Research Findings - Basic Neurosciences Research

Phosphorylation of WAVE1 Regulates Actin Polymerization and Dendritic Spine Morphology

The connectionist theory posits that behavioral changes such as those produced by learning and addiction occur by strengthening the connections between neurons called synapses. A neuron sending a signal to a neighboring neuron releases a chemical neurotransmitter into the synapse, which then diffuses across the synapse and binds to receptors located on the adjacent neuron. The receptors in most cases are located on specialized processes or protrusions called dendritic spines. Drugs of abuse such as cocaine, nicotine, and opiates produce long term changes in the length of dendrites, the number of spines, and the shape of spines. An important molecule implicated by the Greengard laboratory in regulating the morphology of dendritic spines is cdk5, a kinase that phosphorylates proteins, i.e., adds a high energy phosphate to proteins. Nobel Laureate Paul Greengard and his colleagues show that WAVE1 (the Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 1) forms a complex with cdk5 and is phosphorylated by cdk5. Phosphorylation of WAVE1 or the loss of WAVE 1 prevents actin, a major cytoskeletal protein from polymerizing and leads to a loss of dendritic spines. Dopamine, a neurotransmitter implicated in reward, decreases WAVE1 phosphorylation by cdk5 through a cAMP dependent dephosphorylation. The decrease in WAVE1 phosphorylation leads an increase in actin polymerization resulting in an increase in the number of spines. Thus, phosphorylation and dephosphorylation of WAVE1 regulates actin polymerization and dendritic spines. Future research will determine the mechanism by which WAVE1 is dephosphorylated by cAMP. Kim, Y., Sung, J.Y., Ceglia, I., Lee, K-W., Ahn, J-H., Halford, J.M., Kim, A.M., Kwak, S.P., Park, J.B., Ryu, S.H., Schenck, A., Bardoni, B., Scott, J.D., Nairn, A.C., and Greengard, P. Phosphorylation of WAVE1 Regulates Actin Polymerization and Dendritic Spine Morphology. Nature, 442, pp. 814-817, 2006.

Synaptopodin Regulates Cytoskeleton Through RhoA

The initiation and regulation of protruding subcellular structures, such as lamellipodia, filopodia formed during cell migration, and dendritic spines formed during neuronal synaptic growth, involves Rho family of small GTPases, including RhoA, Rac1 and Cdc42. It is through reorganization of the cytoskeleton that cells can move and make connections with other neurons. How these molecules each function differently in regulating cytoskeletons, which support the subcellular structures is not clear. Dr. Mundel and colleagues report that RhoA functions in stress fiber formation by interacting with a novel protein called synaptopodin. First, using wild type podocytes as a model system, they found that knock-down of synaptopodin suppresses RhoA initiated stress fibers, and overexpression of RhoA increases stress fibers in these cells. However, when they looked at podocytes lacking synaptopodin, the mRNA for RhoA does not change. Additional search enabled them to discover that synaptopodin does not affect RhoA transcription, but blocks the targeting of RhoA for degradation by Smurf1, a molecule which mediates ubiquitin induced protein degradation. The researchers further noticed that simply overexpressing RhoA in synaptopodin silenced podocytes does not initiate more stress fibers, since synaptopodin also plays roles in activating RhoA when stress fiber forms. Since RhoA is downstream of many G-protein coupled receptors in memory and reward pathways, this work provides important insights about how such pathways can be activated or manipulated during drug abuse. Asanuma, K., Yanagida-Asanuma, E., Faul, C., Tomino, Y.,Kim, K. and Mundel, P. Synaptopodin Orchestrates Actin Organization and Cell Motility Via Regulation of RhoA Signalling. Nature Cell Biology, 8(5) pp. 485-491, 2006.

