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Director's Report to the National Advisory Council on Drug Abuse - September, 2004

Research Findings - Basic Neurosciences Research

Addiction, Plasticity and the Rate of Cocaine Administration

Moving from periodic to compulsive drug use (with a propensity to relapse) characterizes addiction. Much research has focused on how biological differences (such as genetics), rather than drug characteristics, themselves, may affect the transition to addiction. Although rapid drug entry into the brain appears to be associated with addiction potential, there has been almost no research on how the rate of drug delivery alters the neurobiological impact of drugs. For this reason, Dr. A.N. Samaha and colleagues at the University of Michigan undertook a study examining how the rate of intravenous cocaine administration in rats affects three outcomes: (1) psychomotor sensitization, (2) induction of the immediate early genes, c-fos and arc, and (3) the rate of dopamine uptake. Their results indicated that a single infusion of cocaine (2mg/kg) led to behavioral sensitization only when it was delivered rapidly (5 second duration). In their gene expression studies, they also found that rapidly administered cocaine preferentially engaged components of the mesocorticolimbic system. Additionally, they observed that rapid infusion is necessary to engage enkephalin (Enk +) neurons in the striatum, a finding they say is "...reminiscent of previous studies showing that when cocaine is administered under conditions that promote behavioral sensitization (i.e., a novel environment), this also facilitates its ability to engage Enk+ cells in the striatum." Finally, the authors used in vivo voltammetry to study the effect of intravenous cocaine delivery on dopamine uptake in the nucleus accumbens core. At all the rates they tested, they found increases in the half-life of electrically evoked dopamine. The greatest increase occurred and lasted longer when cocaine was rapidly delivered. For example, at 2mg/kg the half-life peaked within 60-100 sec after an injection over 5-25 seconds. (They observed a half-life for endogenously released dopamine in the range of 50-300msec.) Interestingly, the investigators found that cocaine induced greater immediate early gene expression when it was injected over 25 seconds, rather than 100 seconds, but that differences in dopamine reuptake inhibition lasted for only approximately three minutes. From this observation, they suggest that non-dopaminergic mechanisms also may contribute to the effects of infusion rate on gene expression. The authors postulate that rapid drug delivery may bring about adaptive neurobehavioral changes that relate to compulsive drug use. Samaha, A.N., Mallet, N., Ferguson, S.M., Gonon, F. and Robinson T.E. The Rate of Cocaine Administration Alters Gene Regulation and Behavioral Plasticity: Implications for Addiction. Journal of Neuroscience, 24(28), pp. 6362-6370, 2004.

New Study May Explain How Nicotine Cigarettes Help Smokers Concentrate

Dopamine is essential for attention and learning. The results of a new study indicate that how nicotine affects dopamine (DA) release from ventral tegmental area (VTA) neurons depends on the (baseline) firing pattern of the dopaminergic neurons. There are ordinarily 2 firing patterns of the VTA DA neurons, a low-frequency tonic mode (0.5-8 Hz) interrupted by bursts of phasic activity (about 15-50 Hz) that are matched to salient stimuli in learning and memory experiments. These researchers found that nicotine, at levels experienced by smokers (~300 nM), shifts the firing pattern of dopaminergic neurons from tonic firing to high-frequency burst firing. They used carbon-fiber microelectrodes and fast cyclic voltammetry to measure released DA at sub-second resolution in acutely prepared mouse brain slices. While nicotine curbed DA release at low firing frequencies, it allowed a rapid rise in DA at the higher firing frequencies that are associated with behavioral cues. Such contrast enhancement may underlie nicotine's ability to increase sensory gating and attention, and may help explain the appeal of nicotine use in healthy individuals, nicotine's ability to enhance cognition in disorders such as Alzheimer's and attention disorder, and the very high prevalence of smoking in schizophrenic patients, a disease in which sensory gating is impaired. Smoking is often suggested to be a form of self-medication in schizophrenia. Zhang, H. and Sulzer, D. Frequency-dependent Modulation of Dopamine Release by Nicotine. Nature Neuroscience, 7, pp. 581-582, 2004.

Presynaptic Actions of Dopamine

By recording the activity of individual presynaptic neuron terminals in the striatum, researchers found that dopamine (DA) released by either electrical stimulation of the striatum or by amphetamine inhibited the activity of about 85% of the neuronal inputs from the cortex to the striatum (i.e., the corticostriatal terminals). They accomplished these direct recordings by combining optical monitoring of motor area corticostriatal afferents using the fluorescent vesicle marker FM1-43 with electrochemical recordings of striatal DA release. Further, they found that this presynaptic inhibitory effect of DA is mediated by DA D2 receptors. This inhibition resembles DA input associated with either salient behavioral stimuli or psychostimulant drugs, and suggests how DA modifies striatal activity. They found that the most active terminals were selectively resistant to DA inhibition, while the remaining terminals were inhibited. Thus, DA release associated with salience during motor learning may reinforce specific corticostriatal connections by filtering out less effective inputs. The selective filtering of corticostriatal transmission by amphetamine may indicate how drugs of abuse reinforce drug-taking behavior, leading to the development of addiction. Bamford, N.S., Zhang, H., Schmitz, Y., Wu, N-P., Cepeda, C., Levine, M.S., Schmauss, C., Zakharenko, S.S., Zablow, L. and Sulzer, D. Heterosynaptic Dopamine Neurotransmission Selects Sets of Corticostriatal Terminals. Neuron 42, pp. 653-663, 2004.

