Research Findings - Basic Neurosciences Research

Pregnancy & Cigarette Smoking

Children of women who smoked during pregnancy are at increased risk of dependence when smoking is initiated during adolescence. Studies conducted in experimental animals demonstrate that gestational nicotine exposure attenuated dopamine release induced by nicotine delivered during adolescence. In a recent study, NIDA supported researchers report that exposing pregnant rats to nicotine for a period equivalent to the three trimesters of human brain development period reduced nicotine cholinergic receptor (nAChR) expression in dopaminergic regions during adolescence. This reduction reflects lower nAChR subunits transcript levels and fewer neurons in the VTA, as well as other undefined mechanisms. These data indicate that gestational nicotine exposure affected the developmental regulation of nAChR expression and these effects can endure at least into adolescence. These findings are important from a public health perspective because the down-regulation of nAChR expression during brain development may result in heightened vulnerability to dependence on cigarette smoking that affects adolescent offspring of women who smoked tobacco during pregnancy. Chen, H., Parker, S.L., Matta, S.G., and Sharp, B.M. Gestational Nicotine Exposure Reduces Nicotinic Cholinergic Receptor (nAChR) Expression in Dopaminergic Brain Regions of Adolescent Rats. European Journal of Neuroscience, 22, pp. 380-388, 2005.

Cocaine and Progression of HIV

Cocaine is associated with an increased risk for, and progression of, clinical disease associated with human immunodeficiency virus (HIV) infection. In a recent study, NIDA supported researchers, Dr. Michael Roth and his associates at UCLA report findings of their studies designed to investigate the biological interactions between cocaine and HIV infection. These investigations were conducted using a hybrid human-mouse model in which human peripheral blood mononuclear cells (huPBL) were implanted into severe combined immunodeficiency mice (huPBL-SCID) and then infected with a HIV reporter virus. Systemic administration of cocaine significantly increased the percentage of HIV-infected PBL and viral load in huPBL-SCID mice suggesting a dynamic interaction between drug exposure and viral infection. They also report that despite the capacity for cocaine to increase corticosterone and adrenocorticotropic hormone levels in control mice, the hypothalamic pituitary- adrenal axis was suppressed in HIV-infected animals, and corticosterone levels were further decreased when animals were exposed to HIV and cocaine. Activating huPBL in vitro in the presence of cocaine increased expression of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) co-receptors. Expression of CCR5 was also increased at early time-points in the huPBL-SCID mouse model following systemic exposure to cocaine. This effect preceded the boost in viral infection and waned as HIV infection progressed. In addition, they report that a selective sigma -1 antagonist, BD1047, blocked the effects of cocaine on HIV replication in the huPBL-SCID mouse. This is consistent with a previous finding that cocaine' s immunosuppressive effects in vitro are mediated by sigma-1 receptors. Thus, these findings suggest that systemic exposure to cocaine can enhance HIV infection in vivo by activating sigma-1 receptors and by modulating the expression of HIV co-receptors. Roth, M.D., Whittaker, K.M., Choi, R., Tashkin, D.P., and Baldwin, G.C. Cocaine and _-1 Receptors Modulate HIV Infection, Chemokine Receptors, and the HPA Axis in the huPBL-SCID Model. Journal of Leukocyte Biology, 78, pp. 1198-1203, 2005.

Opiate Modulation of Immunity and Antiviral Action

Numerous investigators have provided information on opiate inhibition of antiviral activity, especially in regards to anti-HIV action. Wang and his collaborators have continued to define this relationship and have provided an in-depth understanding of the mechanism of this action at the molecular level of cellular activity. Opiates have profound effects on the function of human immune cells and are a possible cofactor in the immunopathogenesis of human immunodeficiency virus (HIV) disease. Wang et al. investigated the impact of morphine on CD8(+) T cell-mediated, noncytotoxic, anti-HIV activity in latently infected human immune cells. Morphine inhibited the noncytotoxic, anti-HIV activity of CD8(+) T cells in HIV latently infected cells (U1 and J1.1). Naltrexone abolished the morphine-mediated, inhibitory effect on the noncytotoxic, anti-HIV activity of CD8(+) T cells. Interferon-gamma (IFN-gamma), a potent antiviral cytokine produced by CD8(+) T cells, was partially responsible for CD8(+) T cell-mediated, noncytotoxic, anti-HIV activity. The anti-HIV activity of IFN-gamma was also compromised by morphine treatment. Further, morphine attenuated CD8(+) T cell-mediated suppression of the HIV long-terminal repeat promoter activation. Morphine also inhibited CD8(+) T cell-induced expression of the signal transducer and activator of transcription-1, an important transcriptional factor in the IFN signaling pathway. These data provide additional evidence to support the notion that opioids play a role in impairing the anti-HIV function of the immune system. Wang, X., Tan, N., Douglas, S.D., Zhang, T., Wang, Y-J. and Ho, W-Z. Morphine Inhibits CD8+ T Cell-mediated, Noncytolytic, Anti-HIV Activity in Latently Infected Immune Cells. Journal of Leukocyte Biology, 78, pp. 772-776, 2005.

