Samuel A. Deadwyler - US grants

In vitro and in vivo studies of intracellular and extracellular electrophysiology in hippocampus suggest that the role of opioid substances in this structure is a highly strategic one, in that a small number of opiate receptors and enkephalinergic pathways exert a powerful influence on the excitability of a large proportion of pyramidal and granule cells. This laboratory was among the first to demonstrate the excitatory influence of opiates in the CAl field of hippocampus. We have subsequently reported that opiates (morphine) and opioid peptide analogs produced differential effects on intracellular recordings of CAl cells depending upon the region of the cell to which the opiate is applied (basal dendrites and somal region vs apical dendrites). Additionally, it was shown that inhibitory influences appear to be enhanced in the CAl region of tolerant rats. These findings indicate a selective role for opiates as disinhibitory in the hippocampus. In addition to the above CAl influences, we have shown recently that opiates and opioid peptides enhance the transmission of sensory information into the dentate gyrus via the perforant pathway. Both anatomic and neurophysiologic investigations are proposed to continue to examine these modulatory influences. In addition, newly established techniques will be used to evaluate the responsiveness of the dentate gyrus synaptic pathways in the freely behaving animal during acute and chronic opioid administration in order to determine the functional significance of endogenous opioid substances in opiate tolerant and nontolerant animals.

The specific aims of the research are to: 1. Continue to characterize the acute effects of delta-9-THC on auditory evoked neural activity in the dentate gyrus of the unanesthetized behaving rat. These experiments consist of dose-response analyses of the effects of delta-9-THC on synaptically identified auditory evoked potentials and dentate granule cell single unit discharges during the performance of both simple and complex auditory discrimination tasks. 2. Initiate studies on the effects of repeated daily administration of low and high doses of delta-9-THC on auditory evoked activity in the dentate gyrus, and dentate g-cell activity during prolonged performance of the auditory discrimination task. In these experiments we will determine whether tolerance to the acute effects of delta-9-THC develops with respect to these measures following either low or high doses of the drug when given repeatedly over several days. Specialized analyses of trial-by-trial variations in amplitude of these evoked responses indicate that event related correlates of cognitive processes may be altered by high dose levels of delta-9-THC. 3. Determine the effects of withdrawal from long-term exposure to both low and high doses of THC on the auditory evoked dentate gyrus neural activity. In addition, investigations of residual effects of delta-9-THC on these neurobehavioral correlates will also be examined following prolonged exposure to very high dose levels.

toxin metabolism; psychotropic drugs; learning; neural plasticity; hippocampus; Cannabis; cannabinoids; cognition; sensory discrimination; drug withdrawal; pyramidal cells; corticosterone; behavioral medicine; brain metabolism; biological models; histopathology; laboratory rat; adrenalectomy; drug administration rate /duration; dosage; electrophysiology; auditory discrimination;

There are two main purposes of the proposed research. The first is to continue to investigate the acute effects of single isolated exposure to low concentrations of cannabinoids on central nervous system function in a behaving rat model. Research in the prior funding period has shown that the psychoactive ingredient in marijuana, delta-9-tetrahydrocannabinol (THC) produces marked changes in the sensory responses of the hippocampus in the form of suppressed auditory evoked potentials and unitary discharges to a conditioned tone stimulus following a single injection of 1.0-2.0 mg/kg of delta-9-THC. This disruption coincides with a slowing of behavioral responding and a decreased accuracy of performance in a successive two-tone frequency discrimination task. Evidence was obtained which showed that recovery of both behavior and electrophysiological responses in the hippocampus is fairly rapid occurring within 2-4 hours following initial exposure. In the current research plan these effects will be further investigated both with respect to specificity of cannabinoid action and differential effects on identified neural processes within the dentate action and differential effects on identified neural processes within the dentate gyrus of the rat hippocampus. The second purpose of the proposed research plan is to continue to investigate the effects of long-term exposure to moderate dose levels of delta-9-THC. In prior experiments it was shown that rats developed a rapid tolerance (within 5-7 days) to 10 mg/kg doses of THC in terms of the electrophysiologic and behavioral measures described above. Continued exposure to the compound at the same 10 mg/kg dose for a total of 25-30 days produced no change in tolerance. Abstinence withdrawal effected a "reversal" of tone evoked potential amplitudes relative to tolerant and control levels. However, it was delayed at least 5 days and extended for at least 16 days after termination of chronic THC treatment. This "residual" rebound-like effect on well characterized hippocampal mechanisms resembled closely the acute effects of the compound in terms of the directional changes produced in sensory evoked potential amplitudes. Studies are described in the current research plan to investigate more thoroughly the neurobiological and behavioral implications of these residual occurrences following long-term exposure to the psychoactive ingredient in marijuana.

