Norman Spear - US grants

animal developmental psychology; memory disorders; conditioning; child mental disorders; learning; infant animal; stimulus /response; paired stimuli; stimulus interval; memory; early experience; child psychology; neural information processing; cues; association learning; discrimination learning; stimulus generalization; taste; vibration perception; behavioral /social science research tag; visual stimulus; auditory stimulus; laboratory rat; behavior test; olfactory stimulus;

health science research support; university;

The purpose of this application is to request funds to establish a multi-user facility for fluorescence studies of cellular chemistry and structure. The instrumentation requested for this facility is as follows: 1) a combination spectrofluorometer and microscope excitation system; 2) an inverted microscope; 3) an image intensifying video camera and image processor; 4) a high-resolution video recorder; 5) a micromanipulator and vibration-free table. These items will comprise a single, working unit dedicated to the following projects, all of which require the sensitivity and specificity of fluorescence: 1) The role of Ca++ and H+ ions in ligand activated cellular responses; 2) Pinocytotic regulation in normal and transformed cells and its relation to cell size and microtubular structure; 3) H+ gradients along endocytic pathways in a polarized cell: pH in "endosomal tubules" and "giant lysosomes" of absorptive cells in neonatal rat ileum; 4) Mechanism of cell-matrix interaction during cell migration; and 5) Interaction of matrix molecules with embryonic epithelia. The requested instrumentation has been selected for an overall flexibility and versatility which meets the requirements of the five diverse projects described herein, and which anticipates future facility requirements arising from newly developed fluorescence techniques.

The proposal is to test ontogenetic change in how acute ethanol ingestion affects learning, memory and stimulus selection. Recent surveys indicate substantial consumption of ethanol among children, particularly adolescents, but the consequences for learning and memory when developing children or animals are under the influence of ethanol is unknown. With emphasis on periadolescence, in compariosn with earlier and later periods, we will test with an animal model (the rat) the influence of acute ethanol as an agent of contextual control over the expression of memory, the interaction between effects of ethanol and sources of stress, the effects of ethanol on rate of forgetting, and preliminary indications that ethanol may influence what is learned rather than (or as well as) the learning process itself, perhaps especially at ceartain points in ontogency. Through-out, emphasis will be on the careful derivation of functions relating dose of ethanol with learning and memory, assessed in circumstances in which ontogenetic comparisons may be considered. Equivalency doses will be chosen to equate brain alcohol levels (BrAL) across age. The conditioning and test procedures will be rapidly conducted, equally so across agrees to enable containment of the duration of conditioning and testing within the peak BrAL for all animals at all ages. The proposed experiments address the following questions about the influence of ethanol on learning and retention at different ontogenetic periods: (1) When ethanol produes state dependent retention, how does this effect vary with age? (2) How is learning influenced by ethanol in the presence or absence of isolation stress at different ages? (3) What effect does ethanol have on rate of forgetting in animals of different ages? (4) Does ethanol influence the selection of stimuli attended to and/or learned and does this vary with age? (5) Does ethanol affect learning in the absence of reimforcers that are strong biologically significant stimuli in a manner similar to that observed when reinforcers such as footshock or food are employed, and is the relationship between such effects dependent upon ontogenetic status? (6) Does ethanol have effects on periadolescent animals that differ from those at younger or older ages, as has been observed for other drugs that alter central catecholaminergic activity and do these include effects that alter the learning and/or stimulus selection of these periadolescent animals?

