Predoctoral and Postdoctoral Training Program

Abstract

This NIDA grant supports a successful predoctoral and postdoctoral training program designed to provide training in molecular and cellular aspects of drug abuse research. The University of Washington School of Medicine has strong research programs studying molecular aspects of drug receptor signaling mechanisms in several departments, and this training program has dramatically facilitated the coordination of training and collaboration of research effort among the drug abuse researchers at this institution. We expect that the continued application of increasingly sophisticated biochemical and physiological methods will provide important advances in our understanding of the mechanisms by which specific drugs of abuse act. It is the intent of this program to identify and support three predoctoral students and three postdoctoral fellows interested in studying molecular and cellular mechanisms of drug action of specific abused drugs. Beyond the directly beneficial effects on the careers of the trainees, one of the most significant successes of the previously funded program has been its catalytic effect on the research environment at this institution. A funded Drug Abuse Research Training Program will continue to serve as an important catalyst to focus research effort at this institution on the basic neurobiology of a significant health issue.

A list of the principal faculty members involved:

• Charles Chavkin (director), Pharmacology
• Stan McKnight, Pharmacology
• Richard Palmiter, Biochemistry
• Ilene Bernstein, Psychology
• Sandra Bajjalieh, Pharmacology
• Bill Catterall, Pharmacology
• Bertil Hille, Physiology and Biophysics
• Sheri Mizumori, Psychology
• Neil Nathanson, Pharmacology
• John Neumaier, Psychiatry
• Valerie Olson, Biochemistry
• Paul Phillips, Psychiatry and Behavioral Sciences
• Nephi Stella, Psychiatry and Pharmacology
• Robert Steiner, Physiology and Biophysics
• Dan Storm, Pharmacology
• Jane Sullivan, Physiology and Biophysics
• Greg Terman, Anesthesiology

A list of the trainees supported

Research Areas

Charles Chavkin, Ph.D. [top of list]
D-425 HSC, 206-543-4266
cchavkin@u.washington.edu
Chavkin Web Page
Lab Web Site

The research effort in this lab continues to be focused on 1) the mechanisms by which endogenous opioids regulate synaptic transmission in the mammalian brain, 2) the signal transduction events in brain activated by opioid receptors, and 3) the molecular mechanisms of opioid receptor desensitization. This lab uses a combination of electrophysiological, anatomical, and molecular approaches to the understanding of the role of opioid neuropeptides as neurotransmitters in the brain and spinal cord. We know that these endogenous peptides regulate synaptic plasticity (LTP) in memory events and we know that changes in the physiological role of the endogenous opioids occurs in certain forms of epilepsy. Our work has attempted to rigorously define the anatomical and biophysical properties of this neuropeptide synapse in the mammalian brain.

To understand the biochemical coupling between opioid receptors and ion channels important for opioid actions, we have studied the cellular processes mediating opioid effects in hippocampal slices and cultures. Opioid receptor activation leads to the activation of pertussis toxin sensitive G proteins that then cause an increase in potassium channel conductance and decrease in calcium channel conductance. We are defining the types of potassium channels activated. Changes in the activation of specific ion channels results in a somatic and presynaptic inhibition by molecular mechanisms that we are defining.

To understand how the biochemical coupling between receptor and ion channel is regulated, we are measuring the behavioral and electrophysiological effects of prolonged activation of opioid receptors. The mechanisms responsible for desensitization of the response following extended agonist exposure is a complex mixture of processes including changes receptor phosphorylation, changes in G-protein levels, and changes in the channel. The important steps in this regulation are being identified and defined. We have continued to use the oocyte gene expression system and transfected neuroblastoma cell lines to reconstitute the opioid receptor link to potassium channels as defined in the hippocampus and to study the actions of potential regulators by co-expression. The results of these studies are expected to provide the necessary understanding required to define animal behavior at the cellular and molecular level.