Morphine Withdrawal Contributes to Th Cell Differentiation by Biasing Cells Toward the Th2 Lineage

The consequences of drug withdrawal on immune functioning have only recently been appreciated; however, given the wide variety of use and abuse of opiate analgesics, understanding the decrements to immune function that withdrawal from these drugs cause is of crucial importance. The immune system functions in many ways to combat diseases. There are lymphocytes that combat viruses/bacteria upon entry and others that develop antibody production to combat them. The entry of virus/bacteria into the host is met by Th1-type cells to keep down the proliferation of the invader. Th2 cells and B-cells act together to produce antibodies, a later event in host defense. Numerous investigators are observing a change in the profile of Th1/Th2 type cell when drugs are administered. Many other compounds act similarly in altering the body' s host defense mechanism. Although most investigators look at the early actions of drugs on T-cell maturation, a recent study has observed similar profile alterations when a chronically-administered-drug is withdrawn. Thus, it may be as important to be aware of disease states during drug withdrawal as it is to oversee potential infectious situations following opiate administration following surgery or other drug use situations. Kelschenbach, J., Barke, R.A. and Roy, S. Morphine Withdrawal Contributes to Th Cell Differentiation by Biasing Cells Toward the Th2 Lineage. The Journal of Immunology, 175, pp. 2655-2665, 2005.

Neuroprotection by Pharmacologic Blockade of the GAPDH Death Cascade

NIDA supported investigator, Dr. Solomon Snyder and his colleagues, have identified a key biochemical pathway that plays a role in mediating cell death and may provide potential treatment for neurodegenerative diseases. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is normally involved in sugar metabolism. However, under certain conditions of cell stress GAPDH is nitrosylated by nitric oxide. Nitrosylation of GAPDH abolishes its enzymatic activity and converts it to a cell death molecule by binding to SIAH (sevenless in absentia homolog), a ubiquin E3 ligase. Nitrosylated GAPDH acting as chaperone binds to SIAH causing the GAPDH/SIAH complex to translocate to the cell nucleus where degradation of SIAH targets produces cell death. Now Dr. Snyder and his colleagues show that both deprenyl, a drug used to treat Parkinson' s disease and a related agent, TCH346, block cell death by preventing nitrosylation of GAPDH and subsequent translocation of SIAH to the nucleus in both macrophage cell lines treated with LPS and in dopamine neurons treated with MPTP, a toxin that damages dopamine neurons. In cultures of cerebellar granule cells deprenyl prevented apoptotic cell death elicted by etoposide. This work suggests that deprenyl may not only be effective for treating Parkinson disease but may also be useful in treating other neurodegenerative disorders. Hara, M.R., Thomas, B., Cascio, M.B., Bae, B-I., Hester, L.D., Dawson, V.L., Dawson, T.M., Sawa, A. and Snyder, S.H. Neuroprotection by Pharmacologic Blockade of the GAPDH Cascade. Proceedings of the National Academy of Science of the United States of America, 103, pp. 3887-3889, 2006.

A Nitric Oxide (NO) Signaling Pathway Controls CREB-Mediated Gene Expression in Neurons

Neurotrophins such as BDNF are thought to mediate the actions of drugs of abuse such as cocaine. The actions of neurotrophins are in part mediated by activating the transcription factor, CREB (cyclic AMP Response Element Binding protein) to alter gene expression. In the classical model, phosphorylation of CREB on serine 133 by CREB kinases causes CREB to associate with CBP (CREB binding protein) and bind to CRE to initiate transcription. Recent work by the laboratory of Dr. Solomon Snyder in collaboration with the laboratories of Dr. David Ginty and Dr. Ted Dawson at Johns Hopkins Medical Center suggest that phosphorylation is not required for activation of CREB. Transcriptional activation by CREB remains even when phosphorylation of CREB 133 is blocked. Instead Dr. Snyder and his colleagues show that binding of CREB to CRE is blocked by inhibitors of nitric oxide synthase (NOS) in a calcium dependent manner and that NO is sufficient to induce CREB binding to CRE. Furthermore, CREB dependent transcription is absent in mice lacking the nNOS, the neuronal form of nitric oxide synthase. Because nNOS acts either by activating cyclic GMP or by nitrosylating proteins, Dr. Snyder and his colleagues tested whether CREB binding is mediated by either one of these signaling pathways. Inhibitors of cyclic GMP dependent protein kinase did not abolish CREB binding to CRE in response to BDNF. Although Dr. Synder and his colleagues did not find CREB to be nitrosylated following neurotrophin treatment, they suggest that inactivation of histone deacetylase 2 by nitrosylation permits histone 3 and histone 4 to become acetylated, enabling CREB to bind CRE. These results suggest that the binding of CREB to CRE is independent of the ras/erk pathway and is mediated by nitrosylation of nuclear proteins. Riccio, A., Alvania, R.S., Lonze, B.E., Ramanan, N., Kim, T., Huang, Y., Dawson, T.M., Snyder, S.H., and Ginty, D.D. A Nitric Oxide Signaling Pathway Controls CREB-Mediated Gene Expression in Neurons. Molecular Cell, 21(2), pp. 283-294, 2006.