The objectives of the research to be conducted are directed at establishing the consequences of long-term exposure to delta-9- THC, the psychoactive ingredient in marijuana, on central nervous system function. The neural system to be investigated in these studies is the hippocampus of the awake freely moving animal. Experiments are described which will examine the effects of intermediate (30 days) and very long-term (8 month) daily exposure to delta-9-THC on well characterized electrophysiological correlates of hippocampal function during the performance of both auditory and spatial discrimination tasks. The research plan is designed to systematically evaluate the effects of delta-9-THC and other cannabinoid derivatives on synaptic and cellular responses in the hippocampus during controlled behavioral performance both during and after long- term exposure to these cannabinoids substances. In particular, a portion of this research strategy is designed to evaluate the implications of recently demonstrated pathological changes in the hippocampus resulting from 8 month exposure to delta-9-THC in rats. The two behavioral protocols utilized to assess these changes have been shown to provoke specific changes in hippocampal cell firing.

Research is described in this application to continue investigations of the effects of delta-9-THC, the psychoactive derivative of marijuana, on neurobehavioral mechanisms involved in the perception, registration, and retrieval of trial specific sensory information of rats. Past research accomplished in the last funding period has indicated that behavioral tasks sensitive to destruction of the hippocampus and related brain regions are disrupted in dose-dependent fashion by delta-9-THC. Concomitant recording of hippocampal electrical activity in the form of well characterized sensory evoked potentials and cellular discharges were also disrupted by the same dose levels of delta-9-THC. Experiments are proposed to continue research in two main areas of behavioral influence of delta-9-THC initiated in the last funding period: 1) evaluation of effects of delta-9-THC on perceptual processes utilizing signal detection methodology in order to evaluate delta-9-THC produced deficits in registration of sensory information, and in separate but parallel experiments 2) investigation of the selective influence of delta-9-THC on the registration and retrieval of trial specific information in behavioral tasks which require hippocampal involvement utilizing delayed match to sample and variations of those behavioral paradigms. In these studies, neural correlates of successful behavioral performance have been and will continue to be examined in terms of dose-dependent influences of delta-9- THC. A major purpose of the project is to isolate and characterize neurobehavioral events which are 1) critically involved with information processing as indicated above, and 2) selectively influenced in "dose- dependent" manner by acute doses of delta-9-THC. Further extension of this research initiated in the last funding period includes evaluation of the above described behavioral and electrophysiological endpoints following chronic exposure to delta-9-THC over a 35-day treatment period. Preliminary evidence indicates a differential timecourse of tolerance development in the above behavioral measures. This suggests that there exists a hierarchy of sensitivity of neurobehavioral mechanisms to long term or repeated exposure to delta-9-THC which cannot be predicted from reactivity exhibited following a single acute injection.

cocaine; reinforcer; dopamine; neuropharmacology; neurophysiology; brain electrical activity; self medication; psychopharmacology; neurochemistry; neural information processing; action potentials; nucleus accumbens; tegmentum; frontal lobe /cortex; drug abuse; neuromuscular system; antidromic impulse; stimulus generalization; operant conditionings; behavior test; electrophysiology; laboratory rat; drug delivery systems; videotape /videodisc;

This is a renewal application of a research program in its third year of electrophysiological assessment of the role of cannabinoid receptors in brain. In the first 2.5 years of the project an extensive series of studies has been completed in which the effects of cannabinoids have been characterized in cultured hippocampal pyramidal cells. Several types of investigation including patch clamp recordings of whole cell currents and biochemical assessments of cellular mechanisms on the same type of hippocampal neurons have confirmed that cannabinoids modulate voltage dependent potassium A-current (IA) in these neurons. The effect is dramatic in that the capacity of the A-current channel is greatly altered by cannabinoids binding to the receptor. In neurons exposed to several different types of cannabinoid receptor ligands, the voltage dependence of IA was shifted positive by as much as 20 mV, indicating that cannabinoids are capable of profoundly altering the excitability of neurons and axon terminals. Studies went on to verify that, as required, this process was g-protein mediated (Gj) and was sensitive to concentrations of cannabinoid ligand that produced a corresponding increase in low Km GTPase activity in hippocampal slices. A more important finding was that modulation of the voltage dependence of IA was dependent on decreased cellular levels of cAMP produced by the well known inhibitory effect of cannabinoids on adenylyl cyclase. Increasing cellular cAMP either directly (8-bromo-cAMP) or indirectly through stimulation with forskolin, produced a reciprocal negative shift in IA voltage dependence from resting levels. The negative shift in IA produced by forskolin could be reversed by cannabinoid activation of the receptor that inhibited the forskolin stimulated adenylyl cyclase activity. Further studies showed that the cAMP dependent decrease in IA produced by cannabinoid receptor occupancy was likely mediated by alterations in phosphorylation status of the channel, since protein kinase A inhibitors effected a positive shift in IA voltage dependence and at the same time reduced or blocked the effects of cannabinoids on IA Thus the above studies show that cannabinoids alter the responsiveness of hippocampal neurons in culture via a cAMP dependent change in the voltage dependence of potassium A-current. Studies proposed in the next phase of the project extend these investigations to 1) the use of the newly discovered receptor antagonist SR141716A (Sanofi), 2) the role of the putative endogenous cannabinoid receptor ligand "anandamide" in this process. 3) determination of the interactions between decreased conductance in N-type calcium channels and the alteration in voltage dependence of IA produced by cannabinoids, 4) studies of the effects of cannabinoids on IA in cells from slices of adult hippocampus and 5) the use of antisense probes to determine the type of potassium A channel modulated by the cannabinoid receptor.

