Female rats display higher methamphetamine-primed reinstatement and c-Fos immunoreactivity than male rats.

Methamphetamine (meth) dependence is often characterized by persistent and chronic relapse (i.e., return to drug use). Previous work suggests females may be at greater risk to relapse. In this study, we extended this limited evidence and identified sex-dependent neural substrates related to meth-triggered reinstatement. Male and female Sprague-Dawley rats were implanted with indwelling jugular catheters. Half of the rats were then trained to self-administer meth (0.05 mg/kg/inf); the other half self-administered saline during 21 daily sessions (2 h). Rats were then given 12 extinction sessions. Twenty-four hours after the last extinction session, rats received reinstatement testing. Half of the rats received a meth-prime (0.3 mg/kg, IP) injection and the remaining rats received a saline injection. This design resulted in 4 separate groups for each sex, allowing for careful investigation of brain regions related to meth-triggered reinstatement. Brains were harvested following the reinstatement session and c-Fos immunoreactivity was measured in multiple brain regions. Meth triggered reinstatement in both sexes and this effect was more robust in females compared to males. Significant sex differences were detected. Females showed greater c-Fos immunoreactivity in the cingulate cortex area 1, lateral orbitofrontal cortex, prelimbic cortex, caudate-putamen, nucleus accumbens core and shell, and central nucleus of the amygdala following meth-primed reinstatement.

Conditioned enhancement of the nicotine reinforcer.

This study was designed to assess whether nicotine can acquire additional reinforcing properties through associations with other rewards. To this end, rats self-administered nicotine-alone (0.01 mg/kg) or nicotine paired with access to sucrose during the conditioning phase. In the subsequent challenge phase, we tested the effect of nicotine-sucrose pairings on the reinforcing effects of nicotine using a progressive ratio schedule of reinforcement. Using this approach, we show that (a) rats in both paired and nicotine-alone conditions self-administered similar amounts of nicotine in the initial conditioning phase of the study when intake was limited to 10 infusions per session, (b) nicotine rapidly acquired control over goal-tracking behavior in the paired condition, (c) rats that had a history of nicotine and sucrose pairings worked harder and took more nicotine as measured on a progressive ratio using a distinct response form, and (d) conditioned goal-tracking evoked by nicotine did not show extinction when sucrose was no longer paired with nicotine over the 11 days of nicotine self-administration on a progressive ratio schedule of reinforcement. Overall, our results demonstrate that in addition to the multifaceted nature of nicotine stimulus that includes primary reinforcing effects, conditioned reinforcing effects, and reward enhancing effects, nicotine can also acquire additional reinforcing properties through associations with other rewards. This ability to acquire additional reinforcing properties through associative learning may contribute to the development and perpetuation of tobacco use disorder. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Role of Brain Derived Extracellular Vesicles in Decoding Sex Differences Associated with Nicotine Self-Administration.

Smoking remains a significant health and economic concern in the United States. Furthermore, the emerging pattern of nicotine intake between sexes further adds a layer of complexity. Nicotine is a potent psychostimulant with a high addiction liability that can significantly alter brain function. However, the neurobiological mechanisms underlying nicotine's impact on brain function and behavior remain unclear. Elucidation of these mechanisms is of high clinical importance and may lead to improved therapeutics for smoking cessation. To fill in this critical knowledge gap, our current study focused on identifying sex-specific brain-derived extracellular vesicles (BDEV) signatures in male and female rats post nicotine self-administration. Extracellular vesicles (EVs) are comprised of phospholipid nanovesicles such as apoptotic bodies, microvesicles (MVs), and exosomes based on their origin or size. EVs are garnering significant attention as molecules involved in cell-cell communication and thus regulating the pathophysiology of several diseases. Interestingly, females post nicotine self-administration, showed larger BDEV sizes, along with impaired EV biogenesis compared to males. Next, using quantitative mass spectrometry-based proteomics, we identified BDEV signatures, including distinct molecular pathways, impacted between males and females. In summary, this study has identified sex-specific changes in BDEV biogenesis, protein cargo signatures, and molecular pathways associated with long-term nicotine self-administration.

