Ronald P. Hammer - US grants

Delta-9-tetrahydrocannabinol (THC), the active constituent of marijuana, is a prevalent psychoactive substance of abuse. Although we do know that it is active in the central nervous system, its mechanism of action and the means by which it produces behavioral alteration are unknown. The experiments proposed in this application will examine the metabolic effects of acute THC administration in the rat brain. A range of THC doses, from low (2mg/kg, 4mg/kg) to moderate (8mg/kg) and high (16 mg/kg, 30mg/kg) will be intravenously administered. Tritiated 2-deoxy-D-glucose (2DG), a high resolution marker for autoradiographic localization of brain metabolism, will be given 30 min after the THC dose and the brain will be sectioned and prepared for autoradiography using (3H)sensitive X-ray film. Autoradiographs will be densitometrically analysed using an image processing system; at least 56 brain regions will be individually outlined on Nissl-stained brain sections and their optical densities will be measured from the corresponding autoradiographs. Ratios of this regional optical density to that of a white matter region in the same section will be calculated. These regional optical density ratios will be statistically compared between THC-treated and control groups and between high and low THC-treated groups to assess the THC-induced changes in regional glucose uptake. Drug-induced alterations in glucose uptake have been used to assess the effect of the drug on regional brain metabolism: specifically, on neuronal or synaptic activity in the region. THC-induced changes in 2DG labeling will help elucidate the mechanism of THC action on the central nervous system. These results will suggest brain loci for further investigations into acute THC mechanisms, putative differences between acute and chronic mechanisms, and regional co-localization of THC receptors and brain metabolism.

This project will examine the interaction between sex hormones, opiate receptors and endogenous opioid compounds in the medial preoptic area (MPOA) of the rat during perinatal development and during adult life. Opiate receptors in this region exhibit a sex difference shortly after birth and respond dynamically to hormonal changes during the estrous cycle of females. The development of MPOA opiate receptors may be sex hormone dependent. This project will examine the ontogeny of MPOA opiate receptors by quantitative densitometry of [3H]naloxone autoradiographs of the MPOA from intact male and female rats from the time of birth to 6 days of age (the period during which the sex difference develops), and in castrated or tamoxifen-treated males and testosterone- or dihydrotestosterone-treated females during the early postnatal period. The ontogeny of the endogenous opioid compounds, methionine- and leucine enkephalin and Beta-endorphin, will be examined during the early postnatal period using immunohistochemical procedures. Autoradiographic techniques will also be used to study the influence of various sex hormones which mimic the estrous cycle on MPOA opiate receptor binding in adult gonadectomized females and males. The adult hormonal manipulations will elucidate the hormonal mechanism involved in opiate receptor density variations during the estrous cycle and examine the "fixed" or dynamic nature of opiate receptors in the male hypothalamus. Endogenous opioid systems may be involved in the control of reproductive behavior and/or hormonal cyclicity. These investigations of the ontogeny and adult function of hypothalamic opioid systems represent an initial step in our understanding of the control mechanism. The results may elucidate the mechanism of normal and aberrant reproductive function and of narcotic effects on reproductive function.

chemical structure function; brain metabolism;

This project will quantitatively assess the effect of opiate compounds or opiate antagonists on neuronal development in the rat medial preoptic area of the hypothalamus (MPOA). The results will elucidate the mechanism of normal neuronal growth, the influence of endogenous opiates on brain growth, the consequence of prenatal narcotic exposure on brain development, and putative aberrant developmental mechanisms which could lead to mental retardation. Time-pregnant female rats will be given morphine sulfate (either 20 mg/kg/day, 10 mg/kg/day, or 2 mg/kg/day), morphine and naloxone (10 mg/kg/day and either 20 mg/kg/day (high antagonist dose) or 2 mg/kg/day (low antagonist dose), respectively), naloxone (either 20 mg/kg/day, 5 mg/kg/day, or 2 mg/kg/day) [D-Ala2]Met-enkephalinamide (either 20 mg/kg/day, 10 mg/kg/day or 5 mg/kg/day) or saline vehicle by subcutaneously-implanted, four-week duration osmotic minipump beginning at postinfertilization day 12. In addition, a group of pups born to control mothers will be placed with morphine-treated mothers and killed at postfertilization days 32 and 82 and additional groups of prenatally-morphine-treated pups will be cross-fostered to control mothers and reared to postfertilization days 32 and 82. Thus, animals will have had either gestational, gestation-lactational, or lactational exposure to drugs. Brain samples from both sexes will be removed on postfertilization days 22, 24, 26, 28,32 and 82, and prepared for quantitative microscopy. Analysis of completely impregnated, randomly-selected, Golgi-stained MPOA neurons will utilize a computer-assisted, Quantitative Morphometry System to obtain data on neuronal size and pattern and extent of dendritic arborization. These data will be compared across age, sex, and experimental condition to determine the effect of opiates or opiate antagonists administered during the critical period of sexual brain differentiation on MPOA neuron growth and development. The mechanism of action of opiates will be investigated by assaying steroid hormone levels, MPOA opiate receptor density, and endogenous hypothalamic opiate levels in similarly-treated animals.

