Gordon A. Barr - US grants

The experiments in this project propose to examine the development of the positively reinforcing properties of abused drugs. Two paradigms that are well established in the adult literature and are believed to assess drug abuse potential will be used. These are the conditioned place preference and intracranial electrical self-stimulation paradigms (ICSS). Because of the large literature suggestive of a critical role of the mesolimbic dopamine system in drug abuse and in these two paradigms, the proposed experiments concentrate on the ventral tegmental area of the midbrain (VTA). The effects of morphine and cocaine will be tested in these two paradigms: we will test that ability of these drugs to enhance ICSS responding to stimulation by a VTA located electrode and to support place preference conditioning when injected directly into the VTA. These experiments will determine the development of the reinforcing properties of these two abused drugs and will provide a developmental time frame for in vivo dialysis studies of the effects of these drugs on mesolimbic dopamine function. In a second series of experiments, the effects of early postnatal exposure to morphine and cocaine will be tested in three models of drug abuse vulnerability. The first two are the conditioned place preference and ICSS as described above and the third is self-administration of abused drugs.. Rats dosed chronically during infancy will be tested in these three models; in addition, subjects treated both in infancy and adulthood will be tested. These experiments are unique in that there are no data that speak to the issue of the long term consequences of early perinatal exposure to drugs, despite the large and growing numbers of human infants who have been so exposed. In addition, these treatments serve as independent variables for testing the hypothesis that the action of abused drugs is mediated through the mesolimbic dopamine system. Biochemical studies of in vivo dopamine release and opioid and dopamine receptor autoradiography will pinpoint neurochemical differences caused by the neonatal drug exposure that do or do not correlate with the increased drug abuse vulnerability. The studies therefore provide new and important information about the development of the reinforcing properties of morphine and cocaine, the long term consequences for brain reward mechanisms due to exposure to these drugs, and the neural mechanisms by which these.drugs produce their potent effects on neural systems related to reinforcement.

Large numbers of human infants are exposed in utero to illicit opiate drugs such as heroin and to prescribed opiates such as methadone. It is therefore important to understand the effect of acute and chronic exposure to drugs such as these on the immature organisms. A necessary step in understanding the long term effects of early drug exposure is to describe fully how the drug acts on neurobehavioral systems in the neonate. Because infants typically experience withdrawal from opiates, either in utero or shortly after parturition, a fuller understanding of the consequences of opiate withdrawal in the immature animal is important but is a largely unexplored area of research. Although there has been some question as to whether infants undergo withdrawal, from our previous experiments we have reason to believe that rat neonate experiences clear behavioral changes during opiate abstinence, including the induction of "dysphoric" states on cessation of drug exposure. It is hypothesized that the infant undergoes withdrawal, but that the signs and symptoms experienced differ from those suffered by the adult. In the proposed research we will describe the effects that repeated exposure to and withdrawal from the prototypic opiate, morphine, has on the immature organism. The rat is the model of choice because it is an altricial species and thus quite immature at birth and because there is a large existent database that describes the neurobiology and behavior of this species. Using classic paradigms from the adult literature, and developmentally appropriate measures, we will examine in depth the behavioral and physiological changes that occur during precipitated and spontaneous opiate abstinence. We begin by using observational methods to describe behavioral changes during precipitated withdrawal in the fetus, young infant and older animal, comparing these changes to those describe for the adult. We relate these behaviors to measured levels of morphine in brain and plasma both prior and subsequent to the development of the blood brain barrier. Secondly we explore alterations in physiological systems including cardiovascular, thermoregulatory and sleep/wake states. During withdrawal from morphine, pups increase ultrasonic vocalizations, fail to quiet in the company of a companion, and can learn to avoid stimuli previously associated with precipitated withdrawal. These may be developmental parallels to negative affective states experienced by adults in withdrawal. These behavioral changes will be explored in detail. We will also initiate studies on the neural bases of the development of opiate abstinence syndromes, specifically focussing on two brain areas, the nucleus accumbens and the locus coeruleus, demonstrated in the adult to be involved in separable components of that syndrome. The last series of experiments explores the consequences of spontaneous opiate withdrawal or later mother-infant interactions. The results from these experiments should provide important data on the effects of drugs exposure and withdrawal during development.

