Galen Wenger - US grants

The United States is in the middle of an unprecedented cocaine abuse epidemic. Over 22 million Americans are estimated to have used cocaine and the impact of this dramatic increase is now becoming evident. There is currently a paucity of information on the behavioral effects of the chronic use of cocaine, the degree of tolerance which develops, and the presence or absence of withdrawal signs upon abrupt discontinuation of cocaine. This lack of information extends to the effects of cocaine on cardiovascular function at doses which produce behavioral effects. It is speculated that the cardiovascular effects may contribute to cocaine related medical emergencies and lethality. Frequently, cocaine is abused simultaneously with other behaviorally active drugs. Such combinations as cocaine and narcotics, cocaine and amphetamines, and cocaine and marijuana are frequently used and the resultant effects are essentially unknown. This project will attempt to provide answers to some of these questions by studying the effects of cocaine alone in the squirrel monkey before, during, and after chronic administration of cocaine, and in combination with four other drugs which are likely to be co-abused with cocaine: morphine, d-amphetamine, Delta-9-tetrahydrocannabinol, and caffeine. The effects of cocaine and cocaine combined with selected doses of the other four drugs will be determined on responding under a multiple fixed-interval (food), fixed-interval (escape) schedule of reinforcement. During the behavioral sessions, cardiovascular function (blood pressure, heart rate, and EKG), and core body temperature will be recorded. In addition, the pharmacokinetics of cocaine will be determined before and during the chronic administration of cocaine. These experiments will provide information on the degree of tolerance development or increased sensitivity as a result of the chronic dosing, the extent of withdrawal signs upon the abrupt discontinuation of chronic cocaine administration, and the nature of the interactions which occur as a result of combined administration of cocaine and other drugs of abuse. In addition, it will be possible to determine the contribution of any changes in cocaine disposition kinetics in the changes observed in the cocaine dose-response curves as a result of chronic dosing.

Animal models of memory have largely involved the measurement of short-term memory or more precisely working memory. This can be defined as the memory which is required to perform correctly on a given trial. This is different from long-term or reference memory. Reference memory has been defined as that memory which relates the presentation of the stimulus, the response and the presentation of the reinforcing stimulus. These operational definitions are essentially what is known in operant conditioning as stimulus control and schedule control of behavior. Most animal models of short-term or working memory have utilized discrete trial procedures in which each correct behavioral response was reinforced. Such continuous reinforcement of behavior has essentially ignored the influence of schedules of reinforcement on the control of behavior. It is, therefore, not surprising that it has been difficult to know whether the drug was effecting stimulus control (working memory) or schedule control of the behavior (reference memory). This application proposes to examine the effect of the schedule of reinforcement on the effects of drugs of abuse on. memory function. Animal models of working memory, delayed matching-to-sample and delayed alternation, will be programmed under continuous and intermittent schedules of reinforcement. Specifically, delayed matching-to-sample and delayed alternation will be programmed under either a discrete trial (fixed-ratio 1) schedule, or under different second-order schedules (a second-order fixed-ratio 10, a second-order fixed-interval 300 sec, a second-order variable-interval 100 sec, and a second-order fixed-interval 100 sec) in which the unit of behavior is the discrete trial. In addition, two different approaches will be used to examine whether the drug is disrupting stimulus control (working memory) or schedule control (reference memory). The effect of each drug will be determined under both simultaneous matching and delayed matching, and by examining the effects of schedule-induced bias in responding. Finally, the effects of drugs of abuse will be examined as a function of the degree of stimulus control of behavior by different types of events (position vs. visual stimuli or auditory vs. temporal stimuli). It is hoped that these studies will provide additional insights into the effects of drugs of abuse on cognitive function, as well as, provide information on the role of the schedule of reinforcement in the measurement of cognitive function.

The ability of drugs of abuse to affect cognitive processes in both laboratory animals and humans is now well documented, Yet, the mechanism for this effect is not understood. This project will focus on the effects of three drugs of abuse (diazepam, pentobarbital and phencyclidine) on memory function as determined by two operant schedules of reinforcement: a delayed alternation schedule and a delayed matching- to-sample schedule. These drugs have been shown to bind to specific sites in the brain: diazepam has been shown to bind to the GABA chloride channel complex, pentobarbital has been shown to bind at the GABA channel complex, as well as the AMPA activated channel and to the sigma binding site. However, there is little information available about the relative role of each of these sites in the modulation of memory function by these drugs. Thus, the overall goal of this project is to determine the role of these binding sites in the effect of the drugs on memory function. The behavior of rats, pigeons and squirrel monkeys will be maintained under a delayed alternation schedule and a delayed matching-to-sample schedule. Once responding has stabilized and dose-response curves have been etermined for diazepam, pentobarbital and phencyclidine, the role of the GABA receptor complex in mediating the effects of the three drugs on memory will be explored by using selected doses of RO 15-4513, and picrotoxin. The role of the NMDA activated channel and the PCP receptor will be examined by using selected doses of NMDA and CGS 19755. The role of the AMPA activated channel will be determined by using selected doses of NBQX, and the role of the sigma binding site will be studied by using selected doses of DTG, haloperidol and BMY 14802. These experiments will provide data relevant to determining the locus of action of three drugs of abuse and what if any commonalities exist between them. Understanding the locus/mechanism of action of these drugs of abuse on memory function will provide a more complete picture of the pharmacology of human drug abuse.