Activator of G Protein Signaling 3: A Gatekeeper of Cocaine Sensitization and Drug Seeking

It is known that chronic cocaine administration reduces G (guanine nucleotide binding) protein signaling efficacy. It is now reported that expression of the G protein binding protein AGS3 (activator of G protein signaling 3), which complexes the Gi_ subunit and thus diminishes signaling through Gi_-mediated signal cascades, is upregulated in the prefrontal cortex (PFC) during late withdrawal (as late as 2 months after the end of treatment) from repeated cocaine administration. These researchers then employed a method to mimic elevated levels of AGS3 in the PFC of drug-naive rats by microinjecting a peptide containing the Gi_ binding domain of AGS3 fused to the cell permeability domain of HIV-Tat. Infusion of this peptide mimicked the phenotype of chronic cocaine-treated rats by manifesting (1) sensitized locomotor behavior to cocaine, (2) enhanced drug-seeking behavior when primed with a single injection of cocaine, and (3) increased glutamate transmission in nucleus accumbens. Then, by preventing cocaine withdrawal-induced AGS3 expression with antisense oligonucleotides, signaling through Gi_ was normalized, and both cocaine-induced relapse to drug seeking and locomotor sensitization were prevented. When antisense oligonucleotide infusion was discontinued, drug seeking and sensitization were restored. The involvement of AGS3 in drug reinstatement appears to be selective to cocaine-related reward, as infusion of the antisense into rats resuming lever pressing for food had no effect. Thus, it is proposed that AGS3 gates the expression of cocaine-induced plasticity by regulating G protein signaling in the PFC. Bowers, M.S., McFarland, K., Lake, R.W., Peterson, Y.K., Lapish, C.C., Gregory, M.L., Lanier, S.M. and Kalivas, P.W. Activator of G Protein Signaling 3: A Gatekeeper of Cocaine Sensitization and Drug Seeking. Neuron, 42, pp. 269-281, 2004.

Cocaine, Benzoylecgonine, Amphetamine, and N-Acetylamphetamine Binding to Melanin Subtypes in Hair

The two major melanin types in hair are the black eumelanins and the reddish-brown pheomelanins. The purpose of this study was to assess the binding of cocaine, benzoylecgonine (BE), amphetamine, and N-acetylamphetamine (N-AcAp) to these individual melanin types. These drugs were chosen because cocaine exhibits a known hair color bias whereas its chemical congener BE, has not been shown to exhibit this bias. Similarly, although amphetamine exhibits a hair color bias, its non-basic analog N-AcAp does not exhibit a hair color bias. Experiments were performed to document the in vitro binding of all above drugs to synthetic melanin subtypes. The melanins included in this study were two black eumelanin subtypes and a reddish brown pheomelanin and two mixed melanin copolymers. Results indicated that cocaine and amphetamine bind to eumelanins and mixed eu-/pheomelanins to varying degrees, but not to pure pheomelanin. BE and N-AcAp, net neutral molecules, do not bind to any type of melanin. Overall these findings show that basic drugs, such as cocaine and amphetamine, have a greater affinity for melanin than their net neutral analogues. These data further reveal that melanin types differ when it comes to drug binding, and begin to elucidate what properties of melanin are important for drug binding, and why hair color drug-binding biases exist. Borges, C.R., Roberts, J.C., Wilkins, D.G. and Rollins, D.E. Cocaine, Benzoylecgonine, Amphetamine, and N-acetylamphetamine Binding to Melanin Subtypes. Journal of Analytical Toxicology, 27, pp. 125-134. 2003.