Sandra M. Bajjalieh, Ph.D. [top of list]
D-429 HSC, 206-616-2962
bajjalie@u.washington.edu
Bajjalieh Web Page

The Bajjalieh lab studies the molecular events that produce and regulate neurotransmitter secretion and how changes in those events contribute to changes associated with experience, drug addiction and disease. The strategy is to systematically characterize proteins and protein complexes localized to the synapse with the goal of identifying how each contributes to the synthesis, filling, targeting, priming fusion and recycling of synaptic vesicles. There are currently two projects in progress; identifying the role of Synaptic Vesicle Protein 2 (SV2) and of ceramide kinases in synaptic functioning. The latter project is supported by NIDA.

SV2 is an integral membrane protein present in all synaptic vesicles. We have demonstrated that SV2 is essential for normal neurotransmission by generating SV2 knockout mice. We have shown that in the absence of the most widely expressed SV2 isoform, SV2A, levels of both inhibitory and excitatory neurotransmission are reduced and that calcium is less able to trigger vesicle fusion in neuroendocrine cells. We are currently using neurons isolated from these mice to systematically test hypotheses of SV2 action that could contribute to this phenotype. Biochemical studies of SV2's structure and molecular interactions are guiding this effort. For example, we have found that SV2 interacts with the putative calcium sensor, synaptotagmin and that this interaction is regulated by calcium and phosphorylation of SV2.

The sphingolipids are emerging as a class of signaling molecules involved in cellular functions as disparate as membrane trafficking, proliferation and cell death. Based on our early observation that synaptic vesicles contain a ceramide kinase, we are studying the role of ceramide phosphate generation in vesicle trafficking by characterizing ceramide kinases.

Ilene Bernstein, Ph.D. [top of list]
321 Guthrie, 206-543-4527
ileneb@u.washington.edu
Bernstein Web Page
Biosketch Web Page

Dr. Ilene Bernstein is a Professor of Psychology, and her research is in the area of neural basis of learning and motivation including taste aversion learning, conditioned drug tolerance, dopamine systems and motivation, and alcohol preference and amphetamine sensitization. She brings to this program a high degree of expertise in design and assessment of behavioral protocols. Research in the Bernstein lab has important links to both alcohol and drug abuse, and she is actively involved in the program project grant PO1 DA15916, "Molecular components underlying drug abuse." An additional area of interest has been identifying factors that contribute to vulnerability to alcohol and drug abuse. Genetic factors have been examined in studies in rats, using the P and NP rat lines selectively bred for differential alcohol intake (Thiele et al, 1997). These studies have examined strain difference in patterns of neuronal activation to ethanol. Studies in mice have indicated that knockout and transgenic modification of genes for NPY and subunits of the PKA genes can significantly alter voluntary ethanol intake as well as the physiological response to ethanol (Thiele et al, 1998; Thiele et al, 2000). Experiential factors, particularly a history of sodium deprivation, have been examined in more recent work. Prior experience with strong homeostatic challenges (sodium depletion and the induction of a salt appetite) has been shown to produce changes in neuronal morphology in cells in the nucleus accumbens that are very similar to those reported to occur after drug sensitization (e.g. amphetamine; cocaine) (Roitman et al, 2002). Furthermore, rats with a history of salt appetite show a sensitized psychomotor activation in response to their first experience with amphetamine, suggesting that their depletion history has sensitized them to amphetamine (Roitman et al, 2002).

William A. Catterall, Ph.D. [top of list]
F-427 HSC, 206-543-1925
wcatt@u.washington.edu
Catterall Web Page

Research in this laboratory is focused on the voltage-gated sodium and calcium channels that are responsible for action potential generation in nerve and muscle and for initiation of synaptic transmission and excitation-contraction coupling. We study the structure and function of these ion channels, their regulation by physiological pathways, drugs, and neurotoxins, and their role in coordination of electrical excitability and synaptic transmission in neurons.

