induced reinstatement than lever or lever + cue extinction. These data indicate that active extinction of the drug-taking response may be critical for reduction of relapse proclivity in former cocaine users.

Impact of tobacco regulation on animal research: new perspectives and opportunities.

INTRODUCTION: The Family Smoking Prevention and Tobacco Control Act in the United States and the World Health Organization Framework Convention on Tobacco or Health ratified by over 170 countries render scientific investigations into the abuse liability, harm, and effects of tobacco more critical than ever. A key area to explore relates to the potential regulation of nicotine content in cigarettes. Determining the nicotine content per cigarette below which smokers reliably reduce their consumption of and dependence on cigarettes, an idea proposed almost 20 years ago (Benowitz & Henningfield, 1994), could be a powerful approach to reduce the abuse liability and consequent harm from cigarettes. However, this approach is laden with potentially complex issues. Many of these complications can be studied using animal models, but they require a particular perspective. METHODS: Herein, we review several challenges for animal researchers interested in nicotine reduction as examples of how this perspective dictates new approaches to animal research. These include defining the threshold nicotine dose for maintaining self-administration, evaluating the differential impact of various implementation strategies, assessing the factors that could interact with nicotine to alter the reinforcement threshold, describing the role of cues in maintaining low dose nicotine self-administration, and examining individual differences in response to nicotine reduction. CONCLUSIONS: Researchers who study tobacco using animal models have the opportunity to play a central role in the regulatory science of tobacco and conduct studies that directly inform policy decisions that could impact the lives of millions.

Treatment of cocaine withdrawal anxiety with guanfacine: relationships to cocaine intake and reinstatement of cocaine seeking in rats.

RATIONALE: Successful treatment of cocaine addiction is severely impeded by the propensity of users to relapse. Withdrawal severity may serve as a key predictor of susceptibility to relapse. Therefore, the identification and treatment of cocaine withdrawal symptoms such as anxiety may improve addiction treatment outcome. OBJECTIVES: The current study examined the role of anxiety-like behavior during cocaine withdrawal and anxiolytic treatment in reinstatement of cocaine seeking in an animal model of relapse. METHODS: Male rats experienced daily IV cocaine self-administration. One group of animals received the norepinephrine α-2 agonist, guanfacine, or vehicle prior to anxiety testing 48 h after the last self-administration session. In the second group of rats, relationships between cocaine intake, anxiety-like behavior after withdrawal of cocaine, and reinstatement responding were investigated. The third and fourth groups of animals received guanfacine, yohimbine (norepinephrine α-2 antagonist), or vehicle once per day for 3 days 48 h after cessation of cocaine self-administration, followed by extinction and subsequent reinstatement induced by cocaine injections, cocaine-paired cues, and yohimbine administration. RESULTS: Cocaine-withdrawn rats at 48 h demonstrated higher levels of anxiety-like behavior as measured on a defensive burying task when compared to yoked saline controls, an effect reversed by guanfacine treatment. Cocaine intake was positively correlated with measures of anxiety-like behavior during early withdrawal, and this anxiety-like behavior was significantly correlated with subsequent cocaine-primed reinstatement. Yohimbine treatment during early withdrawal increased reinstatement to conditioned cues, while guanfacine treatment reduced reinstatement to yohimbine. CONCLUSIONS: These studies suggest an important role for noradrenergic mediation of anxiety-like behavior that emerges after withdrawal of cocaine and potential risk of relapse as modeled by reinstatement, and suggest that treatment of anxiety symptoms during early abstinence may reduce the risk of relapse.

Corticotrophin releasing factor (CRF) induced reinstatement of cocaine seeking in male and female rats.

Significant sex differences have been demonstrated in clinical and preclinical studies of cocaine addiction, with some of the most consistent differences noted in regard to the role of stress and craving. The current study examined stress-induced reinstatement of cocaine seeking in male and female rats in an animal model of relapse using corticotropin-releasing factor (CRF) administration. Both male and female rats demonstrated increased cocaine seeking in response to CRF. CRF-induced reinstatement was highly variable across both male and female rats, and further analysis revealed a subpopulation that was particularly sensitive to CRF (high responders). Female high responders displayed significantly increased responding to CRF compared to males. Individual differences in stress responsivity could thus contribute to the likelihood of relapse, with females showing greater heterogeneity to stress-induced relapse.

Inactivation of the bed nucleus of the stria terminalis in an animal model of relapse: effects on conditioned cue-induced reinstatement and its enhancement by yohimbine.