The research program outlined in this proposal supports the application for renewal of a Research Scientist Award to Dr. Sam A. Deadwyler, Professor of Physiology, Bowman Gray School of Medicine. The objectives of the research to be conducted over the five year period of the award are directed toward establishing the acute and chronic effects of exposure to cannabinoids and cannabimimetic substances as well as the neural effects of cocaine and psychomotor stimulants. Four different individual research projects are supported in this program, each of which deals with a separate issue concerning cannabinoid and/or cocaine action in specific brain regions. Two of the projects are concerned with the actions of cannabinoids and recently developed cannabinoid analogs in the hippocampus and related brain regions, under acute and chronic exposure conditions. It has been firmly established in the preceding period of this award that cannabinoid receptors in the hippocampus play a major role in memory processing and that long term exposure to these substances produces a considerable degree of tolerance to the initial disruptive effects. The research strategy in this application seeks to understand the mechanisms of cannabinoid disruption of memory processes in terms of both the cellular and neural circuit components of memory formation in the hippocampus. A third project examines the effects of cannabinoid and cannabinoid analogs on individual cellular mechanisms in order to determine precisely the conductance changes effected by cannabinoid receptor occupancy. In the past period of the award it has been discovered by the candidate that there are at least 2 candidates for this effect, increased potassium conductance, and increased chloride conductance on hippocampal and cortical neurones. A third possibility, decreased calcium conductance was reported also in a separate laboratory. The fourth and most recent project in the research program deals with the electrophysiological investigation of neural correlates of cocaine self-administration in rats. This project applies technology developed in the first two projects on hippocampal investigation in vivo to an analysis of many neurone activity in the nucleus accumbens and ventral tegmental dopamine cells in rats trained to self-administer cocaine. This project is primarily concerned with the elucidation of the neural processes underlying the rewarding properties of psychomotor stimulants and the neurophysiological relationship between these structures and the hippocampus, a major source of afferent projections to the nucleus accumbens. The award will allow Dr. Deadwyler to continue to devote maximal effort to the above research objectives, freeing him from otherwise conflicting departmental duties and teaching responsibilities. In addition it will provide the opportunity for him to make extensive visits to other laboratories for the purpose of establishing new techniques and collaborative arrangements not available in the current environment.

Research is described in this application to continue investigations of the effects of delta-9-THC, the psychoactive derivative of marijuana, on neurobehavioral mechanisms involved in the perception, registration, and retrieval of trial specific sensory information of rats. Past research accomplished in the last funding period has indicated that behavioral tasks sensitive to destruction of the hippocampus and related brain regions are disrupted in dose-dependent fashion by delta-9-THC. Concomitant recording of hippocampal electrical activity in the form of well characterized sensory evoked potentials and cellular discharges were also disrupted by the same dose levels of delta-9-THC. Experiments are proposed to continue research in two main areas of behavioral influence of delta-9-THC initiated in the last funding period: 1) evaluation of effects of delta-9-THC on perceptual processes utilizing signal detection methodology in order to evaluate delta-9-THC produced deficits in registration of sensory information, and in separate but parallel experiments 2) investigation of the selective influence of delta-9-THC on the registration and retrieval of trial specific information in behavioral tasks which require hippocampal involvement utilizing delayed match to sample and variations of those behavioral paradigms. In these studies, neural correlates of successful behavioral performance have been and will continue to be examined in terms of dose-dependent influences of delta-9- THC. A major purpose of the project is to isolate and characterize neurobehavioral events which are 1) critically involved with information processing as indicated above, and 2) selectively influenced in "dose- dependent" manner by acute doses of delta-9-THC. Further extension of this research initiated in the last funding period includes evaluation of the above described behavioral and electrophysiological endpoints following chronic exposure to delta-9-THC over a 35-day treatment period. Preliminary evidence indicates a differential timecourse of tolerance development in the above behavioral measures. This suggests that there exists a hierarchy of sensitivity of neurobehavioral mechanisms to long term or repeated exposure to delta-9-THC which cannot be predicted from reactivity exhibited following a single acute injection.