Purified channel proteins, cloned DNA probes which encode the structure of these ion channels, site-directed mutagenesis and functional expression, and site-directed antibodies that recognize specific peptide segments are used to probe the molecular mechanisms of ion channel function, biosynthesis, assembly, and localization. The sodium channel beta subunits have been found to serve as both modulators of channel activity and cell adhesion molecules that may determine channel localization. Specific protein segments of the pore-forming alpha subunits that form the voltage sensors and inactivation gate of the sodium channel have been defined and their functional roles determined by mutagenesis and biophysical analysis. The three-dimensional structure of the inactivation gate of the sodium channel has been determined by NMR methods and correlated with its physiological function. We are currently investigating the sites and mechanisms by drugs (including psychostimulants) alter the properties of ion channels in order to define the mechanism of drug action at the molecular level and identify common themes that may be important in development of new therapeutic agents.

Bertil Hille, Ph.D. [top of list]
H-409, 206 543-8639
hille@u.washington.edu
Hille Web Page
Biosketch Web Page

We are interested in cell signaling by ion channels, neurotransmitters and hormones acting through G-protein coupled receptors and intracellular calcium. Our overall goal is to define every step and the molecular mechanisms underlying physiologically interesting activation and inhibition in single cells, with emphasis on events that occur in time scales of microseconds to seconds.

The cell membrane, cytoplasm and intracellular organelles form a rapidly interacting signaling network. We study single neurons, endocrine cells, cell lines, and synapses using techniques associated with the patch clamp: voltage clamp, membrane capacitance, optical imaging, and photometry of fluorescent dyes. We have analyzed G-protein coupled modulation of neuronal Ca channels, responses of gonadotropes and somatotropes to their releasing hormones, exocytosis and secretion from vesicles, dynamics of intraorganellar calcium in chromaffin cells, and the acrosomal reaction of sperm. Another part of our work is biophysical, probing the ionic permeability and gating properties of individual ion channels.

Ken Mackie, M.D. [top of list]
BB-1429 HSC,206 616-2669
kmackie@u.washington.edu
Biosketch Web Page

The Mackie lab is interested in how cannabinoids produce their psychoactive effects. These effects appear to be mediated by the CB1 receptor, a G protein-coupled receptor that binds and is activated by cannabinoids. Work in this lab has focused on how the activation of the CB1 receptor by cannabinoids modulates neuronal ion channels. Currently, the lab has a strong interest in understanding how tolerance to the effects of cannabinoids develops, and they are studying this at the level of the receptor.

In addition to exogenous plant or synthetic cannabinoids, endogenous cannabinoids are synthesized by neurons and glia following specific stimuli. These endocannabinoids also activate CB1 receptors. Our lab is interested in understanding the signal transduction pathways activated by CB1 receptors and how these receptors and pathways are regulated. We approach these questions by combining molecular biological, electrophysiological, and imaging techniques. Our goal is that by better understanding the function and regulation of CB1 receptors we will gain a deeper understanding into the role that endogenous and exogenous cannabinoids play in the nervous system.

Stanley McKnight, Ph.D. [top of list]
K-540B HSC, 206 616-4237
mcknight@u.washington.edu
McKnight Web Page
Lab Web Site

The major goal of the McKnight lab is to introduce novel genetic mutations into the Protein Kinase A regulatory system that allow investigations into the major in vivo functions of this signal transduction cascade. The laboratory is focused on the use of mouse genetics since the mouse allows a full complement of genetic manipulations and in most instances provides a relevant model of human physiology and behavior. Since all cells in the body produce isoforms of the cAMP-regulated protein kinase, the McKnight lab has developed novel techniques to produce tissue-specific activation or inactivation of discrete subunits and has also made modifications in the tethering scaffold molecules to determine subcellular localization and specificity coupling to substrates. The lab has also pioneered the application of a combined pharmacologic and genetic approach that will allow drug dependent control of kinase activity in specific cell types in vivo.

The McKnight lab is primarily focused on the function of dopamine receptors and the associated changes in PKA activity that occur in animals as they become addicted to drugs such as cocaine, morphine, amphetamine, and alcohol. A major hypothesis to explain the drug reward and drug seeking behaviors seen in both humans and mice after exposure to a drug of abuse is that chronic adaptation occurs in the PKA signaling system that resets the sensitivity to drug effects and increases the craving for drugs during abstinence. PKA mutations are being produced in specific neuronal pathways in the striatum, nucleus accumbens, and hippocampus to test the hypothesis that PKA activity might be a modifier of drug addiction and drug seeking behavior.