p11: A New Link Between Serotonin and Depression

Therapeutic modulation of serotonin metabolism is used to treat a number of neuropsychiatric disorders. The 5-HT1B serotonin receptor subtype, which regulates serotonin neurotransmission, has been implicated in a variety of behaviors including depression, anxiety, sleep, and drug addiction. Using a protein interaction screen, Dr. Greengard and coworkers discovered that the p11 protein specifically binds to the 5-HT1B receptor. Further experiments indicated that the mRNA transcripts of these two genes are expressed in similar patterns in rat brain, the two proteins bind to one another in vivo, and both proteins co-localize on the cell surface. The p11 protein contains calcium-binding motifs, and can translocate proteins to the plasma membrane. Does p11 play a role in depression? Dr. Greengard found that p11 mRNA levels in the brain were reduced in post-mortem patients who suffered from major depression, as well as in a mouse model of depression. Interestingly, p11 mRNA levels increase in response to imipramine and to electroconvulsive therapy, both of which are treatments for depression. Does experimental manipulation of p11 levels alter behavior with respect to depression? Dr. Greengard used genetic techniques to decrease and increase p11 levels in living animals. In some brain regions, mice with genetic inactivation of p11 function had fewer binding sites for a chemical antagonist which binds the 5-HT1B serotonin receptor. These animals also exhibited higher levels of depression-like states and were less responsive to a sweet reward. In contrast, animals overexpressing p11 had more 5-HT1B serotonin receptors. These mice had decreased levels of behaviors associated with depression and anxiety, and behaved more like animals treated with antidepressants. Dr. Greengard' s work establishes a role for p11 modulation of 5-HT1B serotonin receptor levels, leading to alterations in depression-related behavior. This work reveals that the tricyclic antidepressant imipramine functions through p11 and the 5-HT1B serotonin receptor to ameliorate depression. The identification of p11 and its 5-HT1B regulatory role enhances our understanding of how serotonin receptors are regulated and may lead to the identification of new therapies for the treatment of a variety of diseases of the brain, including depression and drug addiction. Svenningsson, P., Chergui, K., Rachleff, I., Flajolet, M., Zhang, X, El Yacoubi, M., Vaugeois, J-M., Nomikos, G.G., and Greengard, P. Alterations in 5-HT1B Receptor Function by p11 in Depression-Like States. Science, 311, pp. 77-80, 2006.

Opiate Reward is Mediated by the Neurotransmitter Norephinephrine

Opiate reward is the pleasure one experiences upon taking an opiate such as morphine. Over thirty years ago, the neurotransmitters norepinephrine (NE) and dopamine (DA) were linked to opiate reward, and the role of DA in this process has been well characterized. However, a conclusive role for NE in this process has not been established. Dr. Palmiter and co-workers investigated opiate reward in mice lacking NE. NE is normally synthesized from DA by dopamine beta-hydroxylase (DBH), so mice with a genetic disruption of the DBH gene lack NE. DBH mutant animals were tested using a conditioned place preference paradigm, in which the animals were treated with morphine in a specific environmental milieu. If morphine treatment is perceived by the animals as pleasurable, the animals will prefer the environmental conditions that coincide with treatment. If it is not perceived as pleasurable the animals will be indifferent to these conditions, and if the treatment is perceived as unpleasant, the animals will avoid these environmental conditions. Normal mice prefer the environment associated with morphine treatment, but DBH mutant animals show no such preference, suggesting that they do not experience opiate reward. Dr. Palmiter then restored NE to the DBH mutant animals by treatment with a compound called DOPS, which can be converted to NE without the need for the DBH enzyme. DBH mutant mice treated with DOPS now prefer the environment associated with the morphine treatment, indicating that NE is required for the animals to experience opiate reward. Dr. Palmiter then investigated where NE is required in the brain to mediate opiate reward, and found that restoration of norepinephrine signaling only to a specific region of the brain called the Nucleus Tractus Solitarus was sufficient to allow opiate reward. This work conclusively demonstrates a crucial role for the neurotransmitter norepinephrine in mediating opiate reward, and will lead to a deeper understanding of the molecules and brain regions that mediate behavioral responses to drug taking. Olson, V.G., Heusner, C.L., Bland, R.J., During, M.J., Weinshenker, D., and Palmiter, R.D. Role of Noradrenergic Signaling by the Nucleus Tractus Solitarius in Mediating Opiate Reward. Science, 311, pp. 1017-1020, 2006.