Our understanding of the role of hippocampus in learning and memory has undergone many changes in recent years. Of particular interest is the realization that hippocampus is essential to encoding specific relationships between behavioral or cognitively relevant events. For the past 5 years, this project has examined how the process of encoding and recalling information in hippocampal ensembles is utilized in short-term (i.e delayed-non-match/match-to-sample [DNMS/DMS]) memory and how that process is disrupted following exposure to the psychoactive drug, marijuana (i.e. cannabinoids). The latter observation is not trivial since the cannabinoid has a high receptor density in hippocampus and with its endogenous ligands is capable of "sculpting", refining or even blocking hippocampal-dependent memory. In the last funding period several technological and computational methods were developed and applied to hippocampal neuronal recording which allowed insight into how ensembles of hippocampal neurons encode information. This Research Project will continue to investigate hippocampal mechanisms of information processing by: (1) characterizing the transference of information within the ensemble from the encoding to the decision- making phase as required to select the appropriate trial specific behavioral response; (2) examining the basis of two types of behavioral errors which likely result from insufficient encoding of transference of such information; (3) further analyzing the topographical organization within the hippocampus with respect of anatomic distribution of different functional cell types for different types of tasks; (4) determining the significance of cannabinoid receptor modulation if GABAergic interneuron activity in hippocampus and its relation to previously reported dose-dependent attenuation of information encoding in DNMS/DMS tasks by acute cannabinoids, and (5) incorporating electrophysiological analyses in the assessment of behavioral tolerance to the acute effects of cannabinoids on memory that develop following chronic exposure to the drug.

DESCRIPTION: (Applicant's Abstract) The research program outlined in this proposal supports the application for renewal of a Research Scientist Award (K05) to Dr. Sam A. Deadwyler, Professor of Physiology, Bowman Gray School of Medicine, Wake Forest University. The objectives of the research over the next 5 years of the award are delineated in the Research Plan and cover three distinct research areas, each supported by a separate individual research grant. The 3 areas include: 1) effects of cannabinoids on hippocampal memory processes, 2) mechanisms of action of cannabinoid receptors, and 3) neurophysiological assessment of cocaine self-administration. These three major areas of research have been under investigation throughout the duration of the last award period. Each of the grants supporting that research has been renewed and is currently active until the year 2000. The overall strategy of the research proposed is to understand at the cellular and molecular level the effects of cannabinoids and cocaine on neural substrates of behavior and cellular function. With respect to cannabinoids, the effects of both the endogenous cannabinoid (CB1) receptor ligand "anandamide" and the exogenous receptor analogs will continue to be assessed with respect to their influence on memory processes mediated via the hippocampus in the rat. In addition, investigations of the chronic effects of these substances are now underway to determine the mechanisms of tolerance and dependence in the cannabinoid system in the brain. The third area of study focuses on the effects of cocaine on neurons located in the nucleus accumbens (NA), an area of the brain believed to be critical for establishing and maintaining reinforced behaviors. Specifically, these studies will continue to investigate the firing patterns of neurons in the NA in animals self-administering cocaine in order to define those conditions under which the drug alters neuronal activity to support addictive behavior. The renewal of this award will allow Dr. Deadwyler to continue to devote maximal effort to the above research objectives, freeing him from other departmental and teaching responsibilities. It will also provide him with the opportunity to expand his research on the above topics to include more advanced techniques and to make visits to other laboratories to arrange collaborations on topics of mutual interest with other well known researchers in the field.

