Examination of Methamphetamine Reinstatement in Female and Male Rats: A Pre-Clinical Model of Relapse

Methamphetamine (meth) dependence is often characterized by persistent and chronic relapse (i.e., return to drug use). There is growing pre-clinical and human evidence suggesting females are at greater risk to relapse. The set of studies presented in this dissertation extended this limited evidence by identifying sex-dependent neural substrates correlated with meth-triggered reinstatement (Experiment 1) and by examining sex-differences in reinstatement triggered by drugs of abuse that are commonly co-abused with meth (Experiment 2). Female and male rats were trained to self-administer meth, received subsequent extinction sessions, and then tested for reinstatement. In Experiment 1, rats were perfused following reinstatement testing and c-Fos activity was examined as a measure of neural activation. Meth triggered reinstatement in both sexes and this effect was more robust in females compared to males. In the females, c-Fos activity was significantly increased following meth-primed reinstatement in the cingulate cortex area 1, lateral orbitofrontal cortex, prelimbic cortex, caudate-putamen, nucleus accumbens core and shell, and central nucleus of the amygdala. In males, there were no significant differences following meth-primed reinstatement. In Experiment 2, nicotine and cocaine were utilized as drug primes to determine if administration of these drugs could trigger meth-seeking behavior. Nicotine and cocaine reinstated meth-seeking behavior in male and female rats with no difference between the sexes. Females were more sensitive to reinstatement triggered with the original self-administration drug and this effect may not generalize to priming with other drugs of abuse. Advisor: Rick A. Bevin

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 0.35 mg/kg). A second group received the standard protocol of training with a single nicotine dose (0.2 mg/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 dosedependent 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 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 0.35 mg/kg). A second group received the standard protocol of training with a single nicotine dose (0.2 mg/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 dosedependent 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

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 United States 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 7 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) were 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

Laboratory Notes From Behavioral Pharmacologists and Trainees: Considerations for the Discipline

In several laboratory meetings, we discussed the challenges that face trainees in behavioral pharmacology. Major concerns, such as a difficult funding climate and limited academic job prospects were discussed at first. However, we decided to concentrate on ways to meet these challenges; versus focusing on negatives and listing gripes. Within this more constructive framework, we identified the importance of broadening training to aligned areas to enhance the capacity of behavioral pharmacologists to collaborate in multidisciplinary teams. With increased breadth of training comes the concern for a balance that does not cheat trainees out of the depth of training also needed for success. We believe that behavioral pharmacologists trained in this manner will be ideally positioned to be leaders of these translational research teams. Related to the breadth and depth of training is the recent concerns over replicability and reproducibility of published research. Behavioral pharmacologists, with the rigors of training in behavioral analysis and experimental design, can be at the forefront of this conversation. This will be especially true if current training is reinforced with additional experience in the use of cutting-edge statistical tools that address the complex experimental designs and large data sets that emerge from modern multidisciplinary collaborations. Finally, communicating the import and potential societal impact of our research to legislators, other scientists, educators, school children, neighbors, and acquaintances is needed to ensure that our field thrives. In closing, the process of explicitly discussing the challenges and potential solutions with current trainees will enhance their mentoring and training

Laboratory Notes From Behavioral Pharmacologists and Trainees: Considerations for the Discipline

In several laboratory meetings, we discussed the challenges that face trainees in behavioral pharmacology. Major concerns, such as a difficult funding climate and limited academic job prospects were discussed at first. However, we decided to concentrate on ways to meet these challenges; versus focusing on negatives and listing gripes. Within this more constructive framework, we identified the importance of broadening training to aligned areas to enhance the capacity of behavioral pharmacologists to collaborate in multidisciplinary teams. With increased breadth of training comes the concern for a balance that does not cheat trainees out of the depth of training also needed for success. We believe that behavioral pharmacologists trained in this manner will be ideally positioned to be leaders of these translational research teams. Related to the breadth and depth of training is the recent concerns over replicability and reproducibility of published research. Behavioral pharmacologists, with the rigors of training in behavioral analysis and experimental design, can be at the forefront of this conversation. This will be especially true if current training is reinforced with additional experience in the use of cutting-edge statistical tools that address the complex experimental designs and large data sets that emerge from modern multidisciplinary collaborations. Finally, communicating the import and potential societal impact of our research to legislators, other scientists, educators, school children, neighbors, and acquaintances is needed to ensure that our field thrives. In closing, the process of explicitly discussing the challenges and potential solutions with current trainees will enhance their mentoring and training

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

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 nicotinelike). 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

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 nicotinelike). 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

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