This proposal requests funds to upgrade facilities and equipment in the Laboratory Animal Service (LAS) of the University of Hawaii at Manoa. Implementation of the project will extend our ability to monitor and improve the quality of animal care and health in LAS facilities. Ongoing peer-reviewed research and training projects involving animals at the University of Hawaii at Manoa total $5.5 million, most of which is derived from NIH grants. Investigators are pursuing various exciting research ideas such as development of vaccines in non-human primate models, regulation and function of the immune system and mechanisms of brain growth and development. These programs are dependent on the animal resource for a steady supply of healthy experimental animals. Training programs for advanced and professional degrees at the University of Hawaii are complementary to these research programs and are dependent on the quality and efficiency of LAS procedures. Biological research is one of several target areas defined by the University of Hawaii Strategic Plan as an area of excellence, and the University has allocated substantial funds during recent fiscal years to assure that adequate resources are available. LAS animal facilities have been upgraded and enhanced by University allocations of nearly $1 million in recent years; LAS has become an independent entity headed by a veterinary Director trained and certified in Laboratory Animal Medicine. Future funding and staffing commitments by the University enable LAS to meet and exceed NIH Guidelines for animal health and care. Nevertheless, certain improvements, additions and replacements are necessary to create optimal conditions for animal care in LAS facilities. Specifically, funds are requested to replace faulty animal caging throughout the facilities, ensure proper cleaning/sterilization capabilities by purchasing rackwashers for Snyder Hall and Biomedical Building and a sterilizer for Woodlawn Small Animal Facility, providing proper refrigerator/freezer and laundry equipment in core LAS facilities, replacing damaged animal food preparation and cleaning sinks and cabinetry throughout the facility, repairing and upgrading existing cagewasher and sterilizer facilities, and renovating HVAC systems in Snyder Hall and Biomedical Building. The benefits derived from the proposed renovation and consolidation of the animal resource include improved efficiency, reliability and containment as well as enhanced public safety.

The long-term objectives of the project are to assess and determine the mechanism underlying the metabolic and pharmacologic effects of cocaine in neuronal circuits related to the rat nucleus accumbens (NAc), a critical dopaminergic reward region, during chronic treatment and early abstinence. The therapeutic potential of dopamine agonists on metabolic recovery during abstinence will be evaluated, allowing a better understanding of beneficial therapies for cocaine abuse. The specific aims of the project are (1) to characterize the effect of chronic cocaine treatment on functional activity in NAc using markers for glucose utilization and pharmacologic receptors affected by cocaine in this region, (2) to determine the mechanism of chronic cocaine-induced differential activation of NAc, (3) to assess the magnitude and time course of selective reduction of functional activity in NAc during cocaine abstinence, and (4) to study the putative enhancement of metabolic recovery induced by dopamine agonists during this period. The quantitative [14C]2-deoxyglucose (2DG) autoradiographic method will be employed to examine the effect of chronic cocaine treatment on glucose utilization in brain circuits related to the NAc, and following selective neuronal and neurochemical lesions in brain regions, such as medial prefrontal cortex, which are demonstrated to project to the affected portions of NAc. Thus, the underlying mechanism of these metabolic effects of cocaine will be elucidated. Correlation of these regional metabolic data with the results of studies of chronic cocaine treatment on regional dopamine and opiate receptor levels using in vitro receptor autoradiography in adjacent sections from the same brain tissue will further elucidate the underlying mechanism. The effect of pharmacotherapy with dopamine agonists during cocaine abstinence on NAc metabolism will also be investigated using the 2DG method. Together, these studies will determine how chronic cocaine treatment causes NAc metabolic alteration, and will test pharmacotherapeutic efficacy following chronic cocaine use.

sex hormones; reproduction; hormone regulation /control mechanism; opioid receptor; preoptic areas; gonads; progesterone; neuroanatomy; estrus; circadian rhythms; hypothalamus; steroid hormone; estrogens; neurons; neuropeptides; receptor expression; autoradiography; laboratory rat; in situ hybridization;

