Christopher Evans Laboratory
[Publications]

One major research objective of Dr. Evans' laboratory is to gain insights into the mechanism of action of opioid drugs and their receptors, with the ultimate goal of discerning molecular processes that contribute to the effects of opiate drugs on pain, the immune system, memory processes, and reward.

The laboratory investigates the signaling and regulation of receptors both in vitro and in vivo utilizing cell biology and molecular methodologies. Current on-going projects in the laboratory include investigations of the differential signaling abilities of opioid drugs, the role of receptor trafficking in the regulation of opioid receptors and the development of methodologies to detect plasticity processes following opioid receptor modulation.

A second, more recent line of research is the analysis of catecholamine systems and the HPA axis in reward and immune regulation using a zebrafish model.

Laboratory Phone: (310)206-7883


Laboratory Staff:

Shoshana Eitan Assistant Researcher   shoshy@ucla.edu	Camron Bryant Graduate Student   cdbryant@ucla.edu	Nazli Saliminejad Staff Research Associate   nsalimi@ucla.edu
Sara J. Levitt Lab Assistant   slevitt@ucla.edu	Andrew Bokarius Lab Assistant   andrew@bokarius.com	Kristofer Roberts Staff Research Associate   kristo@ucla.edu
Arnaud Lacoste Postdoctoral Fellow   alacoste@ucla.edu	Sandeep Gyawali Graduate Student   gyawali@ucla.edu	Louise Cosand Research Technician lcosand@mednet.ucla.edu

Christopher J. Evans, Ph.D.
Stefan Hatos Professor
(310)206-7884
cevans@ucla.edu

Cui-Wei Xie Laboratory
[Publications]

Our primary research interests are the modulation of synaptic plasticity in the hippocampus and its alterations under pathological conditions. Current projects investigate the effects of opioid peptides and Alzheimer’s amyloid ß (Ab ) peptides on hippocampal long-term potentiation (LTP), long-term depression (LTD) and function of NMDA receptor channels. Our objectives are to determine how these peptides affect the long-term synaptic plasticity, what are the intracellular signaling pathways transducing their effects, and ultimately how these processes contribute to the development of opioid addiction or memory loss in Alzheimer's disease. Another aspect of our research is the modulatory effect of opioids on voltage gated ion channels in sensory neurons and its desensitization. The major approaches used in the lab are electrophysiological recordings from brain slices and cultured neurons, in combination with calcium imaging, immunocytochemical and molecular techniques through collaborations with other labs in the Center.

Laboratory Phone: (310)206-2946


Laboratory Staff:

Miao Tan, Ph.D. Postdoctoral Fellow   mtan@ucla.edu

Danyun (Alice) Zhao Staff Research Associate   azhao@mednet.ucla.edu

Cui-Wei Xie, M.D., Ph.D.
Associate Professor

xiexy@ucla.edu
(310)206-0083  

Jim Boulter Laboratory
[Publications]

The long-term research objective of our laboratory is to use a molecular genetic approach to understand the role of nicotinic acetylcholine receptors (nAChRs) in vertebrate central and peripheral nervous system function. Although considerable molecular data regarding the structure and function of the ten known nAChR subunit genes is available, far less is known about neural circuitry or roles subserved by the individual receptors assembled from the various subunits. Moreover, little is known about the physiological consequences of mutations within these genes, the precise roles of receptor subtypes in the normal ontogeny and modulation of central nervous system synapses, or in the establishment of nicotine-induced dependence, tolerance, and withdrawal among habitual tobacco users.

As a first step towards addressing some of these issues, our lab is conducting experiments which use homologous recombination in mice to introduce null or altered-function mutations in selected nAChR subunit genes. The rationale for such studies rests on the assumption that deficits or alterations in targeted genes can, upon analysis of subject animals, reveal or clarify the function of the mutated gene.

Ongoing projects include construction of a transgenic mouse containing a deletion of the nAChR alpha 6 subunit gene. Since alpha 6 is actively transcribed in catecholaminergic nuclei (ventral tegmental area, substantia nigra, and locus coeruleus), it is anticipated that null mutants will help to define central cholinergic circuits which modulate locomotion. Likewise, an alpha 6 null mutation is anticipated to have profound effects on behaviors which originate in the mesolimbic dopamine system and are relevant to the reinforcing properties of nicotine, or to the basic motivational processes which underlie learning and cognitive behavior.

A second genetically engineered mouse will harbor a specific mutation (Ser248Phe) in the nAChR alpha 4 subunit gene. In vitro this mutation potentiates onset and slows recovery from receptor desensitization, while in vivo this mutation is responsible for a human disorder known as autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The purpose of this set of experiments is to provide an animal model to analyze the molecular pathology of partial epilepsy, and to offer a paradigm for the development and evaluation of cholinergic therapeutic strategies.