Suppression of Dynorphin by DeltaFosB Results in Opioid Addiction

Chronic exposure to drugs of abuse like cocaine induces upregulation of the transcription factor deltaFosB, which acts as a molecular switch for addiction in the nucleus accumbens (NAc). Whether morphine addiction shares the same molecular mechanism as cocaine, and if it does, how this mechanism functions in morphine addiction is not clear. Nestler and colleagues at the University of Texas Southwestern Medical Center tackled this question in a NIDA funded research project. Using a bitransgenic mouse line that inducibly overexpresses deltaFosB in the NAc and dorsal striatum and using viral-mediated gene transfer to specifically express it only in the NAc, Nestler demonstrated that deltaFosB is necessary and sufficient to mediate all aspects of the addictive effect of opioids. Thus, deltaFosB overexpression in the NAc increased the sensitivity of the mice to the rewarding effects of morphine and led to exacerbated physical dependence, but also reduced their sensitivity to the analgesic effects of morphine and led to faster development of analgesic tolerance. Furthermore, Dr. Nestler and his colleagues report that the endogenous opioid peptide dynorphin seemed to be at least one of the main targets through which deltaFosB produced this behavioral phenotype: first, the action of deltaFosB is dependent on its expression in dynorphin expressing neurons in NAc, but not in other neurons that do not express dynorphin. Second, the induction of deltaFosB within dynorphin neurons in the NAc alone, in response to the chronic opioid administration, is sufficient to cause many of the various types of behavioral plasticity altered by the abuse. Third, deltaFosB has been shown to bind to the dynorphin gene promoter in cultured neurons, and the researchers suggest a direct action of deltaFosB on suppressing dynorphin expression, which results in opioid addiction. Together, these experiments demonstrated that deltaFosB in the NAc, at least partly through the repression of dynorphin expression, mediates several major features of opiate addiction. Zachariou, V., Bolanos, C.A., Selley, D.E., Theobald, D., Cassidy, M.P., Kelz, M.B., Shaw-Lutchman, T., Berton, O., Sim-Selley, L.J., Dileone, R.J., Kumar, A. and Nestler, E.J. An Essential Role for _FosB in the Nucleus Accumbens in Morphine Action. Nature Neuroscience, 9, pp. 205-211, 2006.

Cocaine-induced Dendritic Spine Formation in D1 and D2 Dopamine Receptor-Containing Medium Spiny Neurons in Nucleus Accumbens

Drug addiction is a chronic relapsing disease suggesting that drugs of abuse produce long lasting changes in the brain. The mechanism by which drugs of abuse produce these long lasting changes in the brain even after the drug is no longer present is not clear. In this study, Dr. Lee and colleagues examined nerve cells in a region of the brain called the nucleus accumbens (NAcc) either 2 days or 30 days after the last exposure to cocaine, which had been administered for 4 weeks prior to removal. They were specifically looking at nerve cells, medium spiny neurons (MSN) that contain several projections called spines. There are two primary classes of MSNs in the NAcc: the dopamine receptor D1-containing cells and the D2 receptor-containing cells. In their analysis, about 50% of the MSNs contain only D1 receptors, 35-40% contain only D2 receptors and 10-15% contain both types of receptors. Using innovative analytical techniques, they found that while there was an increase in the number of spines in both D1 and D2-containing cells two days after the last administration of cocaine, only the increase seen in D1-containing cells persisted to day 30, although at a reduced level than was seen at two days. Another change that they saw in the D1-containing cells that coincided with the persistence of spines was the induction of another important molecule that has been implicated in longer-term effects of cocaine, deltaFosB. Not only did they see an induction, but deltaFosB persisted to a greater extent in the D1-containing cells after 30 days than it did in the D2-containing cells, much like the pattern they see with the spine persistence. Further study is needed to determine whether deltaFos B directly regulates cell morphological changes. Lee, K-W., Kim, Y., Kim, A.M., Helmin, K., Nairn, A.C. and Greengard, P. Cocaine-induced Dendritic Spine Formation in D1 and D2 Dopamine Receptor-containing Medium Spiny Neurons in Nucleus Accumbens. Proceedings of the National Academy of Sciences of the United States of America, 103, pp. 3399-3404, 2006.

Mechanism of Synaptic Plasticity Modulated by Postsynaptic Events

Long term synaptic modulation requires presynaptic structural changes induced by postsynaptic activity. The molecular mechanisms by which postsynaptic modifications lead to precisely coordinated changes in presynaptic structure and function are unknown. To address this issue, NIDA funded researchers at Stanford University, led by Craig Garner and Robert Malenka, examined the presynaptic consequences of postsynaptic expression of members of the membrane-associated guanylate kinase family of synaptic scaffolding proteins, by postsynaptic expression of synapse-associated protein 97 (SAP97). Previous studies suggested that SAP97 can influence synaptic AMPA receptors when overexpressed postsynaptically in culture, and perhaps also affect presynaptic function. In the present study, the investigators engineered overexpression of SAP97 in vivo and probed the cellular events that alter presynaptic structure. They found that compared with other SAP family members, namely SAP90/PSD-95 and SAP102, postsynaptic expression of SAP97 has the most potent presynaptic effect, dramatically increasing levels of the active zone protein Bassoon and the vesicle proteins synapsin and synaptophysin. Recruitment of these presynaptic proteins dramatically affects presynaptic function, as evidenced by a marked increase in N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl) hexatrienyl) pyridinium dibromide (FM4-64) dye uptake. Furthermore, increasing postsynaptic SAP97 also causes a selective increase of several binding partners including the AMPAR subunit GluR1 and the scaffold proteins ProSAP2, Shank1a, and SPAR/SPAL (spine-associated RapGAP/SPA-1-like protein). In addition, they report that the transsynaptic effects of postsynaptic SAP97 require multiple cell adhesion and signaling molecules including cadherins, integrins, and EphB receptor/ephrinB. These results suggest that SAP97 plays a central role in coordinating the growth and functional organization of the presynaptic and postsynaptic components of synapses during both development and synaptic plasticity. Regalado, M.P., Terry-Lorenzo, R.T., Waites, C.L., Garner, C.C. and Malenka, R.C. Transsynaptic Signaling by Postsynaptic Synapse-Associated Protein 97. The Journal of Neuroscience, 26, pp. 2343-2357, 2006.