Sex Differences in the Reward-Enhancing Effects of Nicotine on Ethanol Reinforcement: A Reinforcer Demand Analysis.

BACKGROUND: Alcohol is often consumed with tobacco, and dependence to alcohol and tobacco are highly comorbid. In addition, there are differences in the prevalence of nicotine- and alcohol-abuse between the sexes. Nicotine produces enhancing effects on the value of other reinforcers, which may extend to alcohol. METHODS: Male and female Wistar rats were trained to self-administer 15% ethanol solution in 30-minute sessions. Once ethanol self-administration was established, demand for ethanol was evaluated using an exponential reinforcer demand method, in which the response cost per reinforcer delivery was systematically increased over blocks of several sessions. Within each cost condition, rats were preinjected with nicotine (0.05, 0.1, 0.2, or 0.4 mg/kg base, SC) or saline 5 minutes before self-administration sessions. The effects of nicotine dose and biological sex were evaluated using the estimates generated by the reinforcer demand model. RESULTS: Under saline conditions, males showed greater sensitivity to ethanol reinforcement than females. Nicotine enhanced the reinforcement value of alcohol and this varied with sex. In both sexes, 0.4 mg/kg nicotine decreased intensity of ethanol demand. However, 0.05, 0.1, and 0.2 mg/kg nicotine decreased elasticity of ethanol demand in females, but not in males. CONCLUSIONS: Nicotine enhances ethanol reinforcement, which may partially drive comorbidity between nicotine-abuse and alcohol-abuse. Males showed signs of greater ethanol reinforcement value than females under saline conditions, and nicotine attenuated this effect by increasing ethanol reinforcement value in the females. These findings highlight that a complete understanding of alcohol-abuse must include a thorough study of alcohol use in the context of other drug use, including nicotine. IMPLICATIONS: Nicotine dose dependently enhances the alcohol reinforcement value in a manner that is clearly influenced by biological sex. Under saline baseline conditions, males show lower elasticity of demand for alcohol reinforcement than females, indicative of greater reinforcement value. However, nicotine attenuated this difference by enhancing alcohol reward in the females. Specifically, low-to-moderate doses (0.05-0.2 mg/kg) of nicotine decreased elasticity of alcohol demand in female rats, increasing the perseverance of their alcohol taking behavior. These data indicate that the well-documented reward-enhancing effects of nicotine on sensory reinforcement extend to alcohol reinforcement and that these vary with biological sex.

MicroRNA cluster miR199a/214 are differentially expressed in female and male rats following nicotine self-administration.

Previous research has established sex differences associated with nicotine intake, however a significant gap in knowledge remains regarding the molecular mechanisms that govern these differences at the transcriptional level. One critical regulator of transcription are microRNAs (miRNAs). miRNAs are a family of non-coding RNAs that regulate an array of important biological functions altered in several disease states, including neuroadaptive changes within the brain associated with drug dependence. We examined the prefrontal cortex (PFC) from male and female Sprague-Dawley rats following self-administration (22 days) of nicotine or yoked saline controls using next generation RNA-Sequencing (RNA-Seq) technology and found an array of miRNAs to be significantly and differentially regulated by nicotine self-administration. Of these, we found the expression of miR-199a and 214, which are expressed on the same cluster of chromosome 1, to be upregulated in the female rats exposed to nicotine; upregulation in this group was further validated by real time polymerase chain reaction (RT-PCR). Bioinformatics analysis to assess common targets of miR-199/214 identified Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD)- dependent deacetylase that plays a role in apoptosis, neuron survival, and stress resistance. Using western-blot, we confirmed downregulation of SIRT1 and increased cleaved caspase 3 expression in the brains of nicotine-exposed female rats and no change in expression levels in the other groups. Collectively, our findings highlight a miR-199/214 regulatory network that, through SIRT1, may be associated with nicotine seeking in females which may serve as a potential therapeutic target for sex-specific treatment approaches.