Finally, we are beginning a new project in the lab to determine the possible role of central and peripheral nAChR in the perception of pain. Recent experiments from a number of laboratories have demonstrated that synthetic derivatives of epibatidine (a high affinity, naturally occurring, nicotinic cholinergic agonist) are potent analgesics which show considerable promise for the treatment of chronic, neurodegenerative and inflammatory pain. To determine which nAChRs are involved in peripheral nociception we are examining the repertoire of nAChR genes expressed in spinal cord and dorsal root ganglion neurons (primary sensory afferents) using a combination of in situ hybridization, immunocytochemistry, and reverse-transcription PCR.

Alwin Klaassen Graduate Student   dralwin@hotmail.com

Janet S. Byun Graduate Student   jbyun@ucla.edu

Jim Boulter, Ph.D.
Associate Professor

Nigel Maidment Laboratory
[Publications]

Opioid systems and "hedonic homeostasis"

One area of research in Dr. Maidment’s laboratory is centered round the role of the dopamine and endogenous opioid systems in modulating reward and hedonic homeostasis. A combination of in vivo neurochemical and behavioral approaches is used together with cell culture methods to study the impact of acute and chronic exposure to opiate and psychostimulant drugs on neurotransmitter release along the mesolimbic-ventral pallidal reward axis. The lab is interested in changes that occur in dopamine and opioid peptide transmission as a result of repeated administration of these drugs and, in particular, how sensitization or 'reverse tolerance' in these transmitter systems may be important mechanisms underlying craving and relapse. Recent research has provided evidence for a role of the recently discovered peptide - orphanin FQ or 'nociceptin' - in modulating dopamine neurotransmission and in reversing the rewarding effects of opiates. The lab is currently investigating whether this peptide system, which opposes the analgesic actions of opioid peptides, may serve as an 'endogenous restraint' on the brain's reward mechanisms, by studying mice in which the receptor for this peptide - ORL-1 - has been 'knocked out'.

Viral-mediated opioid receptor gene transfer in primary sensory neurons

Another area of research is utilizing viral-mediated gene transfer to study the regulation of opioid receptor function in primary sensory neurons. Mutated forms of opioid receptors are being expressed in sensory neurons of opioid receptor-deficient mice both in culture, using adenovirus, and in vivo using herpes simplex virus. One goal is to determine the significance of molecular/cellular mechanisms such as receptor desensitization and internalization to the phenomenon of opiate tolerance. Another aim is to explore the feasibility of using viral transfer technologies for "gene therapy" approaches to treatment of chronic pain. In a related study the effectiveness of herpes-simplex viral-mediated expression of nerve growth factor is being tested in a genetic model of diabetic neuropathy.

Targeting the subthalamic nucleus in Parkinson’s disease

The other major focus of the lab is in the area of Parkinson’s disease research. The Maidment lab is a component of the UCLA Udall Center for Parkinson’s Disease Research, directed by Marie-Francoise Chesselet. The research is geared toward understanding the mechanisms underlying changes in the activity of the glutamatergic neurons of the subthalamic nucleus in the disease. To this end, the lab is utilizing biosensor technology to monitor release of glutamate in the major output structures of this brain region – the substantia nigra and entopeduncular nucleus – in animal models of the disease. The hope is that such research will lead to the development of pharmacological therapies targeting the subthalamic nucleus which will mimic the successful surgical deep brain stimulation approach.

Dopamine, serotonin and amino acid release in the human brain

Laboratory Phone: (310)206-7890


Laboratory Staff:

Kelvin Chiu Research Associate   kchiu43@ucla.edu	Eric Walker Graduate Student   ewalker@yahoo.com	Hoa Lam Staff Research Associate   hoalam@ucla.edu
Larry Ackerson Research Technician   lackerson@mednet. ucla.edu	Wendy Walwyn Assistant Researcher   wwalwyn@ucla.edu	Jim Shoblock Postdoctoral Fellow   shoblock@ucla.edu

Nigel Maidment, Ph.D.
Professor

nmaidmen@ucla.edu
(310)206-7767

Our laboratory is interested in the regulation of synaptic transmission, and the relationship between changes in neurotransmitter release and psychiatric illness. We focus on the function of neurotransmitter transporters, the proteins responsible for transporting classical neurotransmitters such as dopamine and serotonin across biological membranes. Using a variety of molecular and genetic techniques, we study how neurotransmitter transport is regulated in cultured neurons and neuroendocrine cell, with a particular focus on the regulation of transporter trafficking to secretory vesicles. We are also using transporters as probes to screen for novel pathways involved in neurotransmitter release in the model organism Drosophila. Finally, we study how dysregulation of transmitter transport may affect neuropsychiatrically relevant behaviors in mice.