DESCRIPTION: (Applicant's Abstract) The long-term objectives of the project are to characterize the neuroadaptive events underlying stress-induced alteration and sensitization following cocaine self-administration in forebrain circuits of the rat. An integrated behavioral, neurochemical and molecular approach will be used to elucidate the regulatory changes underlying the effects of stress exposure on cocaine sensitization. The specific aims of the project are: (1) to characterize the time course and the cellular basis of behavioral sensitization induced by cocaine self-administration in corticostriatal circuits, (2) to examine the impact of a salient social stressor on dopaminergic and glutamatergic response to cocaine challenge in the nucleus accumbens (NAc) during cocaine withdrawal, (3) to determine the neural circuits by which acute or chronic social stress can enhance the response to cocaine challenge, and (4) to assess the influence of glutamatergic input on stress induced alteration of cocaine response by examining changes in the expression of glutamate receptor mRNA and by assessing the effect of NMDA receptor blockade during chronic social stress exposure on the response to a subsequent cocaine challenge. Rats will be implanted with intravenous catheters, then exposed to chronic social stress or cocaine self-administration followed by 0, 3 or 21 days of withdrawal. Cellular response to acute intravenous cocaine challenge with or without prior acute stress will be examined in mesocorticolimbic and other neurons using in situ hybridization histochemistry (ISHH) with oligodeoxynucleotide probes complementary to the sequence encoding c-fos to detect cellular activation, which will be correlated with motor activity data obtained from the same animals. In vivo microdialysis will be used to examine extracellular levels of dopamine, glutamate and cocaine, and their metabolites following cocaine challenge. The influence of chronic stress or cocaine exposure on glutamate receptors will be examined using ISHH to assess NMDA and AMPA receptors in order to differentiate and localize selective regional effects. Together, these studies will examine the functional link in neural circuits between stress and sensitization during cocaine withdrawal, which could result in cocaine craving and relapse to cocaine self-administration. The results will elucidate the mechanism(s) underlying reinforcing and stressful stimuli as well as the cellular and molecular alterations which develop during cocaine withdrawal. This know]edge will permit the design of better approaches to treat cocaine dependence.

DESCRIPTION: (Adapted from applicant's abstract) Symptoms of schizophrenia include sensory flooding and cognitive fragmentation which are thought to be the result of a deficiency in sensorimotor gating. Sensorimotor gating can be measured in humans and animals using a quantitative test that assesses reduction of the startle response to a pulse stimulus after presentation of a weaker prepulse stimuls. Prepulse inhibition (PPI) of the startle response is disrupted in patients with schizophrenia. An identical test has been used in animals to elucidate the mechanisms underlying PPI. Acute administration of dopamine agonists placed into the nucleus accumbens (NAc) disrupts PPI by stimulating dopamine D2-like receptors. This effect is blocked by antipsychotic pretreatment or by local D2-like receptor antagonist administration. Several other neurotransmitter systems are implicated, but the mesolimbic dopamine system remains an important substrate for these effects. The long-range goal of the project is to determine the specific cellular and molecular targets related to sensorimotor gating deficits in schizophrenia. An animal model of sensorimotor gating will be used to examine the neuroadaptive responses leading to recovery of PPI disruption following chronic drug treatment. In contrast to the disrupting effect of acute dopamine agonist treatment, we have shown that repeated treatment attenuates the behavioral disruption by desensitizing dopamine D2-like receptors. The specific aims of the project are (1) to determine whether functional downregulation of dopamine D2-like receptors induced by chronic treatment with a D2-like receptor-selective agonist attenuates disruption of PPI in a time-and dose-dependent manner by actin in the NAc,(2) to ascertain whether reduction of inhibitory G proteins in the Nac is sufficient to attenuate disruption of PPI, and (3) to define and characterize the subset of NAc neurons whose function is altered following chronic D2-receptor agonist treatment using retrograde labeling and immunohistochemical detection of an immediate early gene marker. Together, these studies will produce novel data on the circuitry and signaling mechanisms underlying the recovery of PPI disruption in rodents. The results will advance our understanding of sensorimotor gating deficits win schizophrenia from the regional level to the cellular and molecular levels, and may provide novel therapeutic targets for future development.