The long-term objective of understanding and controlling infantile amnesia will be pursued by continued focus on special dispositions of the infant rat for stimulus selection. This topic is to be studied in the relatively broad framework that incorporates three classes of hypothetical processes associated historically with the ontogeny of knowledge acquisition: differentiation and encoding, integration and organization, and maintenance and expression. In the first category the proposal is to test the notion that infant animals are particularly susceptible to encoding in terms of net intensity, assess the influence of early experience on transfer of conditioning between stimulus compounds and single elements, or vice versa, and identify which attributes of stimuli are actually encoded by infant (and older) animals. To investigate integration and organization, tests at several ages will focus on stimulus-stimulus learning involving a variety of sensory modalities. Of particular interest is the role of inter- and intramodal similarity between stimuli to be learned and the effect of temporal disparity in the presentation of these stimuli. Additional tests will assess the impact of configuring and backward conditioning on integration and organization at different ages. Tests of maintenance and expression will consider both the characteristics of infantile forgetting and the alleviation of such forgetting. For the former there will be tests of age-related differences in retention of incidental vs. target memories, and selected tests involving overshadowing, latent inhibition and sensory preconditioning to help determine whether the encoding disposition during infancy or that at testing (when older) will prevail in determining what is remembered about infancy. Finally, to investigate the alleviation of the forgetting of infantile learning we will test prior cueing treatments and the effects of the distribution of such treatments, the interval between conditioning and prior cueing, and that between prior cueing and test. Alleviation of forgetting by distinctive context will be studied, and the implicit reactivation paradigm will be used to determine how infantile encoding of a particular stimulus affects integration and organization of that stimulus with another at an older stage. Grant=P30CA33572 The Cancer Research Center at the City of Hope is a clearly-defined organizational entity within the City of Hope which brings together in horizontal fashiod the cancer-related elements from each Department and Division. The specific aims of the Cancer Center are to: -Stimulate collaborative research interactions between basic science and clinical investigators and between research groups in different areas of investigation. -Facilitate and enhance established interdisciplinary programs in basic and clinical research. -Develop and support core research facilities which improve the effectiveness and the interdisciplinary nature of the Center's research programs. -Support the development of new investigators and/or new programs of high scientific merit. During the past five years, the Cancer Center at the City of Hope has also continued to mature; its research programs have been extensively re- evaluated, focused, and expanded where appropriate after both external and internal review. A new Director and Associate Directors have been appointed. With the assistance of Core Grant support substantial new Core Facilities also have been developed to meet the needs of the Cancer Center membership. A central mission of the Cancer Center, to facilitate strong interactions between the basic and clinical sciences at the City of Hope, has been realized further with the funding of Program Project-type awards in four of the six Cancer Center Programs. In this application we are requesting support for staff; scientific programs in Molecular Biology, Cell and Tumor Biology, Immunology, Virology, Hematologic Malignancy, and Clinical and Experimental Therapeutics; fifteen shared resources; and developmental and administrative funds to carry out the specific aims of the Cancer Center.

biomedical equipment purchase;

DESCRIPTION: (Adapted from the Investigator's Abstract) The reality of ethanol exposure among children, from gestation through the infant, juvenile and adolescent periods, is supported by epidemiological evidence. The general purpose of this proposal is to study experimentally the nature and basis of early experiences with ethanol that potentiate responsiveness to ethanol later in life. The further purpose is to assess factors that may determine the persistence of these effects and lead, potentially, to increasing susceptibility to ethanol abuse in adolescence and adulthood. Three circumstances of early ethanol exposure will be studied, stated here in the form of three specific aims: (1) to determine the nature and short-term effects of perinatal learning involving ethanol's attributes, and persistence of the consequences of this learning. (2) To understand the nature and limits of short-term effects of a representative amount of ethanol during nursing, and the subsequent persistence of these effects. (3) To understand the nature and short-term effects of early ethanol ingestion (free or forced) as well as mere olfactory exposure to ethanol, and the persistence of these effects. Three sets of consequences of these effects will be assessed with established, largely behavioral tests. The first is alteration in efficacy in detection and perception of ethanol's orosensory attributes, assessed by cardiac orienting and basic behaviors indicating acceptance or rejection of ethanol. The second set assesses altered postingestive and pharmacological consequences of ethanol as well as acceptance of ethanol, in terms of ethanol-induced activation, tolerance to ethanol, and ethanol intake. The third set will determine changes in the reinforcing consequences of ethanol, in terms of conditioned place preference and operant responding for ethanol. The theoretical orientation is based on a model or metaphor of learning and memory, tested directly as part of Study 1, but the value of these studies is independent of the viability of this model. Persistence of the consequences of early ethanol exposure is studied with techniques based on those used previously to promote persistence of the consequences of another form of neuroplasticity, acquired memory. Preliminary studies indicate that persistence of the effects of early ethanol exposure may be promoted by similar principles, as a consequence of re-exposure to ethanol later in life.