Laboratory Phone: (310)206-8323

Laboratory Staff:

David E. Patton Research Technician   dearl@ucla.edu	Christina L. Greer Staff Research Associate cgreer@ucla.edu	Hui-Yun Chang Postdoctoral Fellow   huiyun@ucla.edu
Anna Grygoruk Graduate Student   annagryg@ucla.edu	Rafael Romero Graduate Student   raromer@ucla.edu	Amy E. Shyer Undergraduate Student   asbruin05@aol.com
Lisa Brooks Graduate Student   lisa.brooks@stanfordalumni.org	Anne Simon Assistant Researcher

Brigitte Kieffer Laboratory
[Publications]

Opioid receptors are G protein coupled receptors, which mediate the strong analgesic and addictive activities of opiate drugs. These receptors and their endogenous ligands modulate numerous physiological functions, including the regulation of nociception, mood control and responses to stress. Three receptors classes, mu (MOR), delta (DOR) and kappa (KOR), were described by pharmacological approaches and we have cloned and characterized their genes in mouse and humans. We also have cloned an homologous receptor, ORL-1, considered to be part of an anti-opioid system involved in the development of opioid tolerance. We have analyzed the expression pattern of MOR, DOR, KOR and ORL-1 in the human central nervous system and immune cells. We have screened combinatorial peptide libraries on the four recombinant human receptors and discovered a peptide with dual MOR agonist/ORL-1 antagonist properties of potential therapeutical interest. We have used site-directed mutagenesis to identify determinants for opioid binding, signaling and receptor regulation of DOR and MOR. We also have characterized a signaling-deficient MOR polymorphic variant. We have produced mice lacking MOR, DOR and KOR by homologous recombination to evaluate the molecular mechanism of action of classical, as well as newly developed opiates of clinical interest in vivo and to clarify the specific implication of each opioid receptor in adult physiology. We have exposed MOR-deficient mice to morphine and our results indicate that lack of mu-receptors causes abolition of morphine-induced analgesia, place preference and physical dependence, as well as morphine respiratory depression and immunosuppression. This demonstrates that MOR is the major molecular target for morphine activity in vivo and indicates that mu receptors are essential in mediating both therapeutical and adverse effects of the prototypal opiate. We also have found altered behavior of mutant mice in response to alcohol and cannabinoids, demonstrating that the endogenous opioid system modulates the activity of other drugs of abuse. We have compared behavioral responses of mutant mice in several models of anxiety and depression. Our data show opposing phenotypes in MOR and DOR mutants, which contrasts with the classical notion of alike activities of mu- and delta receptors. They also show consistent anxiogenic- and depressive-like responses in DOR-/- mice, indicating that delta receptor activity contributes to improve mood states. We have compared nociceptive responses of mutant mice to acute pain and data suggest tonic activity of endogenous MOR, DOR, KOR systems with a distinct implication of each receptor to various pain modalities, and a prominent role of kappa receptors in the perception of visceral pain. We have generated triple knockout animals, where the entire opioid system is deleted and the mutant mice are viable and fertile.

On-going projects:

Mu receptors: the gate for opioid tolerance and dependence (C. Contet, D. Filliol, M. Rondeau). We investigate the postulated correlation between MOR desensitization at the cellular level and behavioral adaptations to chronic opiates in vivo, using gene targeting in mice. We also examine MOR polymorphic variants among heroin addicts in a clinical study. Delta receptors: a potential therapeutic target for pain treatment (F. Décaillot, D. Filliol, M. Ballié, G. Scherrer and C. Gavériaux-Ruff). We examine the molecular mechanism of delta receptor activation using random mutagenesis approach combined to a functional screen for the identification of constitutively active mutant receptors. Analysis of the mutants involves signaling studies in transfected cells, inverse agonist pharmacology and 3D-computer modeling. We investigate the role of delta receptors in pain perception as well as in inflammatory and neuropathic pain using mice lacking the DOR gene either permanently, or in a site- and time-controlled manner. Anti-opioids and tolerance to morphine analgesia (F. Simonin and A. Matifas): in collaboration with chemists we develop novel compounds acting at receptors belonging to opioid systems, including ORL-1 and receptors for Neuropeptide FF. Molecular basis for drug addiction (F. Simonin, K. Befort, A. Matifas, D. Filliol): here we aim at identifying novel genes involved in neuroadaptations to drugs of abuse, with an initial focus on opiates. Using genetic screens we identify novel genes encoding proteins that interact with opioid receptors at the level of their C-terminal domains. These proteins potentially modulate the regulation of receptor signaling and play a role in desensitization. We also develop mouse cDNA arrays to study differential gene expression following exposure to opiates and other drugs of abuse. Combined with substractive cloning, this approach will lead to identify molecular mechanisms of tolerance, withdrawal and craving.