RATIONALE: Drug-associated cues and stress increase craving and lead to greater risk of relapse in abstinent drug users. Animal models of reinstatement of drug seeking have been utilized to study the neural circuitry by which either drug-associated cues or stress exposure elicit drug seeking. Recent evidence has shown a strong enhancing effect of yohimbine stress on subsequent cue-elicited reinstatement; however, there has been no examination of the neural substrates of this interactive effect. OBJECTIVES: The current study examined whether inactivation of the bed nucleus of the stria terminalis (BNST), an area previously implicated in stress activation of drug seeking, would affect reinstatement of cocaine seeking caused by conditioned cues, yohimbine stress, or the combination of these factors. METHODS: Male rats experienced daily IV cocaine self-administration, followed by extinction of lever responding in the absence of cocaine-paired cues. Reinstatement of responding was measured during presentation of cocaine-paired cues, following pretreatment with the pharmacological stressor, yohimbine (2.5 mg/kg, IP), or the combination of cues and yohimbine. RESULTS: All three conditions led to reinstatement of cocaine seeking, with the highest responding seen after the combination of cues and yohimbine. Reversible inactivation of the BNST using the gamma-aminobutyric acid receptor agonists, baclofen + muscimol, significantly reduced all three forms of reinstatement. CONCLUSION: These results demonstrate a role for the BNST in cocaine seeking elicited by cocaine-paired cues, and suggest the BNST as a key mediator for the interaction of stress and cues for the reinstatement of cocaine seeking.

Amygdala mechanisms of Pavlovian psychostimulant conditioning and relapse.

Psychostimulant addiction often consists of periods of sustained drug abstinence disrupted by periods of relapse and renewed heavy drug use. Prevention of relapse remains the greatest challenge to the successful treatment of drug addiction. Drug-associated cues are a primary trigger for relapse, as they can elicit intense craving for the drug. These cues become associated with the drug reward through Pavlovian learning processes that develop over multiple drug-cue pairings. The amygdala (AMY) is critical for such drug-related learning. Intrinsic and extrinsic circuitry position the AMY to integrate cue and drug-related information and influence drug-seeking and drug-taking behaviors. Animal models of conditioned drug reward, drug use, and relapse have confirmed the necessary role of the AMY for drug conditioned cues to control motivated behavior. Neurons within the AMY are responsive to the primary effects of psychostimulants, and more critically, they also respond to the presentation of drug-associated cues. The mechanisms by which conditioned cues come to influence drug-seeking behavior likely involve long-term plasticity and neuroadaptations within the AMY. A greater understanding of the associative learning mechanisms that depend upon the AMY and related limbic and cortical structures, and the process by which drug cues come to gain control over behavior that maintains the addictive state, will facilitate the development of more effective addiction treatments.

Footshock stress potentiates cue-induced cocaine-seeking in an animal model of relapse.

Drug-associated cues and stress increase craving and lead to greater risk of relapse in abstinent drug addicts. This risk may be increased when these factors occur simultaneously. The current study examined whether the presentation of three different levels of intermittent footshock would trigger reinstatement or potentiate reinstatement of cocaine-seeking caused by conditioned cues. Male, Long Evans rats underwent daily i.v. cocaine self-administration, followed by extinction of lever responding in the absence of previously cocaine-paired cues. Reinstatement of cocaine-seeking was measured during presentation of cocaine-paired cues, following pretreatment with three levels of intermittent footshock (0.25, 0.5, and 0.75 mA), or after the combination of footshock and cues. Footshock at the 0.5 and 0.75 mA levels led to significant reinstatement when presented alone, and also potentiated the reinstatement triggered by the presentation of conditioned cues. These results demonstrate that while stress and drug-paired cues reinstate drug-seeking when presented in isolation, their interaction leads to potentiated reinstatement. Dual targeting of stress and cues is thus a critical consideration for treatment intervention in abstinent drug users.

Chronic cold stress increases excitatory effects of norepinephrine on spontaneous and evoked activity of basolateral amygdala neurons.

Neurons of the amygdala respond to a variety of stressors. The basolateral amygdala (BLA) receives dense norepinephrine (NE) innervation from the locus coeruleus, and stressful and conditioned stimuli cause increases in NE levels within the BLA. Furthermore, chronic stress exposure leads to sensitization of the stress response. The actions of NE in different structures involved in the stress circuit have been shown to play a role in this sensitization response. Here, we examine how chronic cold stress alters NE modulation of spontaneous and evoked activity in the BLA. In controls, NE inhibited spontaneous firing in the majority of BLA neurons, with some neurons showing excitation at lower doses and inhibition at higher doses of NE. NE also decreased the responsiveness of these neurons to electrical stimulation of the entorhinal and sensory association cortices. After chronic cold exposure, NE caused increases in spontaneous activity in a larger proportion of BLA neurons than in controls, and now produced a facilitation of responses evoked by stimulation of entorhinal and sensory association cortical inputs. These studies show that chronic cold exposure leads to an increase in the excitatory effects of NE on BLA neuronal activity, and suggest a mechanism by which organisms may display an enhancement of hormonal, autonomic, and behavioural responses to acute stressful stimuli after chronic stress exposure.