Kappa-Delta Receptor Association

Previous experiments have suggested the existence of a physical association between the delta (DOR) and kappa (KOR) opioid receptors. The so-called heterodimer of DOR and KOR shows some degree of difference in its trafficking, signaling in neurons and ligand binding, as compared to the properties of separate delta or kappa receptors. For instance, the heterodimer internalizes from the neuron's surface into the cell to a lesser extent than the delta receptor alone, when exposed to the opiate agonist etorphine. The potency of a kappa agonist (U69,593) or a delta agonist (DPDPE) is substantially reduced when interacting with the heterodimer receptor compared to the potency toward the individual receptors. There is also a synergy shown in binding of these compounds to the hetrodimer receptor. That is, there is low affinity for kappa agonists, by the heterodimer receptor, but high affinity for this compound in the presence of the delta agonist. Finally, the heterodimer receptor showed a greater affinity for what are known as partial agonists such as etorphine, as compared to the full agonists U69593 or DPDPE. Recently, Dr. Philip Portoghese has investigated the properties of several bivalent ligands, which contain one pharmacophoric group resembling GNTI (kappa sensitive) separated by a chain of atoms (a "spacer"), linked to a second pharmacophore group (a delta sensitive naltrindole). In particular, one of these bivalent ligands lacked agonist activity, but showed both significant kappa and delta antagonist properties when administered intrathecally to mice. In HEK cells co-expressing kappa and delta receptors, Ki values (displacement of tritiated diprenorphine) were optimal for a "spacer" of twenty-one atoms needed to allow interaction of the bivalent ligand with both receptors. It was also shown that administration of this ligand into the rodent brain antagonized only the delta receptor (DPDPE), but not the kappa (U50488), so that the rodent brain may not possess the same heterodimer organization as observed in the spinal cord. Bhushan, R.G., Sharma, S.K., Xie, Z., Daniels, D.J. and Portoghese, P.S. A Bivalent Ligand (KDN-21) Reveals Spinal δ and κ Opioid Receptors Are Organized as Heterodimers That Give Rise to δ1 and κ2 Phenotypes. Selective Targeting of δ-κ . Journal of Medicinal Chemistry, 47, pp. 2969-2972, 2004.

Antinociceptive Structure-Activity Studies with Enkephalin-Based Opioid Glycopeptides

To develop effective opioid peptides as therapeutic agents pharmacokinetic issues including stability and blood-brain barrier (BBB) permeability need to be considered. The chemical technique of glycosylation of opioid peptides can increase peptide stability and BBB penetration. To further refine and optimize pain-reducing antinociceptive potency, Dr. Hruby and his colleagues were able to synthesize a series of enkephalin-based glycopeptides using a solid phase method. Binding and functional smooth muscle bioassays indicated that these glycopeptides did not significantly affect the opioid receptor affinity or agonist activity. All of the glycopeptides tested produced potent antinociception in mice. Selected compounds were administered to mice to further test for antinociception, assess serum stability and blood brain barrier penetration. All compounds tested produced full antinociceptive effects with calculated A50 values ranging from 2.2-46.4 Ámol/kg. These results provide additional support for the utility of glycosylation to increase CNS bioavailability of small peptides and complement ongoing stability and blood-brain barrier penetration studies. Elmagbari, N.O., Egleton, R.D., Palian, M., Lowery, J.J., Schmid, W.R., Davis, P., Navratilova, E., Yamamura, H.I., Porreca, F., Hruby, V.J., Dhanasekaran, M., Keyari, C.M., Polt, R. and Bilsky, E. Antinociceptive Structure-Activity Studies with Enkephalin-Based Opioid Glycopeptides. Journal of Pharmacology and Experimental Therapeutics, DOI:10.1124/jpet.104.069393, July 7, 2004.

A Single in-vivo Exposure to _9THC Disrupted Functional Plasticity Mediated by Endocannabinoid in the Hippocampus

Individuals who smoke marijuana often show impairments in learning, cognitive abilities, the perception of reward and in emotional well-being. Endogenous cannabinoids and their receptors in the hippocampus and the ventral tegmental area may be important in "tuning" neural activity such as the synaptic activity and plasticity of neurons. Dr. Castillo and his team have shown that the dynamic status of neuronal excitement and the firing pattern of neurons, factors that control the pattern of neurotransmitter release, is tuned by DSI (depolarization suppression of inhibition), a retrograde feedback disinhibition of the inhibitory GABAergic inputs that occurs as a result of the activity of endogenous cannabinoids. These compounds have already been shown to underlie long-lasting synaptic plasticity such as LTP and LTD at excitatory synapses, and the current work shows that endogenous cannabinoids generate LTD at inhibitory synapses in GABAergic neurons within the hippocampus. Both DSI and LTD inhibit GABA neurons, resulting in enhanced activity in stimulated neurons, but they work on different time scales. Activity evoked by DSI lasts less than a minute while the LTD can persist for hours, even days. Coding of neuronal activities and their plasticity depends upon the balance between excitatory and inhibitory inputs in the neuronal network. As GABAergic transmission plays a critical role in the regulation of brain excitability, reduction in the level of inhibition will cause a long-lasting sensitization of the brain to stimulatory inputs and reinforcement of evoked responses, phenomena that could underlie the role of endocannabinoids in learning and memory. Disruption of the endogenous cannabinoids by a single exposure to _9THC, the exogenous cannabis derivative, was enough to produce a long lasting (3 days), reversible occlusion of LTD generation in hippocampus and N. Accumbens. It suggests that the functional disruption may be a use-dependent phenomenon. Chevaleyre, V. and Castillo, P.E. Heterosynaptic LTD of Hippocampal GABAergic Synapses: A Novel Role of Endocannabinoids in Regulating Excitability. Neuron. 38(3), pp. 461-472, 2003. Mato, S., Chevaleyre, V., Robbe, D., Pazos, A., Castillo, P.E. and Manzoni, O.J. A Single in-vivo Exposure to _9THC Blocks Endocannabinoid-Mediated Synaptic Plasticity. Nature Neuroscience 7, pp. 585-586, 2004.