Distinct Modes of Regulated Receptor Insertion to the Somatodendritic Plasma Membrane

One of the most critical factors in determining whether or not two molecules with the potential to interact in a biological system will in fact interact is whether or not they ever "see" each other. It is, of course, essential for interaction between two molecules that the molecules be present in the same place at the same time so they might have the opportunity to interact. Even such co-localization is no guarantee of interaction, but it is guaranteed that they will not interact if they never co-localize. A critical step in ensuring the potential for interaction with other biologically relevant components is delivery of proteins and other cellular component to their site of action. In this study, the researchers have observed just such a delivery. Using cutting edge technology that allowed them to monitor molecular movement at the cell surface using a microscope, they report the observation of a member of the G-protein coupled receptor family inserting into the cell membrane of a primary cultured neuron upon exposure to an agonist of the receptor. They see not only delivery or receptor to the cell membrane at the outer surface of the cell, but have also learned about lateral movement of the receptors once at the surface. One of the intriguing aspects of their observations is that there appear to be two types of insertion events for this receptor. They have named these two events "transient" and "persistent" insertion referring to the length of time a cluster of receptors will remain clustered at the surface once delivered there before spreading out throughout the cell membrane. While both transient and persistent insertion events are observed at the surface of a given cell, continued exposure of the cell to receptor agonist causes a decrease in the frequency of the transient events while causing an increase in persistent events when compared to agonist washout conditions. It is important to note that these studies are consistent with the recycling of already existing receptors to the cell surface rather than delivery of newly synthesized receptors to the cell surface. The opioid receptors are from the same receptor family as the receptor studied here and recycling events of those receptors are believed to be closely linked to drug tolerance. Future studies looking specifically looking at the opioid receptors using this system will be valuable in trying to develop strategies to manage drug tolerance. Yudowski, G.A., Puthenveedu, M.A., and von Zastrow, M. Distinct Modes of Regulated Receptor Insertion to the Somatodendritic Plasma Membrane. Nature Neuroscience, 9(5), pp. 622-627, 2006.

Regulation of DeltaFosB Stability by Phosphorylation

Addiction is a chronic relapsing disease characterized by drug seeking behavior even in the face of adverse consequences. One mechanism thought to mediate addiction is long term changes in gene expression in which genes that encode proteins, the major regulators of cell function, are turned on or off. Addictive drugs acting through receptors on the surface of neuronal cells send signals to the cell nucleus that leads to the activation or inactivation of transcription factors. These transcription factors are proteins that bind to a gene to act as a molecular switch. This in turn leads to increased or decreased transcription of the gene into mRNA. Increases in mRNA lead to increased protein synthesis for a gene encoding a particular protein while decreases in mRNA lead to decreased protein synthesis for a particular protein. In neurons changes in gene expression can lead to changes in neuronal excitability and changes in the strength of connections between neurons. One such transcription factor implicated in addiction is deltaFosB. Once induced, this transcription factor is highly stable and is not degraded for weeks or months. A key question is what mechanism prevents deltaFosB from becoming degraded. In the May 10, 2006 issue of the Journal Neuroscience, Dr. Eric Nestler and his colleagues at the University of Texas Southwestern Medical center provide an answer. They show that phosphorylation of deltaFosB on a serine at position 27 (this serine is the 27th amino acid of deltaFosB) by casein kinase 2 promotes the stability of deltaFOSB. This observation supports the theory that deltaFOSB acts as sustained molecular switch in the brain to promote and maintain long lasting changes in nervous system function. Ulery, P.G., Rudenko, G., and Nestler, E.J. Regulation of _FosB Stability by Phosphorylation. Journal of Neuroscience, 26(19), pp. 5131-5142, 2006.

In Vivo Experience Changes Synaptic Strength and Glutamate Receptor Composition

Sensory experiences such as touch are processed by neuronal circuits within our brains. The neurons in these circuits signal to one another using synaptic connections between the neurons, and these connections can become stronger or weaker depending on the strength and frequency of the sensory experience. This "synaptic plasticity" is critical for learning and remembering sensory experiences, and importantly plays a significant role in the development of addiction to drugs of abuse. Although the molecular mechanisms underlying synaptic plasticity are partially understood, investigating these processes in vivo (in intact organisms) is difficult. Dr. Barth is investigating the molecular mechanisms mediating synaptic plasticity in response to touch experience. She uses mice which have all but one whisker removed, since removal of these touch sensory structures alters the touch experience of the animals, and evokes changes in synaptic plasticity in the neural circuits which can be monitored experimentally. These mice express a piece of DNA containing the promoter of the c-fos transcription factor fused to Green Fluorescent Protein (GFP). After all but one whisker is removed, GFP turns on and green light is emitted in neurons undergoing functional changes in response to the altered sensory experience. These green neurons can then be characterized in a variety of electrophysiological and pharmacological ways to see how their properties have changed as the result of altered sensory experience. Dr. Barth found that GFP neurons in the single whisker animals have stronger synaptic connections. At the molecular level, she found that this is due to the presence of AMPA glutamate receptors (which lack a GluR2 subunit) at the input synapses. Dr. Barth's work expands our understanding of the molecular mechanisms underlying synaptic plasticity, which is critical for gaining a complete understanding of the neuronal basis of addiction. It is hoped that in the future, pharmaceuticals that modulate synaptic plasticity in specific brain regions could be used as therapeutic agents to treat addiction. Clem, R.L. and Barth, A. Pathway-Specific Trafficking of Native AMPARs by In Vivo Experience. Neuron, 49, pp. 663-670, 2006.