This proposal is to assess a new technique for self-administration of ethanol in neonatal rats, when this ethanol constitutes the newborn's first feeding experience. This technique will be examined for its value in testing two issues -- the consequences of early exposure to ethanol for later responsiveness to ethanol, and the basis of ethanol's reinforcing effects. Perinatal exposure to ethanol is known to influence substantially the rat's later responsiveness to ethanol, in accord with recent evidence that amount of prenatal exposure to ethanol is a good predictor of human ethanol abuse during adolescence. Preliminary studies suggest that the proposed self-administration procedure: (1) has advantages over others used for infant rats; (2) might provide an assay for prenatal consequences of ethanol; and (3) could serve as a potential model of human infant ethanol intake during nursing after the mother consumes ethanol or of prenatal ethanol during the third trimester of the human fetus. Preliminary studies have indicated that newborn rats consume substantial amounts of ethanol from a surrogate nipple and subsequent responsiveness to this nipple simulates that observed after intake of milk (but different from that after intake of other fluids such as water or saline). With these procedures ethanol was consumed at relatively high concentrations when delivered in solution with milk. The first specific aim is to establish optimal parameters for self-administration of ethanol by newborns and assess the reinforcing effects of ethanol with these parameters. The second aim is to assess effects of frequency and dose of prenatal ethanol exposure on the newborn's self-administration of ethanol. The third aim is to study consequences of the newborn's exposure to ethanol delivered in either of two ways, -- through a surrogate nipple or intra orally infused -- on later responsiveness to ethanol. The proposed experiments will evaluate the promise of the proposed self-administration procedure in this advantageously simple mammalian model, for use in understanding the reinforcing effects of ethanol and the effects of ethanol exposure on later responsiveness or sensitivity to ethanol.

DESCRIPTION: In both humans and animals prenatal exposure to ethanol increases susceptibility to subsequent ethanol abuse and alters related aspects of responsiveness to ethanol. Postnatal exposure to ethanol also has been found to increase later ethanol intake and related aspects of responsiveness to ethanol in animals. Early onset of ethanol use in adolescence or even pre-adolescence has been shown to be associated with a greater probability of ethanol abuse in adulthood, although causality in these studies with humans has yet to be demonstrated. One determinant of ingestion and related responsiveness to ethanol during early adolescence may be still earlier exposure to ethanol, a possibility to be explored in the present experiments. Toward improvement over previous experimental procedures in which early ethanol exposure occurs in relatively stressful circumstances, we propose to provide early exposure to ethanol in relatively nonstressful, ecologically representative circumstances and assess the consequences for responsiveness to ethanol in adolescence. Our preliminary results indicate that as adolescents, the offspring of dams living with their litter in large enclosures and free to consume ethanol ad libitum chose to drink relatively large amounts of ethanol. The present proposal is to determine the ontogenetic locus of this effect -- whether exposure during gestation, lactation or weaning is differentially effective -- and to determine controlling parameters of this effect. The second proposed step is to assess the breadth of consequences from these forms of early exposure for several key indices of responsiveness to ethanol during adolescence.

DESCRIPTION (provided by applicant): Recent evidence suggests that neonatal and older infant rats readily consume ethanol without initiation procedures (unlike adults) and treat it as an appetitive reinforcer. The proposed experiments test ethanol's ingestion and reinforcing effects throughout infancy. In view of epidemiological evidence for relatively frequent exposure to ethanol among human infants, and clinical evidence that prenatal exposure to ethanol in humans predicts ethanol abuse in adolescence, these experiments are relevant to adolescent and adult abuse of ethanol as well as the experimental study of ethanol's reinforcing effects. The first specific aim is to investigate self-administration of ethanol in infant rats, and the second is to test ethanol's reinforcing effects in infant rats. This will be accomplished separately for each of the developing rat's two systems of ingestion that have been identified and differentiated behaviorally, functionally, and neurophysiologically - suckling and independent ingestion. Our preliminary studies have indicated substantial intake and reinforcing effects of ethanol in both suckling by neonates and independent ingestion by older infants. For ethanol self-administration through suckling, initial experiments would determine the roles of ethanol concentration and duration of exposure, ethanol odor independently of its gustatory consequences, and ethanol flavor separated from ethanol?s pharmacological consequences. Roughly parallel experiments are proposed testing older infants in the context of independent ingestion. The effectiveness of alcohol as a reinforcer (unconditioned stimulus, US) would then be tested for neonates through Pavlovian conditioning in the context of suckling and for older pups in the context of independent feeding. Using two different types of conditioning for neonates, these tests would assess basic determinants such as ethanol concentration and the duration of the CS-US pairing, and analytical issues such as the potentially separable reinforcing consequences of ethanol's pharmacological and gustatory attributes. Related questions will be tested for older preweanlings in terms of conditioning with context as the CS and ingestion of ethanol, or its injection (to minimize gustatory attributes of ethanol), as the US. Finally, parallel experiments testing the intake and reinforcing effects of ethanol in neonatal rats will be conducted with pups from lines genetically selected for ethanol intake (P/NP and HAD/LAD). Understanding of the consequences of maternal ethanol ingestion for prenatal and early postnatal (breastfeeding) human exposure to ethanol could be served by the proposed studies, and correlations between neurophysiological changes during ontogeny and the ontogeny of ingestion and reinforcement in the developing rat should help reveal neurophysiological controls of ethanol intake and its effects such as reinforcement.