Selective breeding has produced strains of rats with either high (P) or low preference (NP) for ethanol. The major focus with these rats has been the documentation of their ethanol preference difference. However, there have been a few studies indicating that the two rat strains may display altered cognitive function. If such cognitive differences exit, it raise the question of whether or not the cognitive differences are genetically linked with the ethanol preference. Such a linkage would be important in understand the role of cognitive processes in alcohol dependence. A first step in examining this question will be to determine if there are clear and unambiguous differences in cognitive function between the P and NP rats. Thus this project will examine the performance of P and NP rats in three tests of different aspects of cognitive function. Prior to the initiation of these tests of cognitive function, the degree of motivation of the P and NP rats to lever press for initiation of these tests of cognitive function, the degree of motivation of the P and NP rats to level press for food presentation will be assessed under a progressive ratio schedule. Once motivation factors have been normalized, the two strains of rats will be tested under a test of short-term memory (delayed matching-to-position), a test of temporal discrimination (ability to discriminate a 3-second versus 10-second delay) and a test of learning (repeated acquisition of behavioral chains). The performance of the rats will be assessed under control conditions and following doses of ethanol, diazepam, scopolamine and methscopolamine. Ethanol was selected because of its reported effect on short-term memory in humans and laboratory animals and because of the reported differences in preference between the two strains. Diazepam was selected because of its known effects on cognitive function and action at the GABA receptor. Scopolamine was selected because it is a prototype amnesic agent believed to act via cholinergic rather than GABA systems. Methscopolamine will be studied to control for peripheral actions of scopolamine. If it can be shown that there are clear differences in the cognitive function of the P and NP rats, future experiments will attempt to determine if the differences in ethanol preference and the differences in cognitive function are genetically linked. If they are, it will provide additional potential insight into the role that cognitive processes play in alcohol dependence.

The possibility that ethanol-induced alterations in attention/vigilance contributes to an increase in accidents and lost productivity in the workplace is an important area of concern in today's society. This research project is based upon the following hypotheses: the effect of ethanol on safety and productivity is at least partly a result of drug- induced alterations in vigilance or attention, the effect of ethanol on vigilance is proportional to the length of time that the subject is required to remain vigilant and inversely proportional to the lack of predictability and the frequency of the signal presentation, the effect of ethanol on vigilance is magnified in the presence of a distracting task, and the finally, the effect of ethanol is altered by changing the cost: benefit ratio to the subject or reporting/or failing to report a signal presentation. Vigilance will be monitored by evaluating the ability of subjects to detect a brief stimulus change and to respond accordingly. To test the stated hypotheses, both the frequency and predictability of the signal presentation will be systematically changed, the relationship between the reporting of a signal presentation and the presentation of food will be changed by altering the schedule of reinforcement, the cost of reporting or failing to report a signal presentation will be examined punishing incorrect responses and false alarms with a mild electric shock or by the postponement of future signal presentations, and the influence of a distracting task will be examined by training the subjects to respond upon one response lever/key for food while simultaneously remaining vigilant to the presentation of a signal and responding upon a different lever/key for food following signal presentation. These studies will be conducted in both rats and pigeons trained to respond following a signal presentation under both a continuous trials and a discrete trials procedure. The continuous trials procedures was selected because it requires a more sustained attention than the discrete trials procedure and therefor may be more analogous to the workplace. However, a discrete trials procedure will also be studied because of the ability to incorporate trials which do not contain a signal. Consequently, the full power of signal detection analysis can be employed in the analysis of the data which is particularly important in addressing questions about change in the bias of responding. Finally, the utilization of the two procedures of the two procedures will permit a direct comparison of the results obtained under both procedures and should contribute significantly to the area of animal models of attention/vigilance. It is hoped that these studies will lead to a further understanding of the relationship between ethanol usage and vigilance and the impact of ethanol use on performance and safety in the workplace.