Prenatal Cocaine Exposure and Behavioral Tolerance

Repeated exposure to cocaine during sensitive periods of forebrain development produces specific, long-lasting changes in the structure and function of maturing neural circuits. Similar regimens of drug exposure in adult animals with mature, homeostatically regulated nervous systems produce neuroadaptations that appear to be quite different in nature and magnitude. In a recent paper, Dr. Pat Levitt and his associate, Dr. Gregg Stanwood, Vanderbilt University School of Medicine, report that specific adaptive changes in neural signaling and/or circuitry that occur in response to repeated exposure to psychostimulants are highly dependent upon the maturational state of the brain. Their studies were designed to investigate the ability of cocaine to induce behavioral sensitization and/or tolerance following repeated administration of cocaine to pregnant rabbits during the period of peak differentiation within the rabbit cerebral cortex (embryonic day 16-25). Offspring and the mothers were later tested following acute administration of amphetamine challenge. The offspring, having received cocaine during the prenatal sensitive period, showed profound behavioral tolerance to the amphetamine challenge. In contrast, the mothers of these offspring, who received cocaine at the same dose and duration, and experienced the same period of withdrawal, exhibited robust behavioral sensitization. Stanwood, G.D. and Levitt, P. Repeated I.V. Cocaine Exposure Produces Long-lasting Behavioral Sensitization in Pregnant Adults, But Behavioral Tolerance in their Offspring. Neuroscience 122, pp. 579-583, 2003.

Vesicular Glutamate Transporters 1 and 2 Target to Functionally Distinct Synaptic Release Sites

Glutamate is the major excitatory neurotransmitter in the brain and in a number of studies has been shown to be important in mediating drug seeking behavior. Like many other neurotransmitters glutamate is stored in synaptic vesicles and is used to communicate with other neurons. Central to the process of synaptic transmission is the means by which neurons concentrate and transport neurotransmitters like glutamate in synaptic vesicles in the nerve terminal. In the April 29, 2004 issue of Science, Dr. Robert Edwards and his colleagues describe two glutamate transporters on nerve terminal (VGLUT1 and VGLUT2) that are targeted to two distinct synaptic release sites. Expression of VGLUT1 is observed in the hippocampus, cerebral cortex, and cerebellar cortex while VGLUT2 is expressed in thalamus, brain stem nuclei, and deep cerebellar nuclei. Synapses expressing VGLUT1 show a lower probably of transmitter release while synapses expressing VGLUT2 exhibit a greater probability of transmitter release when a nerve impulse invades the synaptic terminal. To test directly the role that VGLUT1 plays in synaptic transmission, Dr. Robert Edwards and his colleagues created a mouse lacking the VGLUT1 transporter. The mice lacking the VGLUT1 transporter appear normal for the first two weeks, but then began to show progressive neurological defects resulting in incoordination, blindness, and enhanced startle response. This phenotype is associated with VGLUT1 independent synaptic transmission in the cerebellar cortex and hippocampus that precipitously declines to nothing during the first two months after birth. Analysis, using in situ hybridization, suggests that VGLUT2 is co-expressed with VGLUT1 in the hippocampus and cerebellum during the first two weeks followed by rapid decline of VGLUT2 expression. The overlapping pattern of expression is different from the non-overlapping patterns of expression. So this raises the question of whether the VGLUT1 and VGLUT2 have unique functions within the same neuron. Both electrophysiological analysis and immunohistochemistry shows that VGLUT1 and VGLUT2 are targeted to distinct synaptic release sites instead of being localized to the same synaptic vesicle or different synaptic vesicles at the same release site. Dr. Edwards and his colleagues suggest that differences in vesicles recycling may explain the differences in the probability of release at synapses where VGLUT1 and VGLUT2 are expressed. Synapses that show lower probability of release such as in the hippocampus and cerebellum are more likely to show plasticity. Thus, analysis of the roles that glutamate transporter play may help understand the mechanisms underlying synaptic plasticity. The next key question is whether VGLUT1 and VGLUT2 contribute to differences in the probability of release or are simply a marker for this difference. Fremeau, R.T. Jr., Kam, K., Qureshi, T., Johnson, J., Copenhagen, D.R., Storm-Mathisen, J., Chaudhry, F.A., Nicoll, R.A. and Edwards, R.H. Vesicular Glutamate Transporters 1 and 2 Target to Functionally Distinct Synaptic Release Sites. Science, 304(5678), pp. 1815-1819, 2004.