Reduced Nicotine Reward in Obesity: Cross-Comparison in Human and Mouse

Recent estimates suggest that 20% of current smokers are obese, and there are no studies that have examined differences in nicotine reward between obese and non-obese smokers. Dr. Julie Blendy and her colleagues investigated factors that maintain smoking behavior in obese individuals and to explore the underlying molecular mechanisms in a mouse/human cross-validation model of nicotine reward in lean and obese subjects. In humans, a cigarette choice paradigm was used to examine the relative reinforcing value of nicotine in obese and non-obese smokers. Conditional place preference (CPP) for nicotine was assessed in mice fed standard low fat rodent chow and mice rendered obese by a high fat diet. This study showed that in humans, obese smokers self-administered nicotine via cigarettes significantly less often than non-obese smokers and showed attenuated hedonic effects of nicotine-containing cigarettes compared to denicotinized cigarettes. Similarly, mice exposed to a high fat diet did not exhibit nicotine CPP, compared to control mice. mRNA levels for the m-opioid receptor and the leptin receptor were also downregulated in the ventral tegmental, a key brain reward region, of these mice. Together, these studies provide the first evidence for reduced nicotine reward in obese subjects and suggest that this may be mediated by dietary influences on the endogenous opioid system. Blendy, J.A., Strasser, A., Walters, C.L., Perkins, K.A., Patterson, F., Berkowitz, R., and Lerman, C. Reduced Nicotine Reward in Obesity: Cross-Comparison in Human and Mouse. Psychopharmacology, 180, pp. 306-315, 2005.

Cannabinoids Directly Inhibit Peripheral Capsaicin-Sensitive Nociceptive Neurons

Cannabinoids can reduce pain at a peripheral site of action. However, the signaling pathway mediating this effect is not clearly understood. NIDA grantee Kenneth Hargreaves (University of Texas Health Science Center, San Antonio) and his colleagues tested the hypothesis that certain cannabinoids directly inhibit peripheral capsaicin-sensitive nociceptive neurons by desensitizing the transient receptor potential vanilloid 1 (TRPV1) receptor. Application of the cannabinoid WIN 55,212-2 (WIN) to cultured trigeminal (TG) neurons or isolated skin biopsies rapidly and significantly inhibited capsaicin activated inward currents. The inhibitory effect did not involve the activation of G protein-coupled cannabinoid receptors. Rather, the application of WIN produced a dephosphorylation of the TRPV1 receptors. These data demonstrate that cannabinoids such as WIN directly inhibit TRPV1 functional activities and represents a mechanism of cannabinoid actions at peripheral sites. Patwardhan, A.M., Jeske, N.A., Price, T.J., Gamper, N., Akopian, A.N., and Hargreaves, K.M. The Cannabinoids WIN 55,212-2 Inhibits Transient Receptor Potential Vanilloid 1 (TRPV1) and Evokes Peripheral Antihyperalgesia Via Calcineurin. Proceedings of the National Academy of Science, NAS, 103(30), pp. 11393-11398, 2006.