[unreadable] DESCRIPTION (provided by applicant): The proposed experiments will continue to assess consequences of early exposure to ethanol for responsiveness to ethanol later in development. In view of epidemiological evidence for relatively frequent exposure to ethanol among human infants as well as fetuses, and clinical evidence that prenatal exposure to ethanol in humans predicts ethanol abuse in adolescence and early adulthood, the proposed experiments are relevant to issues of adolescent initiation to ethanol as well as more proximal consequences of early ethanol exposure. Three circumstances of early ethanol exposure to be studied are in the form of three specific aims: (1) To examine consequences of fetal processing of ethanol's chemosensory attributes and those of a learned association between them and ethanol's pharmacological effects; (2) To determine consequences of postnatal nursing experience involving ethanol for subsequent responsiveness to ethanol's chemosensory attributes, sensitivity to ethanol's postabsorptive effects and self-administration of ethanol; (3) To assess longitudinally the interactive consequences of prenatal and early postnatal exposure to ethanol for subsequent ethanol sensitivity and learning about ethanol during the preadolescent and adolescent periods. Experiments will apply techniques developed and previously employed in our laboratories, providing a combination of relatively new methods of treatment and assessment with methods used for many years. The experiments will include systematic assessment of: fetal associative learning supported by the reinforcing effects of ethanol during prenatal ethanol exposure and the consequences for postnatal responsiveness to ethanol; the consequences of ethanol exposure during nursing for later responsiveness to ethanol, novel environments and motor challenges; and conditioned and unconditioned effects of combined ethanol exposure as a fetus and as a nursing infant on later learning about ethanol, acceptance of ethanol and response to socially induced acceptance of ethanol. Theoretical orientation for considering the consequences of early ethanol exposure is focused on learning induced by that exposure and motivational content of related early memories. Special concern is given to specific temporal windows following ethanol exposure that yield memories of specific motivational associates, and the effect of multiple, potentially interactive exposures to ethanol for later patterns of ethanol recognition and acceptance [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Use of ethanol during or just before adolescence clearly predicts ethanol abuse later in life. Is this ethanol use in adolescence due to still earlier exposure to ethanol? Epidemiological evidence in humans and experiments with rats indicate that ethanol acceptance in adolescence is potentiated by prenatal exposure to ethanol. The present proposal tests the hypothesis that a few moderate, nonteratological doses of ethanol during late gestation alters later responsiveness to ethanol, including ethanol acceptance and reinforcement from birth to adolescence and activating effects of ethanol during adolescence. This hypothesis is encouraged by preliminary studies indicating that this prenatal ethanol exposure not only increases later ethanol acceptance but also increases the range of appetitively reinforcing ethanol doses. In each of the proposed experiments postnatal responsiveness to ethanol will be assessed for animals given 0, 1 or 2 g/kg on each of gestational days 17-20 or a control treatment. Specific Aim 1 is to test ethanol intake and ethanol reinforcement soon after birth, in terms of three different reinforcement paradigms. Specific Aim 2 is to test effects of .prenatal ethanol on ethanol acceptance and ethanol reinforcement in adolescence and at a point midway between birth and adolescence (postnatal day 15, P15). Specific Aim 3 is to test the consequences of prenatal ethanol exposure for an established activating effect of ethanol during adolescence, ethanol- induced social facilitation. In view of the apparent role of the endogenous opioid system in ethanol ingestion, ethanol reinforcement and adolescent social behavior, Specific Aim 4 is to test the effects of prenatal ethanol on beta-endorphin gene expression, beta-endorphin brain levels and ?-opioid receptor binding associated with ethanol ingestion, ethanol reinforcement, and ethanol-induced social behavior. These tests will occur soon after birth, on P15 or during adolescence. The intention is to clarify the ontogeny of risk from moderate exposure to prenatal ethanol and determine mechanisms underlying subsequently altered responsiveness to ethanol. *Proposed experiments will test the observation that individuals exposed to ethanol as fetuses are more likely to abuse ethanol as adolescents and adults, whether this fetal exposure makes ethanol more rewarding and acceptable later on, and how these effects might be mediated by the opiate system. [unreadable] [unreadable] [unreadable]