MuLK: A Novel Eukaryotic Multi-substrate Lipid Kinase

One of the fundamental questions in biology is how components of cells communicate within a cell and between cells. One class of molecules that act as cellular messengers is lipids or fatty molecules. The addition of phosphates to these lipid messengers by protein kinases or removal of phosphate molecules by protein phosphatases from the lipid molecules such as mono- and diacylglycerols, sphingosine and ceramides produces signaling molecules that can regulate their function and in turn alter cellular processes such as the secretion of a neurotransmitter from the nerve terminal. A calcium dependent ceramide kinase has previously been found to be associated with synaptic vesicles that store neurotransmitter. In an attempt to identify this ceramide kinase Dr. Bajjalieh and her colleagues searched the human genome database for sequences that resembled the sequence for sphingosine kinase 2, a kinase they hypothesized to share some sequence similarity with the ceramide kinase they sought. As a result of their search Dr. Bajjalieh and her colleagues discovered a unique lipid kinase that phosphorylates not only ceramides, but also other lipids. Typically, the kinases that phosphorylate different lipid types, such as diacylglycerols, ceramides and sphingosine belong to different molecular families that are selective for a specific lipid type. Because of its ability to phosphorylate multiple substrates, they have named the new kinase MuLK for multi-substrate lipid kinase. Based on its sequence, one would conclude that MuLK is expressed in the cytosol or in the nucleus of cells, but localization studies performed in fibroblast cells suggest that MuLK is associated with intracellular membranes. The sequence also suggests that MuLK itself may be a kinase substrate, although the researchers did not conduct those experiments as part of this study. While phosphorylation and dephosphorylation through kinase and phosphatase activities are ways to regulate the activity of molecules in a cell, the kinase activities are also affected by the conditions of the cell. In this case, they found that MuLK is stimulated by calcium when magnesium concentrations are low and inhibited by calcium when magnesium concentrations are high. While the significance of this mode of regulation has not been elucidated, calcium is a regulator of several cellular processes including the secretion of neurotransmitter. One of the other things the researchers observed was low-level activity of MuLK toward the phosphorylation of 2AG, a lipid and endocannabinoid, the endogenous ligand for the cannabinoid receptor. Since phosphorylation often affects molecular interactions, it is likely that phosphorylation of 2AG and other lipid molecules by MuLK results in modification of those molecules with other molecules in the cell. Modification of the molecular interactions often results in changes in cellular activity. What started as a database query to look for ceramide kinases, has led to the beginning of an understanding of the regulation of the first identified multiple substrate lipid kinase. The characterization of this multi substrate lipid kinase and similar molecules may help to elucidate fundamental cellular processes as well as shedding light on how the biochemical pathway involved in processing endogenous cannabinoids is regulated. Waggoner, D.W., Johnson, L.B., Mann, P.C., Morris, V., Guastella, J., and Bajjalieh, S.M. MuLK: A Novel Eukaryotic Multi-substrate Lipid Kinase. Journal of Biological Chemistry, Jul 13 [Epub ahead of print], 2004.

MPDZ Is a Quantitative Trait Gene for Drug Withdrawal Seizures

About 50-60% of drug dependence is genetically determined and its effects include influence on physiological dependence and associated withdrawal from sedative hypnotics, including alcohol, benzodiazepines, inhalants and barbiturates. Buck and colleagues used robust behavioral models of drug physiological dependence in mice and identified an addiction-relevant quantitative trait gene, called the multiple PDZ domain gene (Mpdz), among five candidate genes. PDZ domains are modular protein interaction domains that bind in a sequence-specific manner to short C-terminal peptides or internal peptides. They are typically involved in the assembly of supramolecular complexes that perform localized signaling functions at particular subcellular locations. The Mpdz gene was the only one of the five candidates to show genotype-dependent differences in coding sequence. Mpdz expression was significantly associated with severities of withdrawal from alcohol and pentobarbital, such that lower expression of Mpdz was genetically correlated with greater withdrawal seizure susceptibility. They speculate that Mpdz protein expression and/or structure both affect drug dependence and withdrawal by altering the rate and/or fidelity of signal transduction mediated by one or more of its interaction partners. One mechanism may be that the Mpdz protein interacts with GABAB receptors, which regulate glutamate and GABA release. Findings like these may help to identify new targets for pharmacotherapies for withdrawal. Shirley, R.L., Walter, N.A.R., Reilly, M.T., Fehr, C. and Buck, K.J. MPDZ Is a Quantitative Trait Gene for Drug Withdrawal Seizures. Nature Neuroscience, 7, pp. 699-700, 2004.