Cell Signaling Events in Neural Circuit Associated with Appetite in the Brain

Hedonic or motivated aspects of feeding are mostly mediated by perifornical lateral hypothalamic (LH) neurons. Endogenous cannabinoids and anorexigenic hormone leptin have opposing effects on these neurons, with opposite effects on feeding behavior; but the cellular mechanisms of these endogenous food intake modulators are not known. Using electrophysiology on acute brain slice preparation and aided by single cell RT-PCR and immunohistochemistry, NIDA funded researchers at Columbia University led by Lorna Role report that depolarization of LH neurons elicits a cannabinoid receptor CB1-mediated suppression of inhibition in local circuits thought to be involved in appetite and "natural reward." The depolarization-induced decrease in inhibitory tone to LH neurons is blocked by leptin. Leptin inhibits voltage-gated calcium channels in LH neurons via the activation of janus kinase 2 (JAK2) and of mitogen activated protein kinase (MAPK). Importantly, these researchers further suggest that these signaling cascades activated by leptin suppresses motivated feeding through the reduction of endogenous cannabinoids in the brain. The present study provides mechanistic evidence on how the integration of endocannabinoid and leptin signaling regulates the excitability of lateral hypothalamic neurons in appetite-related circuits. Jo, Y-H., Chen, Y-J.J., Chua, S.C. Jr., Talmage, D.A. and Role, L.W. Integration of Endocannabinoid and Leptin Signaling in an Appetite-Related Neural Circuit. Neuron, 48, pp. 1055-1066, 2005.

Allelic Expression Imbalance of Human mu Opioid Receptor (OPRM1) Caused by Variant A118G

The mu opioid receptor is the primary target for opioid drugs and plays a key role in addiction and pain perception. The single nucleotide polymorphism A118G, leads to an Asn40Asp substitution with uncertain functions, and an allele frequency range of 10-32%. Wolfgang Sadee and his colleagues have measured allele specific mRNA expression of OPRM1 in human autopsy brain tissues, using A118G as a marker. In 8 heterozygous samples measured, the A118 mRNA allele was 1.5-2.5 fold more abundant than the G118 allele. Inserting C118 or T118 into OPRM1 failed to affect mRNA expression compared with the wild-type A118. These results demonstrate that substitution with G118 alone, in the absence of any other regulatory regions, causes a significant change in mRNA expression. No differences in mRNA stability between the two variants were detected, indicating partial defects in transcription or mRNA maturation. Western blotting also demonstrated a much lower protein yield for the G118, indicating that the variant appears to affect translation or post-translational processing and turnover of OPRM1 protein. This study indicates that the OPRM1-G118 is a functional variant with deleterious effects on both mRNA and protein yield. Clarifying the functional relevance of polymorphisms associated with susceptibility to a complex disorder such as drug addiction and pain provides a foundation for clinical association studies. Zhang, Y., Wang, D., Johnson, A., Papp, A. and Sadee, W. Allelic Expression Imbalance of Human mu Opioid Receptor (OPRM1) Caused by Variant A118G. Journal of Biological Chemistry, 280, pp. 32618-32624, 2005.

Cocaine-Seeking Behavior and AMPA Receptor Trafficking in the Nucleus Accumbens

Work in Yavin Shaham's lab at the NIDA IRP demonstrated that cocaine-seeking behavior increases progressively over the first three months of withdrawal from cocaine self-administration in rats, a phenomenon termed "incubation of cocaine craving." NIDA researcher Marina Wolf and her colleagues sought to determine if increased cell surface expression of glutamate AMPA receptors in the nucleus accumbens (NAc), leading to increased excitatory drive to NAc neurons, might underlie the behavioral changes observed during the incubation of cocaine craving. AMPA receptor subunits on the cell surface were distinguished from those inside the cell, where they are not active, using a membrane-impermeant cross-linking agent that selectively modifies surface proteins. Rats were trained to self-administer cocaine (6 hrs/day/10 days) and tested for drug-seeking behavior on day 1 or 45 of withdrawal. Rats tested on withdrawal day 45 responded significantly more than those tested on withdrawal day 1. Other rats were treated identically for protein cross-linking studies showed a robust increase (~500%) in total GluR1 protein levels (surface + intracellular) on day 45 compared with day 1, but no change in the surface/intracellular ratio. This indicates that NAc neurons are producing more GluR1 and transporting the same proportion of GluR1 to the surface, resulting in an increase in absolute levels of GluR1 on the surface. This would be expected to enhance the responsiveness of NAc neurons to glutamate inputs from cortical and limbic regions that trigger drug-seeking behavior. Interestingly, the same cross-linking assay showed that behavioral sensitization to cocaine is accompanied by different changes that also result in a net increase in AMPA receptor surface expression. Boudreau, A.C. and Wolf, M.E. Behavioral Sensitization to Cocaine Is Associated with Increased AMPA Receptor Surface Expression in the Nucleus Accumbens. Journal of Neuroscience, 25, pp. 9144-9151, 2005. A goal of future studies is to determine if AMPA receptor trafficking represents a "link" between locomotor sensitization and incubation of cocaine craving. Conrad, K.L., Marinelli, M. and Wolf, M.E. Cocaine-Seeking Behavior and AMPA Receptor Trafficking in the Nucleus Accumbens. Society for Neuroscience Abstract, Program No. 1030.1, 2005.