DESCRIPTION: (Applicant's Abstract) Continued investigations of the neurophysiological basis of the role of the cells in the nucleus accumbens in cocaine substance abuse are proposed in this application to extend support of a research program previously incorporated in the NIDA Center for Substance Abuse at BGSM. Due to the success of this Project it can no longer be adequately supported within the Center and is being submitted as an R01 application to pursue a more extensive research program. The proposed research program is based on several prior findings regarding the activity of neurons in the rostral pole, shell and core of the nucleus accumbens recorded in rats during either appetitive (water) reinforcement or cocaine self-administration. Having determined in prior studies that there are differential firing patterns of accumbens neurons related specifically to different aspects of the reinforced behavioral response in both reinforcement contexts (water and cocaine), the current research plan will provide a means of further differentiating accumbens cell firing with respect to different types of reinforcers. This is a critical issue because it is not known whether some aspects of cocaine self-administration behavior is maintained by aversive as well as positive reinforcing stimuli. The studies proposed here will therefore make a thorough assessment of the firing characteristics and behavioral correlates (via spike-triggered digitized video) of the same accumbens neurons within 2 of 3 distinctly different operant reinforcement paradigms (appetitive, aversive and cocaine self-administration). In each of the three contexts the motor components of the response will be nearly identical, with the exception that the operant response (lever press) will be an avoidance or escape response in the aversive motivational paradigm. It is important that the same neurons in the accumbens be recorded in at least two of these three conditions in order to perform population analyses on moderate to large numbers (15-40) of accumbens cells within each paradigm. Ensemble analyses will be performed to extract facets of accumbens neural network organization specific to and or in common with each reinforcement context. These analyses will also be performed on spike-triggered digitized video images of behavioral sequences associated with each of the previously identified types of accumbens neurons. The program will provide important information regarding positive and/or negative motivational factors involved in cocaine self-administration.

Project 0003 in the Center for Neurobiological Investigation of Drug Abuse will investigate the role of nucleus accumbens (NA) and other brain regions in the control of cocaine self-administration in non-human primates. These studies are designed explicitly to determine whether encoding of different phrases of the internal between drug infusions in the monkey provokes similar patterns of discharge in NA cells as demonstrated in the rat-self administration model in the prior funding period of the Center. The extensions of those studies to the non-human primate provides for a more informative means of assessing and characterizing single and multi-neuron activity in several different brain regions simultaneously and under more complex circumstances of stimulus and context control than could be achieved in the rodent. Experiments are designed to determine what brain regions beside the NA are critically activated or inhibited in terms of single neuron firing changes, during cocaine self-administration and during assessment of potent cocaine analogs (tropanes) in similar paradigms. Candidate brain regions to be explored are those maximally affected by either acute cocaine injections or self-administration of cocaine as shown by imaging of 2-DG metabolic maps in monkeys. These include, NA (shell and core), dopaminergic cells in the ventral tegmental area (VTA), the amygdala (medial and basal), the hippocampus (CA1 and subiculum) the cingulate and orbital frontal cortex. These areas in most cases supply afferent projections to the NA in the monkey and therefore will be examined simultaneously with the NA for evidence of a functional underlying substrate. Further studies will be conducted to determine extent to which NA and other regional cell firing patterns are controlled by the associative properties of stimuli that have been paired with drug delivery. In particular studies will investigate whether the stimuli, or the contexts in which stimuli occur, are more relevant to the associations formed by pairing with drug versus appetitive reward. In addition when such associations are formed, are those stimuli paired with drug reinforcers more capable of modifying behavior outside the original reward context in comparison to non-drug related reinforcers. Such differential reinforcing capacity should be reflected as differential cell firing patterns in the implicated brain regions, providing further insight into how drugs like cocaine affect motivational processes and gain control over behavior.

Chronic administration of cannabinoids (delta 9-THC) result in an unusua l degree of tolerance in which physiological and behavioral functions revert to near normal (control) levels but important second messenger pathways remain altered Understanding of the cellular mechanisms involved in the development of tolerance to chronic cannabinoid treatment will provide insight into processes responsible for tolerance to other frequently administered or abused drugs. Investigation of these processes can benefit from implementation of new strategies to quickly examine potentially altered expression profiles across large numbers of genes in order to determine those that might be involved in producing tolerance. Preliminary data using large scale cDNA microarray screening technology revealed that a significant number of genes related to various aspects of cannabinoid system function showed altered expression patterns across a 21 day treatment period. These patterns were assessed in two different brain regions, hippocampus and cerebellum and revealed similar time courses to cannabinoid (CBl) receptor and second messenger signaling changes. It is hypothesized that, common systems and their respective molecular counterparts may participate in adaptation to chronic drug exposure but this may differ across particular brain regions specific cell types. We propose to adapt and extend use of cDNA microarrays to delineate further the changes in altered gene expression during chronic exposure to cannabinoids. Specific Aim 1 will characterize the differential gene expression patterns in these and 3 other brain regions at different time points (i.e. days) during chronic cannabinoid treatment. Specific Aim 2 will take the analysis one step further by assessing changes in gene expression profiles during precipitated withdrawal from chronic cannabinoid exposure. Specific Aim 3 will assess the specific brain regions in which these gene expression changes occur utilizing in situ hybridization to determine the regional and cellular distribution of genes whose expression is changed during chronic exposure and during precipitated withdrawal (Specific Aims 1 and 2).