Dopamine Transporter Inhibitors Decrease Cocaine Self-Administration in Rhesus Monkeys

The dopamine transporter (DAT) is thought to play a critical role in mediating the reinforcing efficacy and subjective effects of cocaine. It has been known for many years that the affinity of drugs for the DAT is positively correlated with their capacities to maintaining self-administration behavior in animals. There has been an interest in DAT inhibitors as medications to treat cocaine dependence. Dr. Leonard Howell and his associates at Yerkes National Regional Primate Research Center investigated a series of DAT inhibitors with varying affinities for other monoamine transporters for their ability to block cocaine self-administration in rhesus monkeys. In addition, the behavioral effects of the drugs were compared to their DAT occupancy using PET imaging, both in awake animals and in animals under isoflurane anesthesia. Doses of GBR 12909 and RTI-177 that reduced cocaine self-administration by 50% were associated with DAT occupancies of approximately 67 and 73%, respectively. Both of these compounds were also self-administered by the animals at doses that produced DAT occupancies of 57 and 92%, respectively. RTI-112 is a mixed action monoamine transporter inhibitor that is about equally potent at the DAT and the serotonin transporter (SERT). This compound also inhibits cocaine self-administration in monkeys at a dose that does not produce detectable changes in DAT occupancy. It does, however, occupy approximately 90% of SERT, suggesting that RTI-112's contributions were due to its serotonergic properties and not its dopaminergic ones. Additionally, RTI-112 was not self-administered by monkeys and therefore not likely to have any abuse liability. Lindsey, K.P., Wilcox, K.M., Votaw, J.R, Goodman, M.M., Plisson, C., Carroll, F.I., Rice, K.C. and Howell, L.L. Effects of Dopamine Transporter Inhibitors on Cocaine Self-Administration in Rhesus Monkeys: Relationship to Transporter Occupancy Determined by Positron Emission Tomography Neuroimaging. Journal of Pharmacology and Experimental Therapeutics, 309(3), pp. 959-969, 2004.

Menthol Pharmacology and its Potential Impact on Cigarette Smoking

Mentholated cigarettes are disproportionately smoked by African American smokers which, as a group, are the least likely "ever smokers" to quit smoking. However, since cigarette smoke consists of over 4000 substances, it has been difficult to study the effects due to the menthol additive. This article reviews the research to date on menthol, cigarettes, and smoking behavior. Menthol has cooling and local anesthetic effects, which may be mediated by action on agonist action on a thermally responsive receptor. In addition to thermal effects, menthol has been found to augment nicotine's reinforcing effects, possibly due to a decrease in nicotine and cotinine (a nicotine metabolite) metabolism, increased lung exposure to tobacco smoke constituents, and/or stimulant and depressant effects on the central nervous system. Menthol may also possess reinforcing effects independent of nicotine. There may also be differences in some of menthol's effects across racial and ethnic groups, as illustrated by African American smokers showing typically higher levels of cotinine as compared to Mexican American and Caucasian smokers. Future research directions are also discussed. Ahijevych, K. and Gerrett, B.E. Menthol Pharmacology and its Potential Impact on Cigarette Smoking Behavior. Nicotine Tobacco Research, 6 (supple 1), S17-S28, 2004.

Cannabinoids Activate Sensory Pain Fibers

Delta9-tetrahydrocannabinol (THC), the psychoactive component of marijuana, is known to activate cannabinoid receptors in the brain and periphery to produce many of its effects. However, research supported by NIDA has demonstrated that in sensory nerve fibers, THC also opens the ANKTM1 channel, a calcium channel that has recently been implicated in the detection of noxious cold stimuli. Likewise, mustard oil (allyl isothiocyanate), which produces pain with topical application, also was found to activate the ANKTM1 channel. The activation of the ANKTM1 channel by either THC or mustard oil was blocked by ruthenium red, a ANKTM1 channel blocker. These findings demonstrate a novel site of action of cannabinoids, where activation of this site appears to be pro-nociceptive (pain inducing). Jordt, S-E., Bautisa, D., Chaung, H., McKemy, D.D., Zygmunt, P.M., Hogestatt, E.D., Meng, I.D. and Julius, D. Mustard Oils and Cannabinoids Excite Sensory Nerve Fibers through the TRP Channel ANKTM1. Nature, 427, pp. 260-265, 2004.