Sheri Mizumori, Ph.D. [top of list]
Guthrie 333A, 206 543-2699
mizumori@u.washington.edu
Lab Web Site

Understanding the mechanisms of neuroplasticity as related to learning and memory is fundamental to our understanding of the causes of a variety of cognitive disorders as well as the developmental changes in learning that occur across one’s lifespan. Our laboratory attempts to address these issues by using a rodent model of spatial navigation to investigate the dynamic responses of single neurons in the brain, as well as the complex interactions between cells located in different memory-related structures. These studies employ techniques involving recording extracellular signals from many cells simultaneously as animals perform memory tasks. Our studies have examined many processes that contribute to successful and adaptive navigation: 1) the evaluation of sensory input during active locomotion (e.g. Mizumori & Williams, 1993; Cooper et al., 1998), 2) the integration of current sensory information with past knowledge about an environment (e.g. see Mizumori et al., 1999a,b, Cooper et al., 2001) and with internal state information (e.g. Leutgeb & Mizumori, 1999, 2002), and 3) the behavioral implementation of highly processed spatial information (e.g. Mizumori et al., 1999b, in press). More recently, we have been considering these processes in terms of the flexibility of underlying neural representational systems during shifts in cognitive strategy or task demands. Other studies have evaluated spatial performance and properties of neural representational systems of aged and brain-damaged animals (e.g. Mizumori et al., 1995, 1996). We are also interested in evaluating the relationship between neural and behavioral plasticity from computational perspectives.

Neil Nathanson, Ph.D. [top of list]
K-536A HSC, 206 543-9457
nathanso@u.washington.edu
Nathanson Web Page
Lab Web Site

Our laboratory is interested in the regulation of expression and mechanisms of action of muscarinic acetylcholine receptors (mAChR). Interactions between dopaminergic and muscarinic pathways are thought to be involved in sensitization and reward mechanisms involving psychostimulants such as cocaine. The role of the cholinergic system and muscarinic receptors in the regulation of function and of dopaminergic transmission in the basal ganglia has been intensively studied by many investigators. For example, muscarinic antagonists inhibit the catalepsy induced by dopamine D2 antagonists by haloperidol and inhibit the D2 antagonist-mediated induction of immediate early genes (such as c-fos) and the preproenkephalin gene in striatopallidal neurons. Muscarinic drugs can inhibit amphetamine-induced locomotor activity, as well as amphetamine-induced induction of dynorphin, enkephalin, and substance P expression. Genetic disruption of the M1 receptors has been shown to result in increased dopamine release in the striatum and an increased locomotor response to amphetamine.

Our laboratory has generated M1 receptor knockout mice, and we have recently obtained M3 and M5 knockout mice as well. We will use these mice to determine if the loss of individual mAChR subtypes results in altered development and expression of behavioral sensitization to the locomotor-enhancing effects of cocaine and amphetamine. We will also determine if these strains of mice will exhibit defects in the ability to associate the rewarding effects of cocaine and amphetamine to a particular chamber by conditioned place preference. Finally, since the mAChR regulate dopamine release, we will determine if loss of any of these mAChR subtypes alters the expression of or dopamine receptor subtypes in the nucleus accumbens.

John Neumaier, M.D., Ph.D. [top of list]
R&T 303C HMC, 206 341-5803
neumaier@u.washington.edu
Biosketch Web Page
Molecular Neuroscience Web Site