Delta Opioid Receptor Ligands Modulate Anxiety-Like Behaviors in the Rat

Dr. Unterwald of Temple University School of Medicine examined the role of the delta opioid receptor in regulating anxiety-like behavior in male Sprague-Dawley rats. Using an elevated plus maze, the effects of the selective delta opioid receptor antagonist naltrindole (1 or 5 mg/kg) and agonist SNC80 (1, 5 or 20 mg/kg) on anxiety-like behavior were measured. Anxiety was also measured following administration of diazepam (3 mg/kg) and amphetamine (1 mg/kg) and compared to the effects of SNC80. Locomotor activity following administration of naltrindole, SNC80, diazepam, and amphetamine was measured. Finally, the defensive burying paradigm was used to confirm the findings from the elevated plus maze. Results demonstrated that SNC80 produced dose-dependent anxiolytic effects similar to that of the classical antianxiety agent, diazepam. Administration of naltrindole caused anxiogenic behavior in rats further supporting the involvement of the delta opioid receptor system in regulating anxiety. Naltrindole also blocked the anxiolytic effects of SNC80. Amphetamine had no effect on anxiety-like behavior. SNC80 induced hyperactivity similar to amphetamine at the doses tested, while naltrindole and diazepam did not significantly affect locomotor activity. Although SNC80 can increase locomotor activity, control experiments reported herein indicate that hyperlocomotion is not sufficient to produce an anxiolytic response on the elevated plus maze. Together with the results from the defensive burying paradigm, this suggests that the effects of SNC80 on reducing anxiety are independent of its effects on locomotion. Collectively these data show that the delta opioid receptor system can regulate anxiety-like behavior in an anxiolytic (agonist) and anxiogenic (antagonist) manner. Perrine, S.A., Hoshaw, B.A. and Unterwald, E.M. Delta Opioid Receptor Ligands Modulate Anxiety-like Behaviors in the Rat. British Journal of Pharmacology, pp. 1-9, 2006 [Epub ahead of print].

Endocannabinoids Act on Inhibitory Interneurons and Modulate an Intrinsic Population Rhythm of Hippocampal Neurons

Assessment of single neuronal behavior reveals that endocannabinoids (eCB) fine-tune the dynamic status of synaptic activity, strength and plasticity of hippocampal neurons. Upon afferent activation, the principal neuron discharges an action potential. By releasing eCBs, it sends a retrograde feedback signal to inhibitory interneurons, which in turn affects the discharge pattern and intensity of the principal neurons they innervate. This phenomenon, known as depolarization suppression of inhibition (DSI), is observed wherever the eCBs affect the theta rhythm wave, an intrinsic neuronal population rhythm of hippocampal neurons. Theta rhythms are behaviorally relevant electrical oscillations in the mammalian brain, particularly in the hippocampus, and metabotropic glutamatergic and/or cholinergic inputs often drive these oscillations via the activity of inhibitory postsynaptic potentials (IPSPs). Dr. Alger' s recent work shows that perisomatic-targeting interneurons, whose output is inhibited by endocannabinoids, are the likely source of theta IPSPs. Under conditions that block all glutamate receptors, theta rhythm IPSPs induced by muscarinic acetylcholine receptors in hippocampal CA1 region can be transiently interrupted by action potential-induced, retrograde endocannabinoid release. Simultaneous recordings from pyramidal cell pairs reveal synchronous theta-frequency IPSPs in neighboring pyramidal cells, suggesting that these IPSPs may help entrain or modulate small groups of pyramidal cells. With the capability of rapidly switched-on by afferent inputs and switched-off by the eCB-mediated retrograde feedback DSI, the intrinsic neuronal network may constitute the fundamental mechanism for temporal coding and decoding in the hippocampus. Disruption of theta rhythm might be one mechanism by which cannabinoid drugs cause cognitive dysfunction. Reich, C.G., Karson, M.A., Karnup, S.V., Jones, L.M. and Alger, B.E. Regulation of IPSP Theta Rhythm by Muscarinic Receptors and Endocannabinoids in Hippocampus. Journal of Neurophysiology, 94, pp. 4290-4299, 2005.

Estradiol Selectively Reduces the Stimulated Release of GABA in Rat Striatum

NIDA-supported research has shown that females (rats and humans) appear to be more sensitive to the effects of psychostimulants than males. Research conducted in Dr. Jill Becker' s laboratory suggests that the naturally occurring higher concentrations of estrogen in females relative to males may be related to this observation. Becker and her colleagues ovariectomized female rats, replaced the estradiol in some of them, and monitored the efflux of GABA, taurine, and glutamate in the striatum after local application of 75 mM K+. They found that GABA and taurine were both enhanced in the striatum after the K+ challenge, but that the increase in GABA was much less in rats that also were given estradiol; glutamate did not change after challenge in either group. As GABA is the predominant inhibitory transmitter in the brain, this finding may be important for our understanding of how estradiol can alter neurotransmission and how that may be related to the differential effects of stimulants on males and females. Hu, M., Watson, C.J., Kennedy, R.T. and Becker, J.B. Estradiol Attenuates the K+ -induced Increase in Extracellular GABA in Rat Striatum. Synapse, 59, pp. 122-124, 2006.