Dopaminergic Supersensitivity in G Protein-Coupled Receptor Kinase 6-Deficient Mice

Brain dopaminergic transmission is a critical component in numerous vital functions, and its dysfunction is involved in several disorders, including addiction and Parkinson's disease. Responses to dopamine are mediated via G protein-coupled dopamine receptors (D1-D5). Desensitization of G protein-coupled receptors is mediated via phosphorylation by members of the family of G protein-coupled receptor kinases (GRK1-GRK7). Dr. Richard Premont of the Department of Medicine, Duke University Medical Center and his colleagues show that GRK6-deficient mice are supersensitive to the locomotor-stimulating effect of psychostimulants, including cocaine and amphetamine. In addition, these mice demonstrate an enhanced coupling of striatal D2-like dopamine receptors to G proteins and augmented locomotor response to direct dopamine agonists both in intact and in dopamine-depleted animals. The present study indicates that postsynaptic D2-like dopamine receptors are physiological targets for GRK6 and suggests that this regulatory mechanism contributes to central dopaminergic supersensitivity. Gainetdinov, R.R., Bohn, L.M., Sotnikova, T.D., Cyr, M., Laakso, A., Macrae, A.D., Torres, G.E., Kim, K.M., Lefkowitz, R.J., Caron, M.G. and Premont, R.T. Dopaminergic Supersensitivity in G Protein-coupled Receptor Kinase 6-deficient Mice. Neuron, 38(2), pp. 291-303, 2003.

Cyclin-Dependent Kinase 5 Phosphorylates the N-terminal Domain of the Postsynaptic Density Protein PSD-95 in Neurons

PSD-95 (postsynaptic density 95) is a scaffolding protein that links NMDA receptors to the cytoskeleton and to its signaling molecules. The N-terminal domain of PSD-95 is involved in the synaptic targeting and clustering of PSD-95 and in the clustering of NMDA receptors at synapses. There are consensus phosphorylation sites in the N-terminal for cyclin-dependent kinase 5 (cdk5), a serine-threonine kinase that is necessary for brain development. This kinase is also implicated in synaptic plasticity, dopamine signaling, cocaine addiction, and neurodegenerative disorders. Dr. Maria Morabito of Harvard Medical School and her research team report that PSD-95 is phosphorylated in the N-terminal domain by cdk5 both in vitro and in vivo. Furthermore, this phosphorylation is not detectable in brain lysates of mice that lack cdk5 (cdk5-/- mice). The phosphorylated product was found in postsynaptic densities together with cdk5 and its activator, p35, suggesting that phosphorylated PSD-95 may have an important action at synapses. In heterologous cells, coexpression of active cdk5 with PSD-95 reduces the ability of PSD-95 to multimerize and to cluster neuronal ion channels. When cdk5 was omitted from the cultures, there were larger clusters of PSD-95/NMDA receptors. In cortical neurons from mice that did not express cdk5 (-/-) cortical neurons, there were more PSD-95 immunostained clusters than were observed in wild-type neurons. In hippocampal neurons, expression of the inactive form on cdk5 (DNcdk5) or of full-length PSD-95 with the triple alanine mutant (T19A, S25A, S35A), the size of the PSD-95 cluster was increased. These results are important because they identify cdk5-dependent phosphorylation of the N-terminal domain of PSD-95 as a novel mechanism for regulating the clustering of PSD-95/NMDA receptors and support the possibility that cdk5-dependent phosphorylation of PSD-95 dynamically regulates the clustering of PSD-95/NMDA receptors at synapses. Such an activity would provide a possible mechanism for rapid changes in density and/or number of receptors at synapses. Morabito, M.A., Sheng, M. and Tsai, L.H. Cyclin-dependent Kinase 5 Phosphorylates the N-terminal Domain of the Postsynaptic Density Protein PSD-95 in Neurons. Journal of Neuroscience, 24(4), pp. 865-876, 2004.

Morphine-induced Alterations of Immune Status are Blocked by the Dopamine D2-like Receptor Agonist

In is known that morphine administration produces profound effects on the immune system, including reductions in natural killer cell activity, mitogen-induced lymphocyte proliferation, and cytokine production. Although it has been established that the activation of central nervous system (CNS) mu-opioid receptors by morphine induces immunomodulation, little is known about the neural mechanisms underlying such processes. Interestingly, it has been shown that the dopamine (DA) D-2-like receptor agonist (7-OH-DPAT) blocks the effect of morphine on a number of behaviors that are mediated by central dopamine pathways. The present study examined whether dopamine is involved in the immunomodulatory effects of morphine. In separate experiments, 7-OH-DPAT was administered either systemically or in the brain prior to morphine treatment in male Lewis rats. The results demonstrate that both systemic and central administration of 7-OH-DPAT attenuate the suppressive effect of morphine on several measures of immune status. Overall, these findings provide the first evidence that brain dopaminergic mechanisms are directly involved in morphine-induced immunomodulation. Saurer, T.B., Carrigan, K.A., Ijames, S.G. and Lysle, D.T. Morphine-induced Alterations of Immune Status are Blocked by the Dopamine D2-like Receptor Agonist 7-OH-DPAT. Journal of Neuroimmunology, 148, pp. 54-62, 2004.