DESCRIPTION: (Adapted from the Investigator's Abstract) The cannabinoid receptor/transmitter system is under intense investigation as to the delineation of its many roles in modulating and controlling neural processes which range from analgesia to memory (Howlett 1998). The research proposed in this application is a continuation of an ongoing program to study the electrophysiological and neurobiological factors responsible for cannabinoid receptor mediated effects in the CNS and in particular in the hippocampus. The current research program is investigating three major areas of cannabinoid receptor function, 1) which specific subtypes of potassium channels are responsible for the types of currents previously shown to be modulated by cannabinoid receptor-G-protein coupling to different cellular signaling processes, 2) to determine the interaction between GABAergic mechanisms and cannabinoid receptor activation in culture and in slices of hippocampus and 3) to investigate in further detail the protective effect of cannabinoids with respect to NMDA and mitochondrial models of neurotoxicity. Each of the above aims is directly related to a fourth objective of the program which is to evaluate and characterize the electrophysiological consequences that occur from tolerance to cannabinoids following chronic exposure. In prior studies this laboratory it was shown that repeated administration of cannabinoid agonists resulted in desensitization of the cannabinoid receptor-to-G-protein coupling as evidenced by decreased receptor stimulated GTP-gamma-S binding (Sim et al., 1996). In this project we propose to examine the changes in the above cannabinoid mediated processes (K-channel and GABA-B receptor modulation) in a cellular model of tolerance using primary cultures of hippocampal neurons. These effects will then be compared with recordings made in hippocampal slices obtained from tolerant animals. Information obtained from those studies will be applied to investigations of the protective actions of cannabinoids in cellular models of neurotoxicity in which hippocampal cells in culture are protected if exposed to 2 different cannabinoid receptor agonists (WIN 55,212-2, CP55940).

This subproject will continue to investigate cellular correlates of motivation and cognition in nonhuman primates as they relate to influences of cocaine on performance and behavior under different training paradigms. The subproject has established the means to record single and multiple single unit activity in different brain regions while rhesus macaque monkeys are engaged in complex behavioral and cognitive tasks. The first task is a Go-Nogo paradigm in which both juice and cocaine are utilized as reinforcers for either initiating or withholding responses to specific stimuli within the trial. There are three variations of reward conditions that are employed in the task each testing a different aspect of juice vs cocaine rewarded responding. Neuronal activity is recorded from the ventral and dorsal striatum, frontal cortex and substantia nigra/VTA complex in monkeys that are well trained to perform the tasks. In the ventral striatai area we have found that neurons segregate into distinct classes to encode all the features of the task, including specific neurons that encode trials on which cocaine is the reinforcer and not those trials in which juice is delivered. The second major task is a cognitive test of shortterm memory in which stimuli are presented in a delayed-match-to-sample paradigm. Monkeys are trained to respond to identify a sample object from 2-6 other stimuli in the match phase at delays of 1-30 sec. Performance decreases both as a function of length of delay and number of items to choose from during the match phase of the task. Neurons are recorded from the hippocampal formation and frontal cortex and juice or cocaine presented as rewards on a trial-to- trial basis. Changes in discharge pattem as a function of cocaine vs juice reinforcement will be assessed. In addition, the effects of long-term exposure to cocaine (100 days) in the above two types of behavioral paradigms will be assessed with respect to changes in firing patterns of neurons with task-relevant firing tendencies. Monkeys will be withdrawn for a period of 30 or 60 days and saline delivered instead of cocaine on drug rewarded trials to extinguish prior task-associated cues. After withdrawal, cocaine reinstatement will be implemented in which drug trials (and the original stimuli associated with drug rewards) will be re-invoked. Alterations in neuronal firing patterns of identified cell types in the above structures will be assessed relative to initial exposure to cocaine to determine changes in system sensitivity following long-term exposure.