The effect of N-acetylcysteine or bupropion on methamphetamine self-administration and methamphetamine-triggered reinstatement of female rats.

N-acetylcysteine and bupropion are two promising candidate medications for treatment of substance use disorder. The effects of N-acetylcysteine or bupropion on methamphetamine self-administration of female rats are not well understood. To fill this gap, this study assessed the effects of N-acetylcysteine (0, 30, 60, or 120 mg/kg) and bupropion (0, 10, 30, and 60 mg/kg) on methamphetamine self-administration of female rats across the natural estrous cycle. Following a completed dose-response curve, responding for methamphetamine self-administration was extinguished and the effects of N-acetylcysteine or bupropion on methamphetamine-triggered reinstatement was evaluated in separate experiments. N-acetylcysteine did not decrease responding maintained by methamphetamine or methamphetamine-triggered reinstatement. Bupropion significantly decreased methamphetamine self-administration and methamphetamine-triggered reinstatement in female rats with highest dose (60 mg/kg) also significantly decreasing general chamber activity. In a companion experiment, testing the effect of bupropion on responding maintained by sucrose, we confirmed non-specificity of bupropion's effects as bupropion also decreased responding for sucrose. Considered together, our findings suggest that while N-acetylcysteine has considerable promise for treatment of cocaine dependence it may not generalize to other stimulants like methamphetamine. Furthermore, although bupropion has been shown to effectively decrease methamphetamine self-administration, and presently methamphetamine-triggered reinstatement, its locomotor and reward suppressing effects warrant further investigation including both sexes.

Nicotine- and cocaine-triggered methamphetamine reinstatement in female and male Sprague-Dawley rats.

Preclinical studies have demonstrated a return to methamphetamine (meth)-seeking behavior (reinstatement) induced by injections of meth administered by the experimenter (drug-prime). Notably, females tend to be more sensitive to drug-prime; often displaying more reinstatement behavior when compared to males. While meth-primed reinstatement of meth-seeking behavior has been established, little is known about the ability of other drugs of abuse to substitute for meth during drug-primed reinstatement; nicotine and cocaine were the focus of the present work. We also examined if self-administration and/or reinstated meth-seeking behavior was affected by repeated nicotine administration. Male and female Sprague-Dawley rats were trained to self-administer meth during daily sessions. During this self-administration phase, rats were placed into 1 of 2 groups: saline or repeated nicotine exposure. Rats in the repeated nicotine group received nicotine injections 4h after meth self-administration sessions, whereas the remaining rats received saline. Following self-administration was extinction in which meth was no longer available and nicotine was no longer administered. After extinction, rats were tested to determine if 0 (saline), 0.2, and 0.4mg/kg nicotine reinstated meth-seeking behavior. Three days of re-extinction followed nicotine testing. Finally, rats received reinstatement tests with 0 (saline), 5, and 10mg/kg cocaine. Nicotine and cocaine reinstated meth-seeking behavior in male and female rats with no difference between the sexes. Repeated nicotine administration potentiated meth reinstatement following the 0.4mg/kg nicotine-prime. While females may be more sensitive to reinstatement triggered with the original self-administration drug, this effect may not generalize to priming with other drugs of abuse.

Double dissociation of the anterior and posterior dorsomedial caudate-putamen in the acquisition and expression of associative learning with the nicotine stimulus.