Proinflammatory Chemokines Can Affect Opioid Receptors

Pain is one of the hallmarks of inflammation. Opioid receptors mediate anti-pain responses in both the peripheral nervous system and the brain. The present study showed that pretreatment of the CCR1and mu-opioid receptor combination in a HEK293 cell preparation with the chemokine CCL3 (MIP-1alpha) induced internalization of mu-opioid receptors and severely impaired the mu-opioid receptor-mediated inhibition of cAMP accumulation. Further analysis using immunohistochemical staining showed that CCR1 and mu-opioid receptors were co-expressed on small to medium diameter neurons in rat dorsal root ganglion, and that both types of receptors were functioning. Pretreatment of neurons with CCL3 impaired the mu-opioid receptors. Other chemokines, such as CCL2, CCL5, and CCL8, exhibited similar inhibitory effects. These data indicate that proinflammatory chemokines are capable of desensitizing mu-opioid receptors on peripheral sensory neurons, providing a novel potential mechanism for peripheral inflammation-induced hyperalgesia. Zhang, N., Rogers, T.J., Caterina, M. and Oppenheim, J.J. Proinflammatory Chemokines, Such as C-C Chemokine Ligand 3, Desensitize Mu-opioid Receptors on Dorsal Root Ganglia Neurons. Journal of Immunology, 173, pp. 594-599, 2004.

Neuromolecular Activation in cAMP Response Element-binding Protein (pCREB) is Important in Stress-induced Relapse

Relapse to drug addiction is often precipitated by exposure to stress. Using animal models, relapse can be precipitated by administering the drug itself (i.e., drug-induced relapse), by exposure to a stressor (i.e., stress-induced relapse), or through exposure to the environment where drug taking previously took place (i.e., cue-induced relapse). To investigate the neurobiological changes associated with stress-induced relapse, Drs. Kreibich and Blendy from the University of Pennsylvania, exposed mice to a forced swim stressor (FS), which has been successfully used to test the efficacy of putative anti-depressants in animal models. In this experiment, exposure to FS induced relapse to cocaine preference in mice. Importantly, relapse was accompanied by an increase in the phosphorylated cAMP response element-binding protein (pCREB) in discrete brain regions that were distinct from the pattern observed after cocaine-induced relapse. Activation of pCREB mediates several aspects of addiction, depending on the specific region of the brain involved. For example, morphine-induced activation of CREB in the a region of the brain known as the locus coeruleus (LC) can lead to some of the symptoms underlying physical opiate dependence and withdrawal. Furthermore, chronic exposure to opiates, cocaine, and alcohol also activates pCREB in the nucleus accumbens (NA), a brain region associated with drug reward. Increased activation of CREB in this brain region is associated with a decrease in the rewarding effects of drugs of abuse such as cocaine. CREB may also affect the release of stress hormones such as corticotrophin-releasing factor, which can increase the likelihood of relapse. In this study, researchers further demonstrated that the alterations seen in CREB are a necessary molecular change for stress-induced relapse because the researchers showed that genetically altered mutant mice that lack CREB were not as susceptible to stress-induced relapse as wild-type mice. In contrast, however, these mutant mice lacking CREB were still susceptible to cocaine-induced relapse. Overall these results suggest that CREB's involvement and its pattern of activity in drug- and stress-induced relapse is different. Developing pharmacotherapies that regulate CREB may be useful for reducing and treating stress-induced relapse. Kreibich, A.S. and Blendy, J.A. cAMP Response Element-binding Protein is Required for Stress but not Cocaine-induced Reinstatement. Journal of Neuroscience, 24(30), pp. 6686-6692, 2004.

Homer Proteins Regulate Sensitivity to Cocaine

The consequences of addiction to cocaine include changes in brain chemistry and behavior. One consequence is reduced Homer protein. Szumlinski et al. demonstrate that reducing Homer1 or Homer2, but not Homer3, in drug-naive mice causes increased sensitivity to the locomotor activating effects of cocaine and PCP, but not heroin or caffeine. Furthermore, reduction of these two genes also enhanced glutamate output in the nucleus accumbens, an outcome usually observed upon withdrawal of cocaine. Restoration of the genes via adeno-associated virally mediated gene transfers reversed the cocaine-induced phenotype, which reulted in a decreased sensitivity to cocaine reward and motor activation, as well as less excitatory drive in the nucleus accumbens. The parallel between animals addicted to cocaine and reduced Homer suggests that Homer may regulate the development of addiction. Szumlinski, K.K., Dehoff, M.H., Kang, S.H., Frys, K.A., Lominac, K.D., Klugmann, M., Rohrer, J., Griffin, W., III, Toda, S., Champtiaux, N.P., Berry, T., Tu, J.C., Shealy, S.E., During, M.J., Middaugh, L.D., Worley, P.F. and Kalivas, P.W. Homer Proteins Regulate Sensitivity to Cocaine. Neuron, 43(3), pp. 401-413, 2004.


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