Noradrenergic modulation of basolateral amygdala neuronal activity: opposing influences of alpha-2 and beta receptor activation.

Substantial data exists demonstrating the importance of the amygdala and the locus ceruleus (LC) in responding to stress, aversive memory formation, and the development of stress-related disorders; however, little is known about the effects of norepinephrine (NE) on amygdala neuronal activity in vivo. The basolateral nucleus of the amygdala (BLA) receives dense NE projections from the LC, NE increases in the BLA in response to stress, and the BLA can also modulate the LC via reciprocal projections. These experiments examined the effects of noradrenergic agents on spontaneous and evoked responses of BLA neurons. NE iontophoresis inhibited spontaneous firing and decreased the responsiveness of BLA neurons to electrical stimulation of entorhinal cortex and sensory association cortex (Te3). Confirmed BLA projection neurons exhibited exclusively inhibitory responses to NE. Systemic administration of propranolol, a beta-receptor antagonist, decreased the spontaneous firing rate and potentiated the NE-evoked inhibition of BLA neurons. In addition, iontophoresis of the alpha-2 agonist clonidine, footshock administration, and LC stimulation mimicked the effects of NE iontophoresis on spontaneous activity. Furthermore, the effects of LC stimulation were partially blocked by systemic administration of alpha 2 and beta receptor antagonists. This is the first study to demonstrate the actions of directly applied and stimulus-evoked NE in the BLA in vivo, and provides a mechanism by which beta receptors can mediate the important behavioral consequences of NE within the BLA. The interaction between these two structures is particularly relevant with regard to their known involvement in stress responses and stress-related disorders.

Opposing influence of basolateral amygdala and footshock stimulation on neurons of the central amygdala.

BACKGROUND: The basolateral complex (BLA) and the central nucleus of the amygdala (CeA) are believed to mediate the expression of affective responses. After affective learning, conditioned stimulus-related information is thought to be conveyed from the BLA to the CeA; the medial CeA (Cem), in turn, projects to hypothalamic and brainstem structures involved with induction of affective responses. Although the conditioned stimulus and unconditioned stimulus both evoke affective responses, the precise response often differs. It is unknown whether this difference is represented by distinct activity patterns of single Cem neurons. Furthermore, the nature of the interaction between the BLA and Cem is unknown. METHODS: Using in vivo extracellular and intracellular recordings, we examined how the BLA affects the Cem and compared this with effects induced by footshock (unconditioned stimulus) in the same neurons. RESULTS: Our results demonstrate that, contrary to conventional views, BLA stimulation primarily inhibits Cem neurons by a polysynaptic circuit, and show that single Cem neurons respond to both BLA input and footshock in an opposite manner. CONCLUSIONS: These results demonstrate the predominantly inhibitory nature of the BLA-Cem interaction. These data further demonstrate the distinct cellular events that might lead to differential modulation of conditioned and unconditioned affective responses.

A room with a view and a polarizing cue: individual differences in the stimulus control of place navigation and passive latent learning in the water maze.

We investigated individual differences in the stimulus control of navigational behavior in the water maze by comparing measures of place learning in one environment to measures of latent learning (via passive placement on the goal platform) in a novel environment. In the first experiment, 12 rats were trained to find a slightly submerged hidden platform at a fixed location in room A for 10 days (4 trials/day). Fast and slow place learners were identified by their mean escape latency and cumulative distance to the goal during acquisition. The same animals were then given a 2-min passive placement on the submerged platform in room B. Latent learning was assessed by the animal's escape latency on a single swim trial immediately following the placement in room B. The results showed that the good latent learners in room B were not necessarily the fast place learners in room A. This weak correlation may be related to the fact that some rats swam near the area in room B that corresponded to the former goal location in room A relative to a common polarizing cue (i.e., the door/entrance to both rooms). When the view of the door was blocked in a second experiment a significant positive correlation between place acquisition and the latent learning test was obtained, although escape performance following passive placement was not improved. These findings suggest that while place navigation and latent learning via passive placement may involve some common cognitive-spatial function, other associative (S-S and/or S-R) processes that occur during place navigation/active movement may be required for animals to exhibit truly accurate navigational behavior characteristic of asymptotic escape performance in the water maze. Additional implications for neurobiological studies using a procedural pretraining design are discussed.