DESCRIPTION (provided by applicant): Until recently the only description of an opiate abstinence syndrome in neonates was for human infants. Yet the complexities of the human setting make it impossible to tease apart the aspects of withdrawal that are due to opiate use, and those that are due to the abuse of other drugs, poor prenatal care, under-nutrition, or any of the myriad of other complications experienced by the mothers of these children. Three groups, including ours, have detailed an opiate withdrawal syndrome in the infant rat. The withdrawal syndrome slowly changes over development to reach, around puberty, the classic constellation of withdrawal behaviors so often described for the adult animal. There are both similarities and differences in the neural substrates underlying withdrawal in infants and adult. Although the neural structures mediating withdrawal behaviors are similar, there are important differences in the role of NMDA receptors mediating these behaviors. NMDA blockers have no effect, or worsen, withdrawal at 7 days of age but ameliorate it at 21 days of age. In contrast, nitric oxide synthase (NOS) inhibitors block withdrawal throughout development. Thus at young ages, NMDA receptors and NOS differentially regulate precipitated withdrawal. Because the abstinence syndrome in the infant is now described in great detail, we are poised to ask a number of questions that could not have been addressed before. In particular little is known of the genetic changes induced by opiate dependence and withdrawal in the adult animal, and nothing is known in the infant. The experiments proposed here define changes in gene expression induced by opiate withdrawal at different stages of development. We test morphine withdrawal at 7 and 21 days of age with and without NMDA blockers and NOS inhibitors to assess specific changes in patterns of gene expression under conditions where withdrawal is or is not expressed. We use gene microarray technology to assess specific changes in patterns of gene expression and advanced bioinformatic methods to analyze and provide access to these data. In each case we assay specific regions of the CNS involved in withdrawal, including the periaqueductal gray of the midbrain, the locus ceruleus, amygdala, nucleus accumbens and spinal cord. By assessing simultaneous changes in expression levels of large numbers of genes, we can identify the nature of the biological responses of specific brain regions to opiate withdrawal. In turn, these data allow a more complete understanding of the mechanisms underlying the differences in drug response between infants and adults, and provide a detailed picture of the developmental changes in patterns of gene expression linked to severity of opiate withdrawal. These results can then direct the development of novel treatments for the opiate withdrawal syndrome in this population of at risk infants.

DESCRIPTION: (provided by applicant) The human infant is often exposed to opiate drugs over extended periods of time, either due to drug abuse of the mother or because of medical conditions. Two serious clinical problems are the development of tolerance to the medical use of opiates for analgesia and dependence to both prescribed and abused opiates. Little is known of the mechanisms that mediate either withdrawal or tolerance in the infant in large part because appropriate animal models have only recently been developed. Although many of the biological mediators of opiate withdrawal in the infant are similar to those of the adult, we have recently discovered that NMDA glutamate antagonists, which are effective antidotes for withdrawal and tolerance to opiates in the adult, are fully ineffective in the infant rat and may indeed exacerbate both withdrawal and tolerance in the young. In contrast, our preliminary data show that both an AMPA glutamate receptor blocker and a metabotropic glutamate receptor (mGluR) agonist completely prevent the expression of morphine withdrawal. We hypothesize specific developmental mechanisms that could explain these data including developmental differences in glutamate receptor subtypes and subunits and alterations in the ability of opiates to up- and downregulate glutamate receptors. The experiments described in this application provide detailed descriptions of the effects of NMDA, AMPA and mGlu receptor antagonists and mGluR Group H agonists on precipitated withdrawal and tolerance to the analgesic effects of morphine both in vivo, measured behaviorally or by c-fos expression, and measured electrophysiologically in the isolated spinal cord preparation. The latter preparation allows us to focus experiments in subsequent aims on the spinal cord and to describe both the normal maturation of glutamate receptors and the ability of morphine to up- or downregulate those receptors. In the final aim, we study mice with genetic disruptions of the normal expression of specific glutamate receptors to determine if the changes in withdrawal and tolerance in the infant mice follow the predictions based on the hypotheses put forth and the pharmacological and biochemical data from the prior aims. The results of these experiments will provide substantial information about mechanisms by which glutamate receptor function is different in the infant than the adult and provide direction for the development of pharmacological tools that are uniquely effective in the human infant.

DESCRIPTION (provided by applicant): One of the difficulties in developing treatments for the seriously ill infant patient has been the limited understanding of how pain processing differs between the infant and the adult. In particular, there are developmental differences in how pain is processed, and in the long-term consequences of tissue insult. Growing evidence both from the clinic and the laboratory demonstrate that there are long lasting changes in sensitivity to subsequent noxious stimuli following injury in the infant. Further, there appears to be a "critical" period, at least in the rat, such than injury in the first two weeks of life, but not later, results in those long lasting changes in pain sensitivity. This clearly has very important clinical implications; yet nothing is known of the mechanisms engaged by the experience of pain in early development. The recent development of microarray methods to assess simultaneously changes in the expression in thousands of genes provides a unique opportunity to define the neural and genetic changes that might be responsible for the differences in pain processing in infants, immediately and in the 24 hours after injury, and into adulthood. Here we propose to use well-characterized oligonucleotide microarrays (Affymetrix) and state of the art analytic methods to define those alterations in gene expression induced by injury. We assay the dorsal horn of the lumbar enlargement of the spinal cord as a model system, largely because of described short and long terms changes in spinal cord function and neurobiology induced by injury. We test rat pups at 3 and 21 days of age and assess gene expression changes at four times after insult over 24 hours. This describes the more immediate changes in gene expression as a function of injury. The second series of studies examine changes in gene expression in the adult spinal cord in pups injured at different ages in early in development. In the putative critical period, during the first two weeks of life, pups are injected with carrageenan. Older pups are tested because they are outside the critical period. Adults treated as pups are tested again, with or without insult, and gene expression is assayed by microarray. The results of these experiments will define in detail, changes in gene expression that are induced by injury early in development when nociceptive processes are distinctly different than the adult, and again later in development, after the end of the critical period. Further, we describe long-term changes in expression that might explain the "permanent" changes in pain perception induced by early injury. These data will provide the basis of rational treatments that might reduce any deleterious effects of pain experience by premature and seriously ill infants.