While many laboratories have focused on the contribution of dopaminergic neurons to addiction, the serotonin system also appears to play an important role. In particular, cocaine increases synaptic serotonin levels in many brain regions to the same extent as dopamine or norepinephrine. This has led to the hope that producing a better understanding of the effects of abused drugs that are mediated by serotonin may point toward new targets for pharmacological treatments of addiction. My laboratory is focusing on the roles of 5-HT1B and 5-HT6 serotonin receptors in psychostimulant addiction. We use a combination of animal models, neuropharmacological approaches, and molecular biology to address these issues. While evidence from most rat brain studies suggest that activating 5-HT1B receptors in the brain increase the rewarding properties of cocaine, knockout strategies have presented more contradictory evidence. We have taken the strategy of using molecularly and anatomically specific techniques to determine the contribution of 5-HT1B receptors in specific populations of neurons to the locomotor stimulant and rewarding properties of cocaine. We recently addressed this problem by using viral mediated gene transfer directly into nucleus accumbens shell (NAccS) neurons to address the role of 5-HT1B receptors in this important component of the brain reward pathways. These GABAergic neurons project to the ventral tegmental area (VTA), a region that has been previously implicated in 5-HT1B effects on dopaminergic function. It was previously known that GABA release in VTA is regulated by 5-HT1B receptors, but we have now provided direct evidence that the 5-HT1B terminal receptors in NAccS projections to VTA are likely to account for these observations. Increased expression of 5-HT1B in the terminals of these neurons sensitized rats to the locomotor stimulating and rewarding properties of cocaine. We have also found that chronic cocaine exposure increases the expression of 5-HT1B mRNA in NAccS. Our plan is to extend these results by systematically dissecting the role of 5-HT1B receptors in the development of cocaine sensitization, to try to determine whether both rewarding and aversive properties of cocaine are altered by increased 5-HT1B expression in NAccS, and whether these effects generalize to amphetamine. We will examine the regulation of endogenous 5-HT1B mRNA following cocaine administration further, and determine whether increased 5-HT1B expression in serotonergic neurons alters the sensitivity of animals to cocaine. We will then turn our attention to the role of 5-HT1B receptors after acute and chronic discontinuation of cocaine. In the longer term we plan to use self administration procedures to study the role of these receptors in drug and stress-induced reinstatement of cocaine self administration. 5-HT6 receptors are heavily expressed in dorsal and ventral striatum, including NAccS, where they are expressed by medium spiny neurons. Based on their signal transduction mechanisms, these receptors are likely to play a role that generally opposes 5-HT1B receptors in these neurons. We have performed some preliminary experiments that indicate that antagonists of 5-HT6 receptors injected directly into NAccS may also sensitize animals to cocaine's locomotor stimulant effects. We plan to pursue these observations in more detail, and have recently constructed a viral vector with which we can increase 5-HT6 receptor expression in these neurons. Since a number of new and selective 5-HT6 agonists and antagonists are becoming available (and we have several), we will use the complementary strategies of selective drug treatment and selective increases in receptor expression to determine the contribution of 5-HT6 receptors to cocaine's addictive features.

Valerie Olson, Ph.D. [top of list]
HSB J 661, 206 543-6090
vgolson@u.washington.edu

Richard Palmiter, Ph.D. [top of list]
J-661F HSC, 206 543-6064
palmiter@u.washington.edu
Palmiter Web Page
Biosketch Web Page

Our lab uses gene knockout techniques in mice to study the role of neurotransmitters in the development and function of the nervous system. Most of the work in the lab centers on the catecholamines norepinephrine and dopamine. For example, we study a line of mice that lack the ability to make dopamine (DA). Considering that enhanced dopamine signaling is implicated in the rewarding properties of virtually all drugs of abuse, the dopamine-deficient mice provide a new model for studying the actions of many different drugs. One would expect that in the absence of dopamine mice would not respond behaviorally to drugs of abuse and maybe not to even to primary rewards such as sweets. In addition to testing this hypothesis in dopamine-deficient mice with a variety of different drugs, we have also perfected means of restoring dopamine to specific brain regions, e.g. just the caudate putamen or just the nucleus accumbens. Thus, if dopamine-deficient mice fail to respond to drugs, we can ask where in the brain dopamine signaling is required for responsiveness. Our experiments so far, have validated this approach. We have shown, for example, that dopamine-deficient mice do not manifest a normal locomotor response to amphetamine, but responsiveness can be restored by viral transduction of the nucleus accumbens. At a more fundamental level, dopamine release in response to rewards (e.g. sweets) is thought to underlie certain forms of associative learning which would be predicted to help animals learn about aspects of their environment that produce a state of well being. As a first step, we are asking whether dopamine-deficient mice show a preference for natural (or artificial) sweets over water, and if so, is dopamine required for associative learning.