Tobacco use is the leading cause of preventable deaths worldwide. This habit is not only debilitating to individual users but also to those around them (second-hand smoking). Nicotine is the main addictive component of tobacco products and is a moderate stimulant and a mild reinforcer. Importantly, besides its unconditional effects, nicotine also has conditioned stimulus effects that may contribute to the tenacity of the smoking habit. Because the neurobiological substrates underlying these processes are virtually unexplored, the present study investigated the functional involvement of the dorsomedial caudate putamen (dmCPu) in learning processes with nicotine as an interoceptive stimulus. Rats were trained using the discriminated goal-tracking task where nicotine injections (0.4 mg/kg; SC), on some days, were paired with intermittent (36 per session) sucrose deliveries; sucrose was not available on interspersed saline days. Pre-training excitotoxic or post-training transient lesions of anterior or posterior dmCPu were used to elucidate the role of these areas in acquisition or expression of associative learning with nicotine stimulus. Pre-training lesion of p-dmCPu inhibited acquisition while post-training lesions of p-dmCPu attenuated the expression of associative learning with the nicotine stimulus. On the other hand, post-training lesions of a-dmCPu evoked nicotine-like responding following saline treatment indicating the role of this area in disinhibition of learned motor behaviors. These results, for the first time, show functionally distinct involvement of a- and p-dmCPu in various stages of associative learning using nicotine stimulus and provide an initial account of neural plasticity underlying these learning processes.

The effects of varenicline on methamphetamine self-administration and drug-primed reinstatement in male rats.

Methamphetamine (meth) addiction is a costly burden to both the individual user and society as a whole. Establishing effective pharmacotherapies to treat meth dependence is needed to help solve this health problem. The study reported herein examined the effects of varenicline, a partial α4ß2 and full α7 nicotinic acetylcholine receptor agonist, on meth self-administration and reinstatement in male Sprague-Dawley rats. Following indwelling jugular catheter surgery, rats were either trained to self-administer meth or saline on a variable ratio (VR) 3 schedule of reinforcement. Self-administration sessions (2h duration; 19 total sessions) were conducted daily. The effect of varenicline pretreatment on meth and saline self-administration was then determined using a within-study design. All rats received varenicline (0.0, 0.3, 1.0, and 3.0mg/kg) prior to 4 different test sessions. Dose order was randomly assigned and each test was separated by 2 standard self-administration sessions to assess stability of responding. Fifteen extinction sessions (no meth available) followed the last test. Extinction was followed by meth-primed (0.3mg/kg IP) reinstatement tests to examine the effect of varenicline on meth-seeking behavior. All rats again received all doses of varenicline over 4 separate reinstatement tests performed on 4 consecutive days. Varenicline did not alter self-administration of meth or saline. Additionally, the 0.3 and 1.0 doses of varenicline non-specifically increased active lever responding during the reinstatement test sessions. This latter finding suggests that varenicline may increase relapse liability and should not be utilized as pharmacotherapy to treat meth dependence.

Sex differences in neurotensin and substance P following nicotine self-administration in rats.

Investigator-administered nicotine alters neurotensin and substance P levels in Sprague-Dawley rats. This finding suggested a role of the dopamine-related endogenous neuropeptides in nicotine addiction. We sought to extend this observation by determining the responses of neurotensin and substance P systems (assessed using radioimmunoassay) in male and female rats following nicotine self-administration (SA). Male and female Sprague-Dawley were trained to self-administer nicotine, or receive saline infusions yoked to a nicotine-administering rat during daily sessions (1-h; 21 days). Brains were extracted 3 h after the last SA session. Nicotine SA increased tissue levels of neurotensin in the males in the anterior and posterior caudate, globus pallidus, frontal cortex, nucleus accumbens core and shell, and ventral tegmental area. Nicotine SA also increased tissue levels of neurotensin in the females in the anterior caudate, globus pallidus, nucleus accumbens core and shell, but not in the posterior caudate, frontal cortex, or ventral tegmental area. There were fewer sex differences observed in the substance P systems. Nicotine SA increased tissue levels of substance P in both the males and females in the posterior caudate, globus pallidus, frontal cortex, nucleus accumbens shell, and ventral tegmental area. A sex difference was observed in the nucleus accumbens core, where nicotine SA increased tissue levels of substance P in the males, yet decreased levels in the females. The regulation of neuropeptides following nicotine SA may play a role in the susceptibility to nicotine dependence in females and males. Synapse 70:336-346, 2016. © 2016 Wiley Periodicals, Inc.