N-Substituted cis-4a-(3-Hydroxyphenyl)-8a-methyloctahydroisoquinolines are Opioid Receptor Pure Antagonists

In continuation of their studies to develop new opioid receptor antagonists as possible treatment drugs for substance abuse, Dr. Carroll and associates have reported that N-substituted cis-4a-(3-hydroxyphenyl)-8a-methyloctahydroquinolines are potent opioid antagonists in the [35S]GTP-(-S functional assay with several analogues showing preferences for the 6 opioid receptor. In this presentation, N-substituted cis-4a-(3-hydroxyphenyl)-8a-methyloctahydroisoquinolines were designed and synthesized as conformationally constrained analogues of the trans-3,4-dimethyl-4-(3-hydroxyphenyl) piperidine class of opioid receptor pure antagonists. The methyl octahydroisoquinolines can exist in conformations where the 3-hydroxyphenyl substituent was either axial or equatorial similar to the 3-hydroxyphenylpiperidines. The 3-hydroxyphenyl equatorial conformation was responsible for the antagonist activity observed in the (3-hydroxyphenyl) piperidine antagonists. Single crystal X-ray analysis of N-methyl cis-4a-(3-hydroxyphenyl)-8a-methyl-octahydroisoquinoline showed that the 3-hydroxyphenyl equatorial conformation was favored in the solid state. Molecular modeling also suggested that the equatorial conformation had lower potential energy relative to that of the axial conformation. Evaluation of N-substituted cis-4a-(3-hydroxyphenyl)-8a-methyloctahydroisoquinolines in the [35S]GTP-g-S in vitro functional assay showed that they were opioid receptor pure antagonists. N-[4a-(3-hydroxyphenyl)-8a-methyl-2-(3-phenylpropyl) octahydro-isoquinoline-6-yl]-3-(piperidin-1-yl) propionamide with a Ke of 0.27 nM at the k opioid receptor with 154- and 46- fold selectivity relative to those of the m and d receptors, respectively, possessed the best combination of the k potency and selectivity. Carroll, F.I., Chaudhari, S., Thomas, J.B., Mascarella, S.W., Gigstad, K.M., Deschamps, J., and Navarro, H.A. N-substituted cis-4a-(3-Hydroxyphenyl)-8a-methyloctahydroisoquinolines Are Opioid Receptor Pure Antagonists. Journal of Medicinal Chemistry, 48, pp. 8182-8193, 2005.

Multiple Mechanisms for the Biosysnthesis of Endocannabinoids

N-Acyl ethanolamines (NAEs) constitute a large and diverse class of signaling lipids that includes the endogenous cannabinoid, anandamide. Like other lipid transmitters, NAEs are thought to be biosynthesized and degraded on-demand rather than being stored in vesicles prior to signaling. The identification of enzymes involved in NAE metabolism is therefore imperative to achieve a complete understanding of this lipid signaling system and control it for potential therapeutic gain. Recently, an N-acyl phosphatidylethanol-amine phospholipase D (NAPE-PLD) was identified as a candidate enzyme involved in the biosynthesis of NAEs. Here, Cravatt's group describes the generation and characterization of mice with a targeted disruption in the NAPE-PLD gene [NAPE-PLD(-/-) mice]. Brain tissue from NAPE-PLD(-/-) mice showed more than a 5-fold reduction in the calcium-dependent conversion of NAPEs to NAEs bearing both saturated and polyunsaturated N-acyl chains. However, only the former group of NAEs was decreased in level in NAPE-PLD(-/-) brains, and these reductions were most dramatic for NAEs bearing very long acyl chains. Further studies identified a calcium-independent PLD activity in brains from NAPE-PLD(-/-) mice that accepted multiple NAPEs as substrates, including the anandamide precursor C20:4 NAPE. The illumination of distinct enzymatic pathways for the biosynthesis of long chain saturated and polyunsaturated NAEs suggests a strategy to control the activity of specific subsets of these lipids without globally affecting the function of the NAE family as a whole. This is a first step toward understanding and controlling this system for therapeutic gain. Leung, D., Saghatelian, A., Simon G.M., and Cravatt, B.F., Inactivation of N-acyl Phosphatidylethanolamine Phospholipase D Reveals Multiple Mechanisms for the Biosynthesis of Endocannabinoids, Biochemistry, 45(15), pp. 4720-4726, 2006.