While less attention is directed towards norepinephrine in studies on drug abuse, there are numerous indications that it may also be involved. For example, blockade of a1-adrenoreceptors with prazosin inhibits amphetamine-induced locomotion, suggesting that norepinephrine signaling through these receptors is required for dopamine that is released by amphetamine to elicit locomotion. We have initiated studies in this area by asking whether chronic norepinephrine deficiency prevents amphetamine-induced locomotion as would be predicted from the observations described above. We discovered, quite unexpectedly, that norepinephrine-deficient mice show an enhanced locomotor response to amphetamine compared to controls. The locomotor response to amphetamine increases with successive administrations of this drug, a phenomenon referred to as sensitization. Our norepinephrine-deficient mice behave as though they are already sensitized. Another situation where norepinephrine has been implicated involves withdrawal from chronic morphine treatment. The unpleasant behavioral syndrome that is precipitated during withdrawal has been ascribed to norepinephrine release from the locus coeruleus. If this hypothesis is correct, we predict that the behavioral response to withdrawal would be attenuated in mice lacking norepinephrine. Studies exploring this possibility are underway.

Paul Phillips, M.D., Ph.D. [top of list]
BB-1538, HSB, 206 543-0121
pemp@u.washington.edu
Lab Web Site

The nucleus accumbens had been proposed as an anatomical substrate of limbic-motor integration. It gathers information from midbrain dopaminergic neurons that are activated by salient sensory stimuli and from glutamatergic afferents from the amydala, hippocampus and prefrontal cortex that encode memory of prior responses, the emotional state and the context. This information is processed and, based on previous learning, an appropriate motor response to the stimulus is generated.

We are particularly interested in the dopaminergic input to the nucleus accumbens. Midbrain dopaminergic neurons burst fire in response to natural reinforcers or to stimuli that have been paired to their delivery. This has been presumed to result in a subsecond, transient increase in dopamine in several forebrain structures. Recently, we confirmed this by making direct chemical measurements of such changes during presentation of natural reinforcers, drug reinforcers or their paired cues. Increases in forebrain dopamine typically result in motor activation. In particular, increases in dopamine in the nucleus accumbens have been implicated in goal-directed movements. Using cocaine self-administration as a model of drug abuse, we provided the first direct evidence that phasic dopamine changes are temporally linked to, and can trigger drug-seeking behavior.

Robert Steiner, Ph.D. [top of list]
G-407 HSC, 206 543-9970
steiner@u.washington.edu
Steiner Web Page
Biosketch Web Page

Research in Steiner lab is primarily directed toward understanding the neuroendocrine mechanisms governing the reproduction and body weight, with focus on the regulation of neuropeptide genes, their receptors, and intracellular signaling pathways [e.g., gonadotropin-releasing hormone (GnRH), galanin, peptides derived from proopiomelanocortin precursor (i.e., alpha-MSH and beta-endorophin), neuropeptide Y (NPY), and galanin-like peptide (GALP)]. Understanding neuropeptide function in brain and how neuromodulators control appetite and appetitive behaviors in general has an obvious relevance to drug abuse research that our lab is interested in developing.