Estrogen Neuroprotection from HIV Protein-Induced Oxidative Stress

Estrogen replacement therapy in older women is associated with improvement of symptoms of dementia and Parkinson' s Disease, so it is believed that estradiol may have neuroprotective qualities in some circumstances. Although the mechanisms of such neuroprotection are unknown, it is possible that estradiol acts as a free-radical scavenger to reduce oxidative stress. Some HIV infected patients experience similar neurological problems, likely involving glutamate and/or oxidative stress-mediated neurotoxicity that may be caused in part by extracellular HIV proteins gp120 and Tat. This study used human neuronal cells grown in culture to test whether estrogen could reverse damage by tat and gp120, as well as the free-radical generator SIN-1. Both tat and gp120, alone and combined, increased oxidative stress as measured by a fluorescent indicator. This increase was greatly diminished in the presence of estradiol but not progesterone or estradiol plus a selective antagonist. When exposed to tat or gp120, synaptosomes made from rat striatal tissue showed reduced dopamine uptake, and this effect was blocked by preincubation with estradiol. Reduced dopamine transporter activity in the striatum is a component of HIV-associated neuropathology; so together these findings suggest that estradiol may be a useful strategy for neuroprotection in the context of HIV-associated neurological disease. Wallace, D.R., Dodson, S., Nath, A. and Booze, R.M. Estrogen Attenuates gp120- and Tat(1-72)-Induced Oxidative Stress and Prevents Loss of Dopamine Transporter Function. Synapse, 59, pp. 51-60, 2006.

Opioid-Induced Tolerance and Dependence In Mice Is Modulated by the Distance Between Pharmacophores in a Bivalent Ligand Series

Given the mounting evidence for involvement of opioid receptors in the tolerance and physical dependence of µ opioid receptor agonists, Portoghese and his collaborators investigated the possible physical interaction between µ and opioid receptors by using bivalent ligands. Based on reports of suppression of antinociceptive tolerance by the antagonist naltrindole (NTI), bivalent ligands (µ- agonist-antagonist series, MDAN series) that contain different length spacers, and pharmacophores derived from NTI and the µ agonist oxymorphone, have been synthesized and evaluated. Chronic i.c.v. studies revealed that MDAN ligands whose spacer was 16 atoms or longer produced less dependence than either morphine or µ monovalent control (MA-19). On the other hand, both physical dependence and tolerance were suppressed at MDAN spacer lengths of 19 atoms or greater. When the spacer length was longer than 22 � (MDAN-19 to -21), neither tolerance nor dependence were observed, which is in harmony with the idea that these bivalent ligands bridge neighboring µ and opioid receptors effectively. The mechanism by which the bridging of the µ- heterodimer suppress tolerance and dependence is not understood. A possible explanation is that the µ- heterodimer is the fundamental signaling unit that mediates tolerance and dependence through specific signal transducer(s) that recognize and couple to the heterodimer but not µ receptor monomers/homomers. The finding that receptor knockout mice do not become tolerant is consistent with this concept. Given these results, it appears reasonable that bridging µ- heterodimers by MDAN ligands would negatively modulate such putative transducers, thus reducing tolerance and dependence. These data also suggest that physical interaction between the µ and opioid receptors modulates µ-mediated tolerance and dependence and MDAN-21 offers an approach for the development of potent, bioavailable analgesics devoid of these side effects. Daniels, D.J., Lenand, N.R., Etienne, C.L., Law, P-Y., Roenig, S.C. and Portoghese, P.S. Opioid-induced Tolerance and Dependence in Mice is Modulated by the Distance Between Pharmacophores in a Bivalent Ligand Series. Proceedings of the National Academy of Sciences of the United States of America, 102, pp. 19208-19213, 2005.

Neurokinin 1 Receptor (NK1R) Internalization is Inhibited Both in vivo and in vitro by mu- and delta-Opioid Agonists

Opioid mu- and delta- receptors are thought to inhibit neurotransmitter release in primary afferents, which results in analgesia. NIDA grantees Drs. Tony Yaksh and Juan Carlos Marvizon and their colleagues have examined the effect of opiates on NK1R internalization in spinal cord slices and in vivo. In slices, pain fiber stimulation induced NK1R internalization that was abolished by the mu agonist [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]-enkephalin (DAMGO) and decreased by the delta agonist [D-Phe(2,5)]-enkephalin (DPDPE). In vivo, hind paw compression-induced NK1R internalization was significantly reduced by morphine, DAMGO, and DPDPE, but not by a kappa-opioid agonist. All effects were reversed by naloxone. These results demonstrate that mu- and delta-opioid agonists produce clear NK1R internalization that is associated with their analgesic properties. Kondo, I., Marvizon, J.C.G., Song, B., Salgado, F., Codeluppi, S., Hua, X-Y. and Yaksh, T.L. Inhibition by Spinal µ- and _-Opioid Agonists of Afferent-Evoked Substance P Release. Journal of Neuroscience, 25, pp. 3651-3660, 2005.