Uterine Anandamide Levels and Pregnancy

Anandamide, an endogenous cannabinoid, plays an important role in implantation of the fertilized ovum. Uterine implantation requires a reciprocal interaction between a blastocyst and a receptive uterus. NIDA supported research in mice has illuminated anandamide's role in mediating this interaction. During early pregnancy, anandamide is at lower levels in both the receptive uterus and at the implantation site. However, the mechanism by which differential uterine anandamide gradients are established is not clearly understood. NIDA researchers Dr. Dey and his associates have recently demonstrated that uterine anandamide levels are primarily regulated by Nape-Pld, the gene encoding N-acylphosphatidyl-ethanolamine-hydrolyzing phospholipase D (NAPE-PLD) that generates anandamide. This suggests that aberrant uterine NAPE-PLD activity may cause implantation failure or defective implantation. These findings may be relevant to human abuse of cannabinoids, since elevated anandamide is associated with spontaneous pregnancy failure. Guo, Y., Wang, H., Okamoto, Y., Ueda, N., Kingsley, P.J., Marnett, L.J., Schmid, H.H.O., Das, S.K., and Dey S.K., N-Acylphosphatidylethanolamine-hydrolyzing Phospholipase D Is an Important Determinant of Uterine Anandamide Levels during Implantation, Journal of Biological Chemistry, 280, pp. 23429-23432, 2005.

Opiates and Apoptosis (Programmed Cell Death)

Opiates have been shown to inhibit cell growth and trigger apoptosis, but the underlying molecular mechanisms remain unclear. Other research has reported that morphine induces Fos expression and promotes Fos-mediated apoptosis. In a recent study, NIDA-supported researchers, Dr. Deling Yin and his associates investigated the mechanisms by which morphine modulates apoptosis in human Jurkat cells, a human T-cell leukemia line. Their study revealed that morphine induced Jurkat cell apoptosis through FADD/p53, anti-apoptotic Pl3K/Akt and NF-kB pathways. They came to these conclusions because they observed that morphine-induced apoptosis was inhibited by transfection with a dominant negative Fos-associated death domain (FADD) plasmid. This suggests that morphine-induced apoptosis is dependent on FADD. Furthermore, suppression of endogenous p53 expression attenuated the morphine-induced apoptosis. In addition, morphine-induced apoptosis appeared to be dependent on the activation of phosphatidylinositol 3-kinase (Pl3K), as Pl3K inhibition significantly enhanced morphine-induced apoptosis. They also noted inhibition of Akt or nuclear factor-kappaB (NF-kB) expression dramatically increased morphine-induced apoptosis. These findings are important as they further our insight in the regulation of morphine-induced immunosuppression. Yin, D.L., Woodruff, M., Zhang, Y., Whaley, S., Miao J.Y., Ferslew, K., Zhao, J., and Stuart, C., Morphine Promotes Jurkat Cell Apoptosis through Pro-apoptotic FADD/P53 and Anti-apoptotic Pl3K/Akt/NF-kB Pathways, Journal of Immunology, 174, pp. 101-107, 2006.

The Properties of in vivo Salvinorin A

Salvinorin A is a non-nitrogenous diterpene produced in the leaves of the sage, Salvia divinorum, along with related salvinorins B-F, and is a potent kappa opioid receptor (KOR) agonist in terms of in vitro binding, despite being structurally unrelated to other known nitrogenous kappa ligands. Its in vivo effects in mice include sedation and antinociception. In the rhesus monkey, intravenous injection of salvinorin A produces sedative effects. In humans, smoking of the leaves and leaf extracts induces hallucinations. Drs. Roth, Pintar, and Rothman, have identified some in vivo properties of salvinorin A based on the development of a KOR knockout mouse model. In brief, wild type mice receiving an intracerebroventricular injection of salvinorin A showed a dose-dependent analgesic response. This effect was absent in the KOR knockout mice. Salvinorin-2-propionate (a chemical derivative of salvinorin A) also produced analgesia, but the salvinorin B did not. Salvinorin A and salvinorin-2-propionate, but not salvinorin B, produced hypothermia in wild type mice. Again the effect was not seen in the knockout mice. Radioligand binding showed that salvinorin A and salvinorin-2-propionate could displace U69,593 at the kappa1 binding site, but not bremazocine, from the kappa 2a/2b binding sites. Binding was not detected in the KOR knockout mice. The data support the idea that most of the behavioral effects of salvinorin A are due to agonist activation of the KOR at the kappa1 binding site. Ansonoff, M.A., Zhang, J., Czyzyk T., Rothman R.B., Stewart J., Xu, H., Zjwiony J., Siebert, D.J., Yang, F., Roth, B.L., and Pintar, J.E., Antinociceptive and Hypothermic Effects of Salvinorin A are Abolished in a Novel Strain of Kappa-Opioid Receptor-1 Knockout Mice, Journal of Pharmacology and Experimental Therapeutics, 318(2), pp, 641-648, 2006.