[unreadable] DESCRIPTION (provided by applicant): The purpose of this research project is to assess the manner in which information processing in brain structures of nonhuman primates is re-organized by the introduction of and sustained exposure to cocaine as a reinforcer for complex cognitive tasks. It has long been implicitly assumed in analyses of human drug addiction that substances which are abused somehow take over normal reinforcement mechanisms in the brain, diverting such "reward pathways" to the control of drug seeking activities. Using a well-characterized short-term memory/executive function paradigm (multi-object delayed match to sample [DMS] task) studies will determine how cognitive processing is affected by acute and longterm exposure to cocaine as a reinforcer in this task. This primate model of cognitive function has been characterized in recent PET imaging and electrophysiological recording studies from this laboratory. On the basis of that work three important brain regions, medial temporal lobe (MTL), dorsal prefrontal cortex (DPFC) and the dorsal and ventral striatum (D/VStr), shown to be engaged during task performance, will be assessed for effects of cocaine on cognitive processing. Aim 1 will determine neuronal firing characteristics in these three brain regions associated with performance of the DMS task and will identify single neuron correlates of low vs. high cognitive load trials. Aim 2 will examine how these neural correlates change when the task is performed for cocaine injections delivered as the trial reinforcer in comparison to normal appetitive (juice) rewards. Aim 3 will extend the above analyses to animals that are repeatedly exposed to conditions in which cocaine and juice reinforcers are implemented in the same random manner during day-to-day testing for a period of six months in order to assess cumulative changes in DMS responding and associated neuronal correlates over a time period in which performance is sustained at criterion levels by both reinforcers. The final Aim 4 will assess the effects of stress on cocaine vs. juice reinforced DMS performance and associated neural correlates of cognitive load (Aim 1), induced by a method of sleep deprivation perfected for nonhuman primates in this laboratory. [unreadable] [unreadable] Relevance: In a society that is evolving more and more toward increased stress and demand on its citizens the individual level of cocaine abuse is a major health care problem. Such behavior eventually results in inability of the addict to cope with the complex nuances of a complex technologically-based work place. Turning to drugs is a natural course of action for pressured, overworked and under employed personnel.How cocaine use advances to addiction in this context is directly related to effects on cognition, reasoning and decision making. Therefore understanding how cocaine modulates and gradually over time eliminates effective cognitive processing, as studied here, is of primary importance in the prevention of drug addiction. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Project will continue to pursue the important issue of defining the role of endocannabinoids in memory and related cognitive processes as determined by the effects on information encoding in hippocampal neurons. The Proposal describes studies that address this question from two different strategic but convergent technologies. Experiments conducted in vitro in hippocampal slices are described that will continue to delineate the minimal, synapse mediated, depolarizing conditions required to release endocannabinoids from hippocampal neurons that act in a retrograde manner to suppress release of inhibitory (DSI) or excitatory transmitters (DSE) onto those same cells. These findings will be related directly to intracellular calcium dynamics as an intermediary step required for release by depolarizing influences. A second complementary strategy has been developed on the basis of our recent discovery that endocannabinoids are involved in the encoding of information by ensembles of hippocampal neurons during performance of a well characterized short-term memory task in rats. This second approach utilizes the fact that hippocampal ensembles have distinct firing patterns which are altered when endocannabinoids are released on some occasions during performance of the task. Experiments will utilize a unique closed loop testing paradigm that ties hippocampal encoding of trial specific information to performance as a function of the length of delay on the same DNMS trial. The influence of released endocannabinoids on this encoding process and its behavioral consequences will be examined using this paradigm in association with pharmacological manipulation of processes involved in endocannabinoid release and CB1 receptor mediated cellular processes. The two strategies will be combined to fully disclose the nature of endocannabinoid actions in hippocampus by employing recently developed patterned multisite electrostimulation that mimics endocannabinoid release in vitro to alter performance in the same manner as when endocannabinoids are released spontaneously in the behaving animal.Relevance: Because cannabinoids are one of the most abused substances in modern society, an understanding of how they produce their well-known memory impairments that occur during recreational and addictive use is of paramount importance to the eventual control of this debilitating influence. The outcomes of these studies will define not only what type of neural processes are susceptible to disruption by cannabinoid use, but also when the encoding of information by those processes is susceptible to such influence.