DESCRIPTION (provided by applicant): Paradoxically, children form strong attachments to their abusive caregiver, and these children are at high risk for psychiatric disorders both during childhood and as adults. Although this is an important ethical /societal and mental health problem, how this paradoxical attachment can occur remains a mystery. Specifically the neurological underpinnings of this paradoxical attachment are not known;however there are substantial data showing that the amygdala has an important regulatory role in fear/aversion learning both in adults and infants. We have modeled the neurobiology of abusive attachment using neonatal rats and fear conditioning (odor-shock pairings). We found odor-aversive shock pairings produces an odor preference during a sensitive period of development in neonatal rats but an aversion in older pups and adults. The naturally occurring elevation in corticosterone that occurs around 10 days of age in the rat pup appears to bring the amygdala "on-line" and switch the infant to the adult response of avoidance from odor-shock conditioning. Specifically, we can either accelerate or retard the end of the sensitive period simply by respectively lowering or raising the pup's endogenous CORT levels either systemically or selectively within the amygdala. Because these learned preferences to aversive stimuli are long lasting they likely engage changes in genes and proteins within the amygdala. We use here microarrays to assess gene expression during and after the "sensitive" period to understand what the mechanisms are within the amygdala. We determine: First, what are the changes in gene expression induced by noxious input when preferences or aversions are learned? This provides a detailed description of how the amygdala responds differently before and after the switch from preference to aversion. Second, what changes in gene expression occur in the amygdala as a result of manipulation of CORT levels that also alter the sensitive period? Third, what are the long term changes in aversion or preference learning that persist into adulthood and modify levels of engagement in other motivated behaviors? The combined use of age-specific learning and CORT (altering the age that preference learning switches to aversion learning), provides a powerful tool to assess amygdala candidate genes in an age dependent and independent paradigm. Our preliminary studies show contrasting changes in dopamine markers during and after the sensitive period and follow-up studies implicate further the role of dopamine in the learned preferences to aversive stimuli.

DESCRIPTION (provided by applicant): Between 50-80% of infants in the neonatal intensive care unit are dependent on opiates by the end of their medical treatment. Best current medical practice is to provide declining amounts of opiates to taper the infant patient off the drugs. As such, this prolongs hospitalization and constitutes a significant burden on the health care system, and an emotional burden on the family whose child remains in the hospital, and has unknown consequences for subsequent development of the newborn. Therefore opiate dependence in the neonate is a substantial clinical and public health problem in need of new therapeutic approaches. We know little about opiate dependence in the neonate, except that it differs in important mechanisms from dependence in the adult, when there is interplay between opioids and the immune system. Whether that interplay is present or not in the infant has not been studied and thus is not known, despite how common infection is in these babies. Our extensive preliminary data in a rat model show that activating the immune system during chronic morphine treatment around weaning worsens both the physical and affective aspects of withdrawal. The same immune activating treatment in very young infant rats has no effect. These behavioral changes are accompanied by age-dependent differences in the expression of immune markers in the spinal cord and brain. In this exploratory R21 application, we propose multiple analytic approaches to define the role of the immune system in morphine dependent/withdrawn infant rats. We propose careful behavioral assays that we have developed over the years to measure the behavioral and affective components of opiate withdrawal. We assess mRNA and protein for immune markers and proinflammatory cytokines in the brain and spinal cord of morphine treated infants. Our working hypothesis is that up- or down-regulating the immune system will not alter withdrawal in the young infant rat (7 days of age; human equivalent is ~full term infant), and that as the animal matures, immune system-opioid interactions begin to function in ways similar to those of the adult. Our mechanistic follow-up hypothesis is that the toll-like-receptor 4 (TLR4) mediates the interactions of opiates and the immune system to modulate opiate dependence in the older infant but has no consequence for the younger infant. To test these hypotheses, we propose pharmacological treatments and tests with mutant mice to understand the role the immune system in general and of the TLR4 receptors in particular in opiate dependent infants. The completion of this work will define how the interactions between opioid and immune systems change as the animal matures. In determining if immune modulation is effective or not in the opiate dependent infant, we will provide preclinical data to direct clinical decisions as to whether or not immune based therapeutics should be developed and implemented for the treatment of opiate dependence in infant patient.