Cocaine Increases Actin Cycling

Cocaine addiction, like other forms of memory, is associated with persistent changes in synaptic function. Peter Kalivas and his colleagues are investigating the response of actin to cocaine. Actin cycling regulates dendritic spine morphology and protein insertion into the postsynaptic density. Kalivas and his colleagues recently reported an increase in filopodia-like actin formation in the nucleus accumbens after cocaine. Following acute cocaine, the increase is short-lived and depends primarily on changes in actin assembly, while after 3 weeks withdrawal from chronic cocaine, the increase is enduring, consistent with filopodia formation, and results primarily from a change in actin cycling. The increased cycling is produced by a reduction in LIM-kinase and a corresponding decreased inactivation (phosphorylation) of cofilin. Further, when increased actin cycling was reversed by intra-accumbens injection of either a Tat-peptide LIM-kinase antagonist (promotes actin de-polymerization and branching) or latrunculin A (inhibits actin polymerization), the reinstatement of cocaine-seeking in rats was augmented. This argues that the increase in actin cycling produced by chronic cocaine is compensatory. Toda, S., Shen, H.-W., Peters, J., Cagle, S. and Kalivas, P.W. Cocaine Increases Actin Cycling: Effects in the Reinstatement Model of Drug Seeking. J. Neurosci., 26, pp. 1579-1587, 2006.

Plasma Concentrations of MDMA that Produce Serotonergic Neurotoxicity in Monkeys Overlap those Reported in Human "Ecstasy" Abusers

George Ricaurte's group examined the pharmacokinetic profile of MDMA in squirrel monkeys after different routes of administration, and examined the relationship between acute plasma MDMA concentrations and subsequent brain serotonin deficits. Oral MDMA administration engendered a plasma profile of MDMA in the monkeys similar to that seen in humans, though the half-life of MDMA in monkeys is shorter (3-4 hr vs. 6-9 hr). As in humans, MDMA was N-demethylated to MDA, and the plasma ratio of MDA to MDMA was 3-5/100, similar to that in humans. MDMA accumulation in the monkeys was non-linear, and plasma levels were highly correlated with regional brain serotonin deficits observed two weeks later. Plasma concentrations of MDMA that produced serotonergic neurotoxic effects in the monkeys overlapped those reported in human "ecstasy" abusers, though their studies did not allow for possible development of tolerance in humans. Their results also indicate that neurotoxic plasma MDMA levels in monkeys are only two to three times higher than those that develop in humans after a single 100-150 mg dose of MDMA in a controlled setting. Since "ecstasy" abusers often use sequential doses hours apart, the findings in the monkeys may be most relevant to such individuals. Mechan, A., Yuan, J., Hatzidimitriou, G., Irvine, R.J., McCann, U.D. and Ricaurte, G.A. Pharmacokinetic Profile of Single and Repeated Oral Doses of MDMA in Squirrel Monkeys: Relationship to Lasting Effects on Brain Serotonin Neurons, Neuropsychopharmacology, 31, pp. 339-350, 2006.

Alpha3beta4 Nicotinic Antagonists, Administered into the Medial Habenula or Interpeduncular Nucleus, Attenuate the Self-Administration of Morphine in Rats

The novel iboga alkaloid congener 18-methoxycoronaridine (18-MC) is a putative anti-addictive agent that has been shown, in rats, to decrease the self-administration of morphine and other drugs of abuse. Previous work established that 18-MC is a potent antagonist at alpha3beta4 nicotinic receptors. Because alpha3beta4 nicotinic receptors in the brain are preferentially located in the medial habenula (MHb) and the interpeduncular nucleus (IPN), a study was conducted to determine if 18-MC could act in these brain areas to modulate morphine iv self-administration in rats. Local administration of 18-MC into either the MHb or the IPN decreased morphine self-administration while having no effect on responding for a non-drug reinforcer (sucrose). Similar results were produced by local administration into the same brain areas of two other alpha3beta4 nicotinic antagonists, mecamylamine and alpha-conotoxin AuIB. Local administration of 18-MC into the ventral tegmental area had no effect on morphine self-administration. These data are consistent with the hypothesis that 18-MC decreases morphine self-administration by blocking alpha3beta4 nicotinic receptors in the habenulo-interpeduncular pathway. Glick, S.D., Ramirez, R.L., Livi, J.M. and Maisonneuve, I.M. 18-Methoxycoronaridine Acts in the Medial Habenula and/or Interpeduncular Nucleus to Decrease Morphine Self-Administration in Rats. Eur J Pharmacol, 537, pp. 94-98, 2006.

