Stephen G. Holtzman - US grants

Opioids comprise a broad range of compounds, from classical morphine-like agonists to pure antagonists. Between those extremes are drugs with varying spectra of agonist and antagonist properties, probably as a consequence of interactions with multiple subtypes of opiate receptors. Abuse potential also varies considerably among the different types of opioids. Experiments are proposed that will continue an ongoing research program aimed at characterizing systematically and comprehensively the behavioral effects of opioids having different spectra of activities in order to identify and study components of drug action relevant to abuse potential, and to gain insights into the neuronal substrates subserving those components of action. Emphasis will be placed on clarifying the role of endogenous opioid peptides in the diverseness of the behavioral effects of opioid antagonists and agonists. Behavioral effects of opioid peptides will be determined following intracerebroventricular (icv) administration, their effects compared to those of prototypic opioid alkaloids, and interactions studied between opioid peptides administered icv and opioid antagonists and agonists administered systemically. Tests will usually be performed in two animal species (rat and squirrel monkey), and in several behavioral procedures in order to assess the generality of experimental findings. Principal studies will include: a) characterizing the discriminative stimulus effects of opioid peptides and their interactions with various opioid antagonists and agonists in animals trained to discriminate saline from prototypic agonists (e.g., morphine, ethylketocyclazocine, SKF 10,047) of proposed receptor subtypes; b) determining if the discrimination of saline from diprenorphine by monkeys is based on stimuli associated with a blockade of endogenous opioid peptide systems; c) assessing the effects of antagonists, agonists and opioid peptides alone and in combination on schedule-controlled behavior (punishment paradigm) in rats and monkeys, and on motor activity in rats. Experiments will also be continued to elucidate the fole of endogenous opioid peptides in the regulation of appetitive behaviors by testing pure opioid antagonists and peptides administered centrally to rats and monkeys.

This is an application for an ADAMHA Research Scientist Award. A program of research is proposed for characterizing the behavioral effects of two types of drugs of major clinical, social, and theoretical importance: opioids, which have long represented a significant abuse problem, and caffeine, which is the most widely consumed behaviorally-active compound in this country. A theme central to the entire research program is that through systematic and comprehensive behavioral studies it will be possible to characterize the bases of drug actions at the neuronal level. Representative opioid alkaloids--agonists, agonist/antagonists, and antagonists--will be studied in several behavioral procedures, notably, drug discrimination, food-reinforced operant responding (punishment paradigm), and locomotor activity, and in at least two animal species, rat and squirrel monkey for optimal assessment of the generality of experimental findings. Emphasis will be placed on clarifying the role of endogenous opioid peptides in the diverseness of the behavioral effects of opioid alkaloids. Behavioral effects of peptides will be determined after central administration, their effects compared to those of prototypic alkaloids, and interactions studied between peptides administered centrally and alkaloids administered systemically. The effects of chronic caffeine administration will be characterized by an integrated behavioral and neurochemical approach. Most studies will be performed on rats receiving caffeine daily by scheduled access to water bottles containing the drug. Matched controls will receive drug-free tap water. Rate and extent of tolerance development, and time course for loss of tolerance to caffeine-induced stimulation of motor activity will be determined as a function of daily drug dosage. Pharmacologic (i.e., alkylxanthine and nonxanthine drugs) and behavioral (i.e., food-reinforced operant responding) variables in tolerance to caffeine will also be assessed. Changes in various measures of the functional state of brain catecholamine and adenosine systems will be determined in parallel with the behavioral changes as a first step toward identifying the cellular mechanisms that underlie tolerance to caffeine.

caffeine; psychopharmacology; central nervous system stimulants; neurochemistry; drug tolerance; cyclic AMP; operant conditionings; dopamine; drug withdrawal; neurotransmitter metabolism; adenylate cyclase; norepinephrine; adenosine; behavior test;

