Deanne M. Buffalari

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.

Noradrenergic Modulation of Basolateral Amygdala Neuronal Activity: Opposing Influences of α-2 and β 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 β-receptor antagonist, decreased the spontaneous firing rate and potentiated the NE-evoked inhibition of BLA neurons. In addition, iontophoresis of the α-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 α 2 and β 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 β 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.

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.

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.

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.

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.

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.

Anxiogenic modulation of spontaneous and evoked neuronal activity in the basolateral amygdala.

The amygdala has a well-established role in stress, anxiety, and aversive learning, and anxiolytic and anxiogenic agents are thought to exert their behavioral actions via the amygdala. However, despite extensive behavioral data, the effects of noradrenergic anxiogenic drugs on neuronal activity within the amygdala have not been examined. The present experiments examined how administration of the anxiogenic drug yohimbine affects spontaneous and evoked neuronal activity in the basolateral amygdala (BLA) of rats. Yohimbine produced both excitatory and inhibitory effects on neurons of the BLA, with an increase in spontaneous activity being the predominant response in the lateral and basomedial nuclei of the BLA. Furthermore, yohimbine tended to facilitate neuronal responses evoked by electrical stimulation of the entorhinal cortex, with this facilitation seen more often in lateral and basomedial nuclei of the BLA. These data are the first to examine the effects of the anxiogenic agent yohimbine on BLA neuronal activity, and suggest that neurons in specific subnuclei of the amygdala exhibit unique responses to administration of such pharmacological agents.

Effects of MAO inhibition and a combination of minor alkaloids, β-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 β-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.

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.