D1 and D2 Dopamine Receptors Form Heterooligomers and Cointernalize Following Selective Activation of Either Receptor

Earlier Dr. Susan George and her research team at the University of Toronto showed that a novel phospholipase C-mediated calcium signal arose from coactivation of D1 and D2 dopamine receptors. In the present study, robust fluorescence resonance energy transfer showed that these receptors exist in close proximity, indicative of D1-D2 receptor heterooligomerization. The closeness of these receptors within the heterooligomer allowed for cross-phosphorylation of the D2 receptor by selective activation of the D1 receptor. D1-D2 receptor heterooligomers were internalized when the receptors were coactivated by dopamine or either receptor was singly activated by the D1-selective agonist (+/-)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) or the D2-selective agonist quinpirole. The D2 receptor expressed alone did not internalize after activation by quinpirole except when coexpressed with the D1 receptor. D1-D2 receptor heterooligomerization resulted in an altered level of steady-state cell surface expression compared with D1 and D2 homooligomers, with increased D2 and decreased D1 receptor cell surface density. Together, these results demonstrated that D1 and D2 receptors formed heterooligomeric units with unique cell surface localization, internalization, and transactivation properties that are distinct from that of D1 and D2 receptor homooligomers. So, C.H., Varghese, G., Curley, K.J., Kong, M.M.C., Alijaniaram, M., Ji, X., Nguyen, T., O'Dowd, B.F., and George, S.R. D1 and D2 Dopamine Receptors Form Heterooligomers and Cointernalize after Selective Activation of Either Receptor. Molecular Pharmacology, pp. 568-578, 2005.

GABA Inhibits NAC Neurons After Intracranial Self-stimulation

Intracranial self-stimulation (ICS) of the median forebrain bundle (MFB) is a motivated behavior that results from contingent activation of the brain reward system. Neurons that course through this pathway elaborate a variety of neurotransmitters including dopamine and GABA. The specific roles each transmitter subserves remain unclear. Cheer, et al., used extracellular electrophysiology and cyclic voltammetry at the same electrode in awake rats to simultaneously examine cell firing and dopamine release in the nucleus accumbens (NAc) during ICS and noncontingent stimulation of the MFB. ICS elicited dopamine release in the NAc and produced coincident time-locked changes (predominantly inhibitions) in the activity of a subset of NAc neurons. Similar responses were elicited with noncontingent stimulations. The changes in firing rate induced by noncontingent stimulations were reversed by the GABAA receptor antagonist bicuculline. Thus, neurons in the NAc are preferentially inhibited by GABAA receptors after MFB stimulation, a mechanism that may also be important in ICS. After inhibition of dopamine release, bicuculline reversed the time-locked inhibitory responses evoked by noncontingent stimulations. GABAergic neurons from the VTA project to the NAc along with the ascending dopamine systems. Thus, their activation by the noncontingent stimulation or during ICS could lead to inhibitions similar to those found in the prefrontal cortex and NAc during electrical stimulation of the VTA. Overall, these findings bolster the view that GABA release occurs during ICS-like stimulations, and suggests that ICS is a behavior involving extensive neuronal circuitry, not solely involving dopamine. Cheer, J.F., Heien, M.L.A.V., Garris, P.A., Carelli, R.M., and Wightman, R.M. Simultaneous Dopamine and Single-unit Recordings Reveal Accumbens GABAergic Responses: Implications for Intracranial Self-stimulation. Proceedings of the National Academy of Science, 102, pp. 19150-19155, 2005.