The effect of switching pharmacological intervention during extinction on nicotine-evoked conditioned responding in rats.

RATIONALE: Pharmacotherapies are often utilized to aid in smoking cessation, and switching medication when treating nicotine dependence has become more commonplace. Although common, little is known about the impact of the initial therapy on the effects of the subsequent therapy. OBJECTIVES: To begin to fill this gap in our understanding, this project determined how switching compounds that share stimulus elements with nicotine during extinction altered extinction responding and generalization of this extinction back to nicotine. METHODS: Rats were trained in a discriminated goal-tracking task where nicotine administration was followed by intermittent sucrose access; sucrose was withheld following saline administration. In experiment 1, nornicotine supplanted nicotine in extinction sessions 1-3 and then a switch to varenicline on extinction sessions 4-6 was examined. In experiment 2, the reverse was investigated; varenicline to start extinction and then a switch to nornicotine. Generalization of extinction back to the nicotine stimulus was then assessed by generating a cumulative dose-effect curve. RESULTS: Generalization of extinction back to the training nicotine stimulus was greater if nornicotine had been received at any point in extinction compared to only receiving varenicline. Whereas, extinction with varenicline alone showed more generalization to lower doses of nicotine. CONCLUSIONS: A switch in cessation pharmacotherapy during extinction did not impede or enhance generalization back to the nicotine-training stimulus. The nornicotine stimulus appears to share more stimulus overlap with the 0.4 mg/kg nicotine stimulus and varenicline may share more overlap with lower nicotine doses.

Behavioral effects of phencyclidine on nicotine self-administration and reinstatement in the presence or absence of a visual stimulus in rats.

RATIONALE: Tobacco use is a serious health problem in the USA, and this problem is potentiated in patients with schizophrenia. The reward system is implicated in schizophrenia and may contribute to the high comorbidity between nicotine use and schizophrenia, but very little research has been done on the topic. The reward-enhancement effect of nicotine has been shown to be important in nicotine use, but there have been no studies on this effect in animal models of schizophrenia. OBJECTIVES: This study was designed to determine the effects of phencyclidine, used to model negative symptoms of schizophrenia, on self-administration of nicotine with or without a co-occurring sensory reinforcer [i.e., visual stimulus (VS)] in rats. METHODS: Phencyclidine (2.0 mg/kg) was administered before each of seven nicotine self-administration sessions (0.01 mg/kg/inf) after which rats (n = 8-9 per group) were given 7 days of extinction without phencyclidine pretreatment. Reinstatement using phencyclidine (2.0 mg/kg), nicotine (0.2 mg/kg), and yohimbine (1.25 mg/kg, a pharmacological stressor) was tested after extinction to determine if previous exposure to phencyclidine would alter reinstatement of active lever pressing. RESULTS: Phencyclidine initially decreased nicotine self-administration but only in the groups with a concurrent VS. This decrease in self-administration dissipated after 5 days. During reinstatement, rats that had previously received phencyclidine during self-administration with a VS were more sensitive to stress-induced reinstatement than any other group. CONCLUSIONS: These results show a transitory effect of phencyclidine on nicotine self-administration. Phencyclidine may induce a potential sensitivity to pharmacological stressors contributing to reinstatement of nicotine.

Interoceptive conditioning in rats: effects of using a single training dose or a set of 5 different doses of nicotine.