Understanding how the entire system works within the context of the intact functioning animal core is a central goal of Neuroscience research. This Core will allow researchers who use genetic and molecular methods to assay the behavioral consequences of drug treatments of mouse mutations. This Core will expand the use of the new Animal Behavior Research Core (ABRC). The ABRC already provides for space (5 behavioral testing rooms), equipment and staff for mouse behavioral testing. However, the single technician slotted for the ABRC cannot provide the support required for the large group of Neuroscientists and thus this Core will be required to hire an additional technician dedicated to the needs of the NINDS-funded investigators. In addition, the application requests funds for some equipment not currently provided for by ABRC or the University (e.g., Ethovision system, MiniMitter monitors with VitalView Software, and a rotarod system). Dr. Ronald Hammer will direct the Core with Dr. Kopin.

DESCRIPTION (provided by applicant): Environmental stressors are thought to trigger the onset or relapse of schizophrenia in vulnerable individuals. Symptoms include sensory flooding and cognitive fragmentation, which are the result of sensorimotor gating deficits. Sensorimotor gating can be measured using a quantitative test that assesses reduction of the startle response to an acoustic pulse stimulus after presentation of a weaker prepulse stimulus. Such prepulse inhibition of the acoustic startle response (PPI) is disrupted in patients with schizophrenia and in rats with dopaminergic abnormalities in the nucleus accumbens and/or prelimbic and infralimbic prefrontal cortex. A stressful social interaction between conspecific animals causes such dopamine dysfunction, and disrupts PPI. The long-range goal of this project is to determine the cellular and molecular mechanisms by which social stress can produce symptoms of schizophrenia. An animal model of sensorimotor gating will be used to examine the neurobiological responses leading to PPI disruption after repeated social stress exposure, initially focusing on the role of prelimbic/infralimbic cortex. The specific aims of the project are (1) to determine the time course of PPI disruption induced by repeated social stress exposure, elucidating the involvement of D2-like receptors in the nucleus accumbens, (2) to ascertain whether tonic dopamine activity in prelimbic/infralimbic cortex is related to social stress-induced PPI disruption, (3) to quantify and characterize the persistent expression of Fos-related antigen(s) in prelimbic/infralimbic cortex following repeated social stress, (4) to characterize the neuroanatomical connections of cortical neurons expressing Fos-related antigens after repeated social stress exposure, and (5) to determine whether social stress-induced elevation of corticosterone level leads to PPI disruption by inhibiting corticosterone synthesis during stress exposure. Together, these studies will produce novel data on the cellular and molecular effects of a salient social stressor, which causes long-lasting sensorimotor gating deficits in rodents, and may trigger the onset or relapse of schizophrenia in patients.

DESCRIPTION (provided by applicant): Symptoms of schizophrenia include various cognitive deficits that are the result of sensorimotor gating deficiency, such as sensory overload, disorganization and thought fragmentation. Sensorimotor gating can be measured using a quantitative test that assesses reduction of the startle response to an acoustic pulse stimulus after presentation of a weaker prepulse stimulus, termed prepulse inhibition (PPI). Normal PPI is disrupted in patients with schizophrenia. An identical test can been used in rats to elucidate the mechanisms underlying PPI disruption, which is produced by dopaminergic abnormalities within the nucleus accumbens. The long-range objective of the project is to determine specific cellular and molecular substrates of PPI regulation and to investigate novel therapies for sensorimotor gating deficits in schizophrenia. An experimental animal model has been used to determine the pharmacology and neural circuitry underlying PPI disruption. This model can predict the efficacy of drugs used to treat schizophrenia. We discovered that repeated treatment with a selective dopamine D2-like receptor agonist reverses PPI disruption in rats, and we described a putative intracellular basis for this PPI recovery. In fact, repeated treatment results in compensatory changes that resemble those produced by atypical antipsychotic drugs. Moreover, this effect occurs selectively in the mesolimbic dopamine system without affecting extrapyramidal brain regions. The proposed efforts will extend our studies of neural substrates underlying PPI regulation by examining the association of molecular changes to the timing of PPI recovery, which will be further characterized using an assay for conditioned avoidance responding. We will also examine the duration of PPI recovery and the effect on phencyclidine-induced PPI disruption. We will investigate the involvement of D2-, D3- and adenosine A2A receptors using selective antagonists, as well as the causative relationship between intracellular cAMP signaling and PPI recovery, using cAMP response element binding protein assays and adeno-associated viral-mediated blockade of cAMP response element binding. Finally, specific target(s) for therapeutic intervention will be identified in characterized neurons of the nucleus accumbens. Together, these studies will elucidate the mechanisms of neural plasticity underlying PPI recovery in rodents, and will provide novel therapeutic targets for schizophrenia.