Dopamine Neurons of the VTA Differ with Respect to the Class of Opiate Receptors They Express and this is Reflected in their Terminal Destinations

The mesolimbic dopamine system, which mediates the rewarding properties of nearly all drugs of abuse, originates in the ventral tegmental area (VTA) and projects to both the nucleus accumbens (NAc) and the basolateral amygdala (BLA). Dr. John Williams' lab distinguished dopamine (DA) neurons within VTA that projected to the BLA from those projected to the NAc based on their expression of kappa and mu opiate receptors. DA neurons originating in the anterolateral VTA project to the BLA and neurons that terminate in the NAc originate in the posteromedial VTA and differed from each other in three important ways. First, differential postsynaptic inhibition by opioids was evident at VTA, where DA neurons that projected to the NAc were sensitive to a kappa-opioid agonist but not the mu/delta opioid agonist [Met5] enkephalin. The opposite action of the opioids was observed in neurons that projected to the BLA. Second, the presynaptic mechanisms mediating GABAergic transmission were also differently affected by the kappa opioid receptor activation. The presynaptic GABAA inputs onto BLA-projecting neurons were more sensitively disinhibited while GABAB input onto NAc-projecting neurons were more sensitively disinhibited. In contrast, activation of the mu/delta receptor equally disinhibited the GABAergic transmission (either GABAA or GABAB) on BLA-projecting neurons and NAc-projecting neurons. Third, the kappa receptor activation disinhibited D2 receptor mediated synaptic activity on these two groups of neurons, while mu/delta receptor activation showed no effect. The DA uptake transporter-mediated properties were identical between these two groups of VTA neurons. These results suggest that the properties of mesolimbic dopamine neurons of the VTA are not homogenous but vary according to terminal location. Behavioral effects of opioids may therefore be the result of inhibition of distinct subpopulations of mesolimbic neurons. Identifying the properties of mesolimbic VTA dopamine projecting neurons is critical to understanding the action of drugs of abuse. These findings warrant further mechanistic investigation of specific signaling between different brain regions. Ford, C.P., Mark, G.P., and Williams J.T. Properties and Opioid Inhibition of Mesolimbic Dopamine Neurons Vary According to Target Location. Journal of Neuroscience, 26(10), pp. 2788-2797, 2006.

Glial Activation Resulting from Combined Morphine and HIV-1 Tat Protein Exposure is Mediated by Inflammatory Chemokine Receptor CCR2 Activation in the Brain

Opiate abuse is believed to exacerbate the neuropathogenesis associated with HIV/AIDS. Laboratory studies have provided compelling evidence for additive or synergistic effects of opioid compounds and neurotoxic HIV proteins such as Tat on glial activation and neuronal dysfunction. In addition, opioid receptor activation can greatly increase the expression (and function) of inflammatory chemokines and their receptors in neural cells (neurons, microglia and astrocytes) as well as leukocytes (lymphocytes, monocytes and macrophages). Glial activation and increased chemokine production in the brain, particularly CCL2 (monocyte chemoattractant protein-1, or MCP-1), are associated with increased neuropathology in multiple diseases including HIV encephalitis, and CCL2 levels are markedly increased by substance abuse in HIV-1 infected individuals. Therefore, this study asked whether increased CCL2 signaling (via the CCR2 receptor) mediated increased astrocyte, macrophage and microglial activation resulting from combined morphine and Tat exposure in mice. The mice received intracerebral injections of HIV-1 Tat into the striatum, or saline/sham injection as controls. Two days following injection, mice received subcutaneous implants of time-release morphine and/or naltrexone pellets, with placebo pellets as controls, to deliver drugs for 5 days. Brains from these mice were examined for expression of the astrocyte marker GFAP, the macrophage/microglia marker F4/80, mu opioid receptor, CCL2 and CCR2. Astrocytes were the predominant contributors of increased CCL2 production following morphine and/or Tat exposure. Systemic morphine increased the proportion of CCL2+ astrocytes at the site of Tat injection, and to a lesser degree saline injection, suggesting that opiates aggravate both focal and Tat-induced inflammatory responses. This increase was completely blocked by naltrexone, and there were no effects of morphine or Tat in the contralateral striatum. Glial changes induced by Tat or morphine + Tat were completely abolished in CCR2(-/-) mice, suggesting that signaling from activation of this chemokine receptor is the principal mechanism involved in this aspect of HIV neuropathogenesis. El-Hage, N., Wu, G., Ambati, J., Bruce-Keller, A.J., Knapp P.E., and Hauser K.F. CCR2 Mediates Increases in Glial Activation Caused by Exposure to HIV-1 Tat and Opiates. Journal of Neuroimmunology, (epub), 2006.