The consequences of positive and negative ethanol reinforcement vary during development. The present proposal tests the hypothesis that positive and negative reinforcements are differentially affected by controlling parameters. Moreover, there is further value in comparing the efficacy of ethanol reinforcement between the second postnatal week and late adolescence. (1) In humans and animals, exposure to ethanol during the early part of this period increases affinity for ethanol during adolescence. (2) As infant rats ingest ethanol in substantial quantities without initiation procedures (like adult rats selectively bred for high initial acceptance of ethanol), they provide an alternative to the use of genetic selection for determining mechanisms of ethanol reinforcement. That is, tests with the developmental model hold the genetic potential constant while varying age, whereas tests with a genetic model hold age constant while varying genetic potential. (3) Although the critical period of adolescence for determining ethanol abuse in humans has become better understood by contrasting the sensitivity of adolescent and adult rats to ethanol, it remains uncertain whether ontogenetic progression into these aspects of adolescence is abrupt, a discrete break from earlier ages, or continuous. Presumably then, younger animals should have vestiges of mechanisms responsible for the adolescent's sensitivity to ethanol and susceptibility to ethanol abuse. Thus, the proposed studies will examine the ontogeny of positive and negative ethanol reinforcement between, the effect of earlier exposure to ethanol on positive and negative ethanol reinforcement in late adolescence, and explore central mechanisms underlying negative and positive reinforcement. Psychopharmacological experiments will examine receptor systems that change ontogenetically and have strong theoretical and empirical links to negative or positive reinforcement. These studies will be supported by collaborations with the CELL/MOLECULAR BIOLOGY CORE, to assess translational and protein expression of localized mu opioid receptors and three GABAA receptor subunits (2, 3, and 5) in response to the ethanol reinforcement;and with the NEUROANATOMY CORE, to determine the number of neurons expressing, in selected neural areas, cFos in response to the early ethanol exposure that is expected to alter ethanol reinforcement in late adolescence. A key feature of these experiments is application of tests for negative and positive reinforcement that are comparable over the range of ontogeny. The proposed experiments will clarify the relationship between early ethanol exposure and the "early debut" effect in humans. Clinical benefits may occur in terms of determining the need for differential treatment for ethanol abuse originating primarily from positive or negative reinforcement sources, the role of pre-adolescent ethanol experience, and the potential interaction between age and "type" of ethanol reinforcement that dominates one's experience.

The consequences of positive and negative ethanol reinforcement vary during development. The present proposal tests the hypothesis that positive and negative reinforcements are differentially affected by controlling parameters. Moreover, there is further value in comparing the efficacy of ethanol reinforcement between the second postnatal week and late adolescence. (1) In humans and animals, exposure to ethanol during the early part of this period increases affinity for ethanol during adolescence. (2) As infant rats ingest ethanol in substantial quantities without initiation procedures (like adult rats selectively bred for high initial acceptance of ethanol), they provide an alternative to the use of genetic selection for determining mechanisms of ethanol reinforcement. That is, tests with the developmental model hold the genetic potential constant while varying age, whereas tests with a genetic model hold age constant while varying genetic potential. (3) Although the critical period of adolescence for determining ethanol abuse in humans has become better understood by contrasting the sensitivity of adolescent and adult rats to ethanol, it remains uncertain whether ontogenetic progression into these aspects of adolescence is abrupt, a discrete break from earlier ages, or continuous. Presumably then, younger animals should have vestiges of mechanisms responsible for the adolescent's sensitivity to ethanol and susceptibility to ethanol abuse. Thus, the proposed studies will examine the ontogeny of positive and negative ethanol reinforcement between, the effect of earlier exposure to ethanol on positive and negative ethanol reinforcement in late adolescence, and explore central mechanisms underlying negative and positive reinforcement. Psychopharmacological experiments will examine receptor systems that change ontogenetically and have strong theoretical and empirical links to negative or positive reinforcement. These studies will be supported by collaborations with the CELL/MOLECULAR BIOLOGY CORE, to assess translational and protein expression of localized mu opioid receptors and three GABAA receptor subunits (2, 3, and 5) in response to the ethanol reinforcement; and with the NEUROANATOMY CORE, to determine the number of neurons expressing, in selected neural areas, cFos in response to the early ethanol exposure that is expected to alter ethanol reinforcement in late adolescence. A key feature of these experiments is application of tests for negative and positive reinforcement that are comparable over the range of ontogeny. The proposed experiments will clarify the relationship between early ethanol exposure and the "early debut" effect in humans. Clinical benefits may occur in terms of determining the need for differential treatment for ethanol abuse originating primarily from positive or negative reinforcement sources, the role of pre-adolescent ethanol experience, and the potential interaction between age and "type" of ethanol reinforcement that dominates one's experience.