DESCRIPTION (provided by applicant): Recent advances in genomics have brought to the forefront the fact that the mouse has been drastically underutilized in the behavioral aspects of drug abuse research. The use of operant schedules of reinforcement has played a significant role in our understanding of drugs of abuse and the addictive process in both humans and laboratory species. However, until recently there have been only a small number of laboratories that have attempted to utilize the mouse in experiments involving operant schedules of reinforcement. Many years of research on the genetics of the mouse has supplied the scientific community with a tremendous number of inbred strains. Many of these strains have well defined genetic differences that result in significant differences in drug effects. For example, it has been shown that marked differences exist between C57Bl/6 and DBA/2 mice in response to many effects of morphine including analgesia, locomotor activity, hypothermia, Straub-tail, and voluntary oral morphine consumption. Similarly, although tolerance to the analgesic and locomotor response to morphine has been observed to be about equal in C57Bl/6 and DBA/2 mice, tolerance to the analgesic effects of morphine has been reported not to occur following chronic administration in 129 mice. Whether strain differences exist in the stimulus or reinforcing properties as measured by drug discrimination or IV self-administration procedures in response to opioid drugs in these strains is not known. Knowledge of such differences may provide significant insight into the role of genetic differences in opioid addiction and the mechanisms of drug action. The present study will use three inbred strains of mice (C57Bl/6J, DBA/2J and 129P3/J). The study will examine the ability of receptor subtype specific opioid agonists to function as discriminative stimuli, the degree to which the drug stimulus generalizes to agonists specific for other receptor subtypes, and the ability of antagonists to block the stimulus properties. Hopefully, by utilizing these well-defined strains, it will be possible to use the known differences in genetics to provide new insight into the abuse of opioids.

DESCRIPTION (provided by applicant): Animal models of human diseases have been of tremendous value in understanding mechanisms of disease and in design of effective treatment protocols. In spite of the success of this approach in certain areas, there has been limited success in development of animal models of diminished mental capacity, especially diminished mental capacity with age. One such model, Ts65Dn, which was developed by Davisson at The Jackson Laboratory, is considered to be the best animal model of human Down syndrome. Although aspects of this model have been studied and parallels to the human condition have been drawn, potential age-related cognitive losses have not been explored. For instance, there are reports of decreased cognitive function in Ts65Dn mice, but most report on a single time period during the first half of the expected life span and most losses are neither robust nor consistently found in all studies. To more fully explore the diminished mental capacity of this model, a longitudinal study over the entire life span of the animal, as we propose here, is needed to determine the extent of additional cognitive loss as this animal model ages. Operant conditioning procedures are uniquely suited for this type of longitudinal study since behavior can be maintained and repeatedly tested in individual subjects for months to years. Recently our group, in collaboration with Dr. Davisson's group, showed a clear deficit in learning in Ts65Dn mice responding under a repeated acquisition of behavioral chains schedule of reinforcement. Specific aims 1-3 will examine the repeated learning, working memory, sustained attention, and the effect of putative cognitive enhancers on the performance of Ts65Dn and littermate controls from 3 to 24 months of age. In specific aim 4, Dr. Griffin's group will analyze the brains of Ts65Dn and littermate control animals to determine the expression of specific proteins that have been implicated in neuropathological changes and in the pathogenesis of age-related decline in cognition in humans and in experimental animals. To our knowledge, the current proposal represents the first attempt to study the behavioral phenotype of the Ts65Dn mouse using operant conditioning procedures, especially over the life span and in conjunction with neuropathological and age-related changes.

A number of studies have shown altered cognitive function in humans who abuse ethanol and other drugs of abuse. However, it is not clear whether these differences existed prior to the abuse or whether the differences are the result of the abuse. An initial approach to answering this question is to examine cognitive function in animals with known differences in preference for an abused substance, alcohol. If differences in memory and learning can be demonstrated and shown to be associated with alcohol preference, it would suggest a possible genetic association between altered memory and learning function and alcohol preference. Such a finding could provide insight into the possible role of cognitive differences observed in humans that abuse ethanol. Selective breeding has produced strains of rats with either high (P, HAD1, HAD2) or low preference (NP, LAD1, LAD2) for ethanol. The major research focus with these rats has been the documentation of their ethanol preference difference. However, there have been a few studies indicating that these rat strains may display altered cognitive function. Thus this project will examine the performance of rats with high alcohol preference (P, HAD1, HAD2) and rats with low alcohol preference ( NP, LAD1, LAD2) rats in an operant model of working memory and two models of repeated learning. Prior to the initiation of these tests of cognitive function, the degree of motivation of the rats to lever press for food presentation will be assessed under a progressive ratio schedule. If motivation differences are detected, an attempt to reduce the differences in motivation will be conducted by adjusting body weights. Once motivational factors have been normalized, the working memory and learning performance of the rats will be examined. The performance of the rats will be assessed under control conditions and following doses of ethanol, diazepam, scopolamine and methscopolamine. Ethanol was selected because of its reported effect on short-term memory in humans and laboratory animals and because of the reported differences in preference between the strains. Diazepam was selected because of its known effects on cognitive function and action at the GABA receptor. Scopolamine was selected because it is a prototype amnesic agent believed to act via cholinergic rather than GABA systems. Methscopolamine will be studied to control for peripheral actions of scopolamine. If it can be shown that differences in the cognitive function of the rats is associated with alcohol preference or non-preference, it will provide additional potential insight into the role that cognitive processes play in alcohol dependence, and possibly open new treatment avenues