Laboratory Staff:

Brigitte Kieffer, Ph.D.
Professor
IGBMC (France)

briki@igbmc.u-strasbg.fr
33 (0)3 90 24 47 48

Tim Hales Laboratory
[Publications]

Repeated stimulation of neuronal pathways leads to long-term changes in cellular function. Lasting changes in function also occur during prolonged or repetitive exposure to drugs, such as opioids, leading to tolerance, dependence and craving. Opioid receptors transduce signals through multiple effectors reducing neuronal excitability. The contribution of specific effectors to the beneficial and detrimental actions of opioids is poorly understood. Opioid receptors modulate ion channels, a phenomenon that cannot be studied in unexcitable cell lines without also expressing additional proteins. Lei Song  is examining the coupling pathways between cloned mu delta- and mu delta-opioid receptors and native K_IR⁺ and Ca²⁺ channels in GH₃ cells. Her observations suggest that distinct pathways are involved that can be preferentially activated by specific opioid ligands.

A role for L-type Ca²⁺ channels?

Former Neuroscience Graduate Student Elemer Piros originally demonstrated that opioid receptors couple to L-type Ca²⁺ channels in GH₃ cells. Under the direction of Jim Boulter, Parsa Safa identified several splice variants arising from the L-type alpha1D gene in GH₃ cells. Using long-range PCR he amplified each variant and established its relative abundance in GH₃ cells and rat brain. Parsa is now studying the recombinant alpha1D channels using the whole-cell patch-clamp technique. Not all of the splice variants form functional channels, but those that do, have important properties that may predispose them to particular cellular functions. Activated G proteins inhibit a specific alpha1D subunit splice variant expressed in GH₃ cells and rat brain. We are looking for colocalization of opioid receptors and this alpha1D splice variant in rat brain using in situ hybridization. Ca²⁺ entry through L-type channels regulates gene expression, our demonstration that opioid receptors can couple to a subtype of this family of ion channels provides a mechanism by which opioids could cause lasting changes in neuronal function.

Opioid tolerance leads to dependence and withdrawal, events responsible for the negative reinforcing qualities of drugs of abuse. By better understanding these processes it may be possible to design compounds that can help prevent or reverse the adaptations that lead to drug dependence. There are few such pharmacotherapies available at present. Recently, ondansetron, an antagonist of a specific subtype of serotonin (5-HT₃) receptor, has shown promise in aiding opioid withdrawal. Ondansetron is used clinically to reduce nausea and vomiting associated with chemotherapy. The fact that opioid and 5-HT₃ receptors are coexpressed by some neurons and the observation that ondansetron can facilitate withdrawal from opioids suggest that there is convergence between the signaling pathways of these two receptors. Ansalan Stewart (Postdoctoral Fellow) is using Ca²⁺ imaging and electrophysiological techniques to determine whether 5-HT₃ and opioid receptor-signaling pathways converge at the level of the voltage-activated Ca²⁺ channel or if opioid receptors inhibit 5-HT₃ receptor activity directly.

Our future studies will focus on the compartmentalization of Ca²⁺ channel subtypes in the brain and how their expression patterns and functional properties are altered by prolonged drug exposure.

Tim Hales, Ph.D.
Associate Professor
George Washington University

phmtgh@gwumc.edu
(202)994-3541

Michael Fanselow Laboratory
[Publications]

Our laboratory focuses on two broad areas related to how opioids and opioid-related behaviors are intertwined with memory and emotional processes. One goal is to unravel the brain mechanisms controlling the formation of associations between opiate drugs and the contexts they are administered in. A second set of questions is how endogenous-opioids, particularly through their analgesic actions, regulate Pavlovian fear conditioning. The laboratory uses rat and mouse models featuring site-specific pharmacological manipulations, focal brain lesions and genetic modifications.

Michael Fanselow, Ph.D.

fanselow@psych.ucla.edu
(310)206-0247

Andrew Charles Laboratory
[Publications]

Investigation of non-synaptic glial, neuronal, and endothelial cell signaling mechanisms with simultaneous imaging and electrophysiology.

Andrew Charles, M.D.

(310)206-9799
acharles@ucla.edu