Salvinicins A and B, New Neoclerodane Diterpenes from Salvia Divinorum

Salvia divinorum is used in traditional spiritual practices of the Mazatecs to produce "mystical" or hallucinogenic experiences. The active ingredients of this plant are Salvinorin A as well as Salvinorin B. A dose of 200- 250 ug, when smoked, produces profound hallucinations lasting up to 1 hour. Salvia divinorum and one of its constituents, Salvinorin A, are gaining popularity as recreational drugs. Young adults and adolescents smoke the leaves and leaf extracts of the plant to induce powerful hallucinations. Currently this plant and its active constituents are not regulated in the United States and are easily available through the Internet. Given its potential for abuse, as well as its unique pharmacological properties as a potent selective kappa opioid receptor agonist, the authors have studied the chemistry and pharmacology associated with constituents of Salvia divinorum. Previous phytochemical investigations of S. divinorum resulted in the identification of several neoclerodane diterpenes present in the leaves. In a recently published paper, NIDA researchers report the identification of two new neoclerodane diterpenes with opioid receptor activity. Salvinicins A and B are unique neoclerodanes that possess a 3,4-dihydroxy-2,5-dimethoxytetrahydrofuran ring. The absolute stereochemistry of these molecules has been assigned through X-ray crystallographic analysis. Harding, W.W., Tidgewell, K., Schmidt, M., Shah, K., Dersch, C.M., Snyder, J., Parrish, D. Deschamps, J.R., Rothman, R.B., and Prisinzano, T.E. Salvinicins A and B, New Neoclerodane Diterpenes from Salvia Divinorum. Organic Letters, 7, pp. 3017-3020, 2005.

FAAH Inhibition

There is considerable interest in understanding the "background" or tonal level of endocannabinoids and their activity in the human brain, in terms of their production on demand, followed by their retrograde activation of the presynaptic cannabinoid receptor CB1 which triggers cannabinoid signaling processes, and their subsequent inactivation by metabolic processes. Levels of one particular endocannabinoid, anandamide, are regulated by the mammalian serine enzyme known as fatty acid amide hydrolase (FAAH), which has been cloned, characterized by crystallography and molecular modeling studies, and its distribution mapped in the human brain. Pharmacological inhibitors of this enzyme can increase the available concentration of anandamide, offering a potential for therapeutic targets of pain management and sleep disorders. Consequently, there are academic and commercial efforts currently directed at designing various chemical classes of inhibitors. One such inhibitor is URB597, developed with NIDA funding by Dr. Daniele Piomelli and Dr. Giorgio Tarzia. This compound inhibits the enzyme with nM affinity; it does not bind the CB1 receptor, does not produce typical THC-like behavioral effects in animals, but does produce anxiolytic effects in rats. There are two recent findings reported by these investigators and their collaborators that contribute to the understanding of URB597 pharmacology. The first is that single intravenous injections of URB597 into rat midbrain produce a significant increase in anandamide concentration after two hours, along with an increase in both serotonin and noradrenergic neuron firing. When URB597 was given intraperitoneally to rodents, it produced behavioral results (decreases in immobility and forced swimming), also produced by the antidepressant desipramine. URB597 did not produce conditioned place preference as did delta-9-THC, and did not generalize to the cannabinoid agonist WIN 55,212. The above neuronal and behavioral effects were blocked by the CB1 antagonist SR141716. URB597 was also shown to be inactive at a battery of receptors, including serotonin, adrenergic, dopaminergic, cannabinoid, nicotinic, and muscarinic, and inactive as well at the SERT, NET, and DAT transporters. Secondly, URB597 has been shown to have a role in reducing mechanical and thermal allodynia, using Freund' s adjuvant in the rat paw to induce an inflammatory pain response. It did not reduce the pain withdrawal threshold fourteen days following sciatic nerve ligation, used as a neuropathic pain model. The response shown in the two inflammatory pain models was dose-dependent, and blocked by co-administration of the cannabinoid antagonists AM 251 and SR144528. Overall, these results suggest an association between FAAH inhibitors and inflammatory pain, as well as with anxiety/mood modulation in animal models. Gobbi, G., Bambico, F.R., Mangieri, R., Bortolato, M., Campolongo, P., Solinas, M., Cassano, T., Morgese, M.G., Debonnel, G., Duranti, A., Tontini, A., Tarzia, G., Mor, M., Trezza, V., Goldberg, S.R., and Cuomo, V. Antidepressant-like Activity and Modulation of Brain Monoaminergic Transmission by Blockade of Anandamide Hydrolysis. Proceedings of the National Academy of Sciences of the United States of America, 102, pp. 18620-18625, 2005; Jayamanne, A., Greenwood, R., Mitchell, V.A., Aslan, S. Piomelli, D., and Vaughan, C.W. Actions of the FAAH Inhibitor URB597 in Neuropathic and Inflammatory Chronic Pain Models. British Journal of Pharmacology, 147, pp. 281-288, 2006.