Caffeine is the most widely consumed behaviorally active drug in the world. Because it has the principal features usually associated with a drug of abuse, caffeine can be a convenient model drug for studying the behavioral and cellular mechanisms of drug reinforcement and related phenomena, such a tolerance and physical dependence. The broad objectives of this project have been to define and quantify effects of acute and chronic treatment wit caffeine at the behavioral and cellular levels. The following specific aim represent a continuation of established lines of investigation as well as new research approaches suggested by recent findings. 1) To test the hypothesis that some behavioral effects of caffeine are due to enhanced postsynaptic dopaminergic mechanism resulting from blockade of adenosine receptors; continuous blockade of adenosine receptors results in a compensatory desensitization of those dopaminergic mechanism and caffeine tolerance. This hypothesis will be tested in rats using the behavioral measures of locomotor activity and unilateral rotation and by in vitro and in vivo measures of postsynaptic dopamine receptor function. 2) To continu to characterize pharmacologically in rats the qualitative differences between the discriminative effects of low and high doses of caffeine and th ability of caffeine to elevate the reinforcement threshold for intracranial self-stimulation. Experiments will be directed at determining the neurochemical bases of those effects and their relevancy to the positive an negative mood states induced by caffeine in humans. 3) To test the hypothesis that caffeine has potent and previously uncharacterized effects on cyclic AMP metabolism and intracellular Ca2+ stores, that these effects are not mediated by blockade of cell-surface adenosine receptors, and that tolerance develops to these effects. Experiments will be performed on a model cell line of CNS origin, the NG108-15 neuroblastome x glioma hybrid cell line, in order to define more clearly these cellular effects of caffeine. Despite the fact that caffeine usually is consumed on a chronic daily basis, there have been relatively few studies of the behavioral and neurochemical consequences of chronic treatment with caffeine. Basic information obtained on the acute and chronic behavioral and cellular actions of caffeine should have broad applicability to many types of drugs of abuse.

A program of research is proposed for studying behavioral effects of two types of drugs of major clinical, social , and theoretical important: 1) opioids, which have long represented a significant abuse problem; 2) caffeine, which is the most widely consumed behaviorally-active compound in the world, and which has many of the characteristics of a drug of abuse. The basic strategy of this research program is to use behavioral methodologies to study interactions of drugs with their neuronal substrates. The objective is to identify and characterize components of drug action that may be relevant to potential for abuse and to related phenomena, such as tolerance and physical dependence. The neuronal substrates of drug action will be characterized with receptor-selective agonists, antagonists, and tolerance and cross-tolerance. Representative compounds will be studied over a range of doses in several behavioral procedures, such as drug discrimination, food-reinforced operant responding, locomotor activity, and autotitration of reinforcement threshold for electrical brain stimulation. Experiments will be performed on rats and, often, on squirrel monkeys. This approach will help in assessing the generality of experimental findings with respect to pharmacological, behavioral, and species variables. The proposed experiments will address a number of hypotheses. Among these are: 1) Endogenous opioid peptides can modulate the behavioral effects of exogenously administered opioid drugs; 2) Similar components of drug action mediate discriminative stimulus, reinforcing stimulus, and subjective drug effects; 3) The discriminative stimulus effects of low and high doses of caffeine differ qualitatively from each other and reflect components of drug action that underlie, respectively, positive and negative mood states in humans.