Interoceptive conditioning contributes to the tenacity of nicotine dependence. Previous research investigating nicotine as an interoceptive stimulus has typically employed administration of a single training dose of nicotine over an extended time. This approach has allowed for careful study of the nicotine stimulus. In humans, the nicotine stimulus is unlikely to be fixed across learning episodes. Thus, from a translational perspective, systematic variation of nicotine dose in training might better approximate interoceptive conditioning in humans. Notably, training with a class or set of discrete exteroceptive stimuli (e.g., different pictures of cars) produces interesting behavioral differences relative to training with a single stimulus. The present study sought to determine whether similar differences would occur if a set of nicotine stimuli were used in place of a single dose. To investigate this question, one group of male Sprague-Dawley rats was trained on a discriminated goal-tracking task with a set of nicotine doses (0.05, 0.125, 0.2, 0.275, and 0.35mg/kg). A second group received the standard protocol of training with a single nicotine dose (0.2mg/kg). On each nicotine session, there was intermittent access to liquid sucrose (26%) in a conditioning chamber. On intermixed saline sessions, sucrose was withheld. We examined acquisition, subsequent extinction, transfer of extinction, nicotine generalization, and mecamylamine blockade. Both groups reliably discriminated between nicotine and saline sessions, were sensitive to non-reinforcement, displayed transfer of extinction, demonstrated dose-dependent nicotine generalization, and responding was blocked by mecamylamine. There were no significant differences between the two groups. The unique nature of an interoceptive pharmacological stimulus and the challenges posed for studying the impact of training with a set of interoceptive stimuli are discussed.

Interoceptive conditioning with a nicotine stimulus is susceptible to reinforcer devaluation.

Pavlovian conditioning processes contribute to the etiology of nicotine dependence. Conditioning involving interoceptive stimuli is increasingly recognized as playing a role in many diseases and psychopathologies, including drug addiction. Previous animal research on diminishing the influence of interoceptive conditioning has been limited to antagonism and nonreinforced exposures to the drug stimulus. The goal of the present research was to determine whether interoceptive conditioning with a nicotine stimulus could be diminished through an unconditioned stimulus (US) devaluation procedure. In two separate experiments, male Sprague-Dawley rats received nicotine injections (0.4 mg base/kg) followed by intermittent sucrose (26%) access in a conditioning chamber. On intermixed saline sessions, sucrose was withheld. Conditioning was demonstrated by a reliable increase in head entries in the dipper receptacle on nicotine versus saline sessions. After conditioning, rats in a devaluation condition were given access to sucrose in their home cages immediately followed by a lithium chloride (LiCl) injection on 3 consecutive days. On subsequent test days, nicotine-evoked conditioned responding was significantly attenuated. Within-subject (Experiment 1) and between-subjects (Experiment 2) controls revealed that the diminished responding was not attributable to mere exposure to the sucrose US in the devaluation phase. Experiment 2 included a LiCl-alone control group. Repeated illness induced by LiCl did not reduce later nicotine-evoked responding. These findings suggest that there is a direct association between the interoceptive stimulus effects of nicotine and the appetitive sucrose US (i.e., stimulus-stimulus) rather than a stimulus-response association.

Disentangling the nature of the nicotine stimulus.

Learning involving interoceptive stimuli likely plays an important role in many diseases and psychopathologies. Within this area, there has been extensive research investigating the interoceptive stimulus effects of abused drugs. In this pursuit, behavioral pharmacologists have taken advantage of what is known about learning processes and adapted the techniques to investigate the behavioral and receptor mechanisms of drug stimuli. Of particular interest is the nicotine stimulus and the use of the two-lever operant drug discrimination task and the Pavlovian drug discriminated goal-tracking task. There is strong concordance between the two methods when using "standard" testing protocols that minimize learning on test days. For example, ABT-418, nornicotine, and varenicline all fully evoked nicotine-appropriate responding. Notably, research from our laboratory with the discriminated goal-tracking task has used an alternative testing protocol. This protocol assesses stimulus substitution based on how well extinction learning using a non-nicotine ligand transfers back to the nicotine stimulus. These findings challenge conclusions based on more "standard" testing procedures (e.g., ABT-418 is not nicotine-like). As a starting point, we propose Thurstone scaling as a quantitative method for more precisely comparing transfer of extinction across doses, experiments, and investigators. We close with a discussion of future research directions and potential implications of the research for understanding interoceptive stimuli.