The laboratory has a special interest understanding the relationships among nutrition, adiposity, and reproduction and revealing the cellular molecular basis of leptin, insulin, and peptide YY's actions on neurotransmitter systems in the brain. The work is principally focused on newly discovered molecule, galanin-like peptide (GALP). In the brain, GALP is expressed exclusively in the arcuate nucleus and median eminence of the hypothalamus, which are nodal points for integration of metabolism and reproduction. Work from the Steiner laboratory has shown that GALP neurons are regulated by the metabolic hormones leptin and insulin and that GALP administration inhibits food intake, reduces body weight and stimulates gonadotropin secretion. These observations suggest that GALP neurons serve as part of the hypothalamic circuitry linking the regulation of body weight to reproduction. The Steiner lab has also discovered that centrally-administered GALP has a profound effect on activity rhythms and the sympathetic nervous system (SNS). The initial effect of centrally administered GALP in mouse is to create a sleep-like state of catalepsy, reminiscent of opiate toxicity. With continued daily infusions, the animals become hyperactive, with dramatic stimulation of sympathetic activity, suggestive of an amphetamine-induced state. These observations suggest that GALP may be involved in arousal states, recalling the effects of other neuropeptides, such as the orexins, which are thought to play a role in sleep and arousal transitions. Studies of GALP's possible role in these behaviors are now underway in this laboratory. Establishing the physiological effects of GALP and learning about the molecular physiology of GALP neurons and the basic circuitry that couples GALP to other neuronal systems are fundamental steps toward the goal of understanding GALP's functional role in arousal and sleep-like states, which may have direct relevance to understanding the mechanism of actions of opiates, amphetamines, and related compounds such as pseudoephedrine, and its extended family of compounds. To explore questions related to these topics, the Steiner group uses various experimental animal models, including the rat, mouse (wild-type as well as spontaneous and engineered mutants), and monkey, and employs techniques such as in situ hybridization, neuroanatomical mapping, gene cloning and targeting to create knockouts, microarray analysis, radioimmunoassays, and neurosurgical procedures. It is hoped that this knowledge will lead to a better understanding of not only certain disorders of reproduction and metabolism but may also prove useful for understanding the mechanisms of action of certain drugs of abuse, which are widely and ill-advisedly used to manipulate arousal, activity rhythms, and body weight.

Nephi Stella, Ph.D. [top of list]
BB-1538C HSC, 206 221-5220
nstella@u.washington.edu
Stella Web Page
Lab Web Site

My laboratory is interested in how the endogenous cannabinoids (anadamide and 2-AG) are synthesized and act in the brain. In addition, we are studying the molecular mechanisms involved in the interaction between brain tumors and microglial cells mediated by endocannabinoids. Several in vitro and in vivo experiments using rodent models of astrocytomas have shown that cannabinoid compounds, such as D9-tetrahydrocannabinol (THC), inhibit and even reverse tumor growth. To address the question of whether THC directly kills astrocytomas or modulate immune responses of microglial cells, we are investigating the cannabinoid receptors and their endogenous ligands (endocannabinoids), in brain tumors and are testing its role in astrocytomas and microglial cells interaction. Our laboratory uses a variety of models to assess the functionality of the cannabinoid signaling system in brain tumors. Among these are astrocytes and microglia in primary culture, cultured astrocytomas (both primary and transformed cell lines), as well as human biopsy tissue and an animal model of injected astrocytomas. We are also characterizing the anti-tumor properties of various cannabinoid compounds in these models. Our broad, long-term goal is to understand the molecular mechanisms underlying immune surveillance in the brain and the biology of microglial cell activation in the presence of brain tumors. This research should provide novel insight into the pathogenesis of brain tumors and open alternative therapeutic avenues.

Dan Storm, Ph.D. [top of list]
J-681F HSC, 206 543-7028
dstorm@u.washington.edu
Storm Web Page
Biosketch Web Page

Our lab is interested in molecular mechanisms underlying neuroplasticity. Recent evidence from our lab and others suggests that the cAMP, Ca2+, and MAP kinase signal transduction pathways may contribute to various forms of neuroplasticity. In fact, cross-talk between these regulatory pathways may be particularly important. The formation of long-term memory requires transcription of specific genes. Our recent data using transgenic mouse model systems indicates that LTP may depend on cAMP-mediated transcription and maximal expression of CRE-mediated transcription in neurons may depend on coactivation of the Ca2+, cAMP, and MAPK signaling systems. Specific studies in the lab include evaluation of the role of type III adenylyl cyclase for signal transduction and the role of the Ca2+ stimulated adenylyl cyclases for learning and memory. Because some long-term changes in neurons and synapses depend upon increased transcription of specific genes, we are also studying mechanisms for regulation of transcription in neurons by cAMP, Ca2+ and the MAP kinase regulatory systems. This lab uses an interdisciplinary approach including molecular biology, electrophysiology and behavioral neurobiology.