DESCRIPTION: (Applicant's Abstract) Results of recent studies on rats indicate a relationship among responsiveness to stress, activity of the mesolimbic dopamine system, behavioral and neurochemical responses to psychomotor stimulant and opioid drugs, and drug-seeking behaviors. Male rats separated from their mothers for 180 min/day (MS180), but not 15 min/day (MS15), on postnatal days 2-14 have an enduring increased responsiveness of their hypothalamic-pituitary-adrenal axis to a variety of stressors. In preliminary experiments, adult MS180 rats had higher locomotor activity than did MS15 rats upon initial exposure to a novel environment; they also were more sensitive to the motor-stimulating effects of morphine upon both acute and repeated administration and had a larger dopamine response in the nucleus accumbens to an IV dose of cocaine. The purpose of this exploratory/developmental project is to determine if early maternal separation results in altered responsiveness to opioid drugs in adulthood, and therefore might provide an animal model for studying factors that contribute to vulnerability to drugs of abuse. Three interrelated hypotheses will be addressed in a circumscribed series of systematic experiments in which a limited number of prototype opioid or psychomotor stimulant drugs will be tested over a range of doses in well-established procedures in animal facility reared, MS15, and MS180 rats: tests of antinociception (analgesia), motor activity, and suppression of drinking. Using morphine, the plasticity of endogenous opioid systems will be assessed in tests of tolerance and naloxone-induced sensitization to its analgesic effects, physical dependence, and sensitization to its motor-stimulating effects. Opioid modulation of effects of drugs that act via brain dopamine systems will examined in tests of cross-sensitization to motor-stimulating effects of amphetamine and cocaine and the interaction of naloxone with those drugs. Results confirming the potential value of this animal model would form the basis for a more comprehensive study of pharmacological, behavioral, and neurochemical variables contributing to vulnerability to opioids and other abused drugs.

DESCRIPTION: (Applicant's Abstract) Continued career development will come from a combination of new research projects and new questions from older projects, leading to new methods and to new as well as continuing collaborations. This will be facilitated by the thriving neuroscience community at Emory University, which includes many principal investigators conducting research on issues related to drug abuse, and by the recent expansion of faculty and breadth of research within the Dept. of Pharmacology. The proposed research focuses on the behavioral pharmacology of opioids and, to a smaller extent, cocaine, and reflects current grant support from NIDA. Specific aims include testing hypotheses about acute opioid dependence in rats and squirrel monkeys and the functional state of endogenous opioid systems in adult rats that had undergone daily maternal separation during the early postnatal period. Trainees at various academic levels - undergraduate, pre- and postdoctoral - will participate in this research program each year. An important goal will be to provide them with the intellectual skills needed for a career of research on the behavioral pharmacology of drugs of abuse. In addition, the Candidate will continue to engage in nonresearch activities that contribute to overall career development, such as teaching neurobehavioral pharmacology, membership on journal editorial boards and government advisory committees, and holding elected office in scientific organizations.

Results of studies on rodents indicate links among reactivity to stress, mesolimbic dopamine system activity, behavioral and neurochemical responses to psychomotor stimulant and opioid drugs, and drug-seeking behaviors. Male rats separated from their mother for 3 hr/day on postnatal days 2-14, as adults, are more reactive to stress than are rats from control rearing groups and have behavioral traits consistent with proposed models of drug vulnerability. They also have altered sensitivity to effects of acute and chronic morphine on pain threshold and motor activity. The principal aim of this project is to characterize further the influence of early maternal separation on opioid sensitivity by extending observations to additional procedures and drugs (e.g., kappa opioids), and to female offspring. We will address the interrelated hypotheses that mother-infant separation results in enduring changes in the functional state of endogenous opioid systems, in their plasticity, and in brain markers of opioid systems. Experiments will focus on phenomena associated with drug-induced neuronal plasticity-tolerance, physical dependence, sensitization-and on measures of drug reinforcement and abuse potential-intracranial self-stimulation, place conditioning, IV drug self- administration. Morphine and other prototypic drugs will be tested over a range of doses in adult rats that, on postnatal days 2-14, had underwent either a) 3-hr maternal separation, b) 15-min separation, or c) no separation. To identify cellular correlates of rearing-induced alterations in behavior and sensitivity to opioid drugs, opioid-related mRNAs will be measured in selected brain regions by solution hybridization and gene chip microarrays. Early mother-infant separation impacts the subsequent behavior and opioid sensitivity of the dams, too. Thus, dams will be tested in some of the same procedures as the offspring. The maternal separation paradigm affords unique animal models that can give new insights into pharmacological, behavioral, environmental, and cellular variables that affect sensitivity to opioid drugs and vulnerability to drug abuse.