DESCRIPTION (provided by applicant): The purpose of this research project is to assess changes in the brains of nonhuman primates (NHPs) during information processing in a cognitive task that involves exposure to cocaine as a reward for successful performance. Evidence is presented that structures in the brain of NHPs are affected by the introduction of cocaine as a reward on signaled trials within a session where appetitive (juice) rewards are also available. The Project will utilize a well-characterized short-term memory/executive function paradigm consisting of a multi- object delayed match to sample (DMS) task. The task provides for testing whether the "cognitive load" on any given trial is directly related to performance in association with the functional neuronal activity in prefrontal cortex (PFC), medial temporal lobe (MTL) and dorsal and ventral striatum (Str) that is imaged from the same behavioral sessions. The proposed studies will determine how cognitive processing is affected by acute and long-term exposure to cocaine in this paradigm and how agents currently utilized in human clinical studies alter the detrimental effects of cocaine on cognitive function. Aims 1 and 2 will assess and characterize PET imaging of 18FDG brain metabolic activity in the above three brain regions and determine the effects of cocaine rewards on performance of the DMS task. Aim 3 will examine the effects of cocaine rewarded cognitive performance as a function of the animal's preference for choosing cocaine vs. juice signaled trials. These analyses will partial out cocaine effects across individual animals in terms of their choice of cocaine vs. juice trials and examine difference in PET imaging of 18FDG brain metabolic activity in the above three brain regions to determine functional differences in animals that prefer cocaine vs. juice rewarded trials in the same DMS paradigm. Aim 3 will also extend the above analyses to animals that are repeatedly exposed to conditions in which cocaine and appetitive rewards are implemented in the same random manner during day-to-day testing for a period of six months. Changes in DMS responding (performance) and preferences for cocaine vs. juice rewards and associated neuronal correlates over this time period will be determined as a baseline for in order to ascertaining the long-term effectiveness of agents administered as part of Aim 4. Aim 4 will examine how the above behavioral and 18FDG imaging correlates of cognitive demand in the DMS task change as a function of prior treatment with two candidate treatment agents, Modafinil and the hypocretin-1 receptor antagonist SB334867, that can alter cocaine's reinforcing effects in self-administration paradigms and are currently considered as possible compounds to treat cocaine addiction in human clinical trials. These actions of these drugs in the early phase of testing will be compared with the effects following long-term exposure to the same paradigm using performance and preference measures as indicants of changes in cocaine's actions in association with altered 18FDG imaging correlates. PUBLIC HEALTH RELEVANCE: The relevance of this Project to public health is directly related to finding agents and drugs that will alleviate the dependence on substances that are abused in society. Primarily the Program will focus on the effects of cocaine on cognition in nonhuman primates which serves as the final testbed for candidate drugs that can lead to therapeutic treatment of cocaine abusers.

DESCRIPTION (provided by applicant): Progressive cognitive impairment can occur in up to 50% of primary and metastatic brain tumor patients surviving e6 months after treatment with fractionated partial or whole-brain irradiation (fWBI); ~200,000 patients/year receive brain irradiation. Although short-term clinical interventions can modulate cognitive impairment, there are no proven long-term treatments or preventive strategies for this radiation-induced morbidity. Rodent models have provided, i] important insights into the pathogenesis of radiation-induced brain injury, and ii] the rationale for anti-inflammatory-based therapeutic approaches, including blockade of the renin- angiotensin system. However, translation of these results to the clinic is limited by concerns about their applicability to humans. Rodents have a brain structure and organization that is very different from humans, and they do not have the higher-order executive functions most often diminished in patients after brain irradiation. Nonhuman primates (NHP) are much less likely to display these limitations when used for preclinical investigations. Indeed, we have developed a NHP model in which fWBI of adult male rhesus monkeys leads to, i] progressive decline in higher-order executive functions, ii] decreased glucose uptake measured by FDG-PET in brain areas involved in task performance prior to fWBI, iii] increased glucose uptake in brain areas previously not involved in the task prior to fWBI, and iv] histopathological and MRI changes that parallel those seen in the irradiated human brain. Thus, we hypothesize that the longitudinal cognitive, intervention, and mechanistic data obtained with this novel NHP model of radiation-induced higher order cognitive impairment will translate faster and more reliably to the clinic than similar rodent data. To test this hypothesis, we propose the following Specific Aims using our NHP model. We will, 1] identify imaging biomarkers and potential mechanisms for the onset and progression of radiation-induced cognitive impairment using FDG-PET and MRI techniques, 2] determine if administration of the angiotensin type 1 receptor antagonist (AT1RA), olmesartan, prior to, during, and for 6 months after fWBI can permanently prevent or ameliorate radiation-induced cognitive impairment and modulate the brain injury assessed by noninvasive imaging techniques during the first year postirradiation, and 3] determine if a 6 month administration of the AT1RA, olmesartan, starting at a postirradiation time when higher-order cognitive function is impaired, can prevent or ameliorate additional radiation-induced cognitive impairment and modulate the brain injury assessed by noninvasive imaging techniques over, i] 6 months of treatment, and ii] an additional 6 months after stopping treatment. Successful completion of these aims should provide new information about the onset and progression of radiation-induced cognitive impairment, and enable us to translate these findings faster and more reliably into clinical trials designed to enhance the long-term survival and QOL of cancer patients receiving fWBI.