We have had a long-standing interest in the effects of cannabinoids on memory formation and the role that cannabinoid regulation of adenylyl cyclase activity may play in memory. These studies focus on the Ca2+-stimulated adenylyl cyclases because these enzymes are required for long-term memory and support CRE-mediated transcription during memory formation. Recently, we discovered that CB1 receptors are coupled to inhibition of the type 1 Ca2+-stimulated adenylyl cyclase. In addition, we are also analyzing the contribution of the Ca2+-stimulated adenylyl cyclases (AC1 and AC8) for cannabinoid withdrawal symptoms because cannabinoid withdrawal is associated with a significant increase in AC1 activity. Because of the central role played by CRE-mediated transcription in memory formation, we have also been analyzing the effects of amphetamine, morphine-withdrawal, and antidepressant treatment on CRE-mediated transcription in collaboration with Eric Nestler and Ron Duman.

Jane Sullivan, Ph.D. [top of list]
G-413 HSC, 206 543-4038
jmsull@u.washington.edu
Sullivan Web Page
Biosketch Web Page

The central goal of Sullivan lab is to identify cellular and molecular mechanisms underlying modulation of synaptic transmission in the mammalian central nervous system by combining electrophysiology with pharmacology and molecular biology (site-directed mutagenesis) to investigate the role that specific proteins, and specific domains within proteins, play in modulating synaptic transmission. We are funded to understand the neurobiological basis for cannabinoid effects on synaptic function. Marijuana and its constituent cannabinoids have been shown to have detrimental effects on cognition and memory that are likely to be mediated by alteration of the normal synaptic functioning of the hippocampus. We are studying the cellular and molecular basis for the effects of cannabinoids on four aspects of hippocampal synaptic function, and on four forms of synaptic plasticity - some of which are believed to form the cellular basis for learning and memory. The role of calcium and potassium channel modulation in cannabinoid-mediated effects are also being studied.

Greg Terman, M.D., Ph.D. [top of list]
BB1433 HSC, 206 616-2668
gwt@u.washington.edu
Biosketch Web Page

This lab is studying mechanisms responsible for opiate analgesic effects and changes in drug response following sustained opiate exposure. In addition the lab group studies the cellular mechanisms responsible for central sensitization to noxious stimulation. Long term potentiation (LTP), the best-studied cellular model of neuroplasticity within the CNS, involves a seemingly permanent increase in neuronal excitation following repeated activation of afferent input to that neuron. We have recently developed a model of LTP in the spinal cord slice preparation and begun to investigate its pharmacological and physiological characteristics using visualized whole cell voltage clamp recordings. Mu opiates inhibit induction of spinal LTP. Both exogenous and endogenous kappa opiates also inhibit LTP but act primarily by inhibiting maintenance mechanisms. This lab studies of LTP in the spinal cord slice by: 1) using imaging techniques to specifically target dorsal horn nociceptors (i.e., selecting back-filled spinothalamic cells with three dimensional morphologies characteristic of nociceptors) for further experiments on kappa modulation of spinal LTP. 2) Examining animals previously sensitized to noxious stimuli by inflammation (with resultant long-term anatomical and physiological changes in spinal pain circuitry) to correlate behavioral evidence of sensitization with electrophysiological evidence of spinal LTP including sensitivity to kappa opioids. 3) Studying the dose related effects of dynorphin in modulating Lamina I neurotransmission and LTP, including differentiation of its kappa opiate and NMDA receptor activities. Such investigations of LTP in the spinal cord will lead to a better understanding of CNS neuroplasticity, in general, and nociceptive sensitization, in particular, and may ultimately lead to better pharmacological means of managing or preventing certain pain states.

A list of the trainees supported:
Currently supported fellows are listed in bold type

Contact C Chavkin for additional information, cchavkin@u.washington.edu