Research Findings - Basic Neuroscience Research

Multimodal Fast Optical Interrogation of Neural Circuitry

Karl Deisseroth, M.D., Ph.D., a NIDA grantee and an awardee for PECASE (Presidential Earlier Career Award for Scientists and Engineers) as well as NIH Director's Pioneer Award, and his research team at Stanford University and collaborators at Max-Plank Institute have published a groundbreaking photogenetic technology work in the April 5, 2007 issue of Nature, describing how genetically engineered proteins can be used to activate or silence very precise groups of neurons at a flick of a light switch (ms time scale), allowing real time, direct control and interrogation of neural circuits dynamics in the brain, a dream that has long been sought by neuroscientists but has been impossible until now. The technology involves a pair of light-activated membrane proteins present in nature, that is, the ChR2 (Channelrhodopsin-2)-NpHR (Natronomonas pharaonis halorhodopsin) system. The ChR2 is a light-activated ion-channel protein present in the green alga Chlamydomonas reinhardtii. When neurons were genetically engineered to express ChR2, blue light activates ChR2 and rapidly and selectively depolarizes the cell and triggered action potentials, giving a genetically encodable, remote-control "on" switch for neurons. NpHR, on the other hand, is a chloride ion pump present in the archaeum Natronomonas pharaonis. When it is expressed in neurons, yellow light activates NpHR and causes rapid and reversible hyperpolarization, thus preventing spikes, allowing a genetically encodable, remote-control "off" switch for neurons. The ChR2- NpHR system, although like all opsins, requires a vitamine-A-based chromophore cofactor in order to be activated by light, needs no addition of such cofactor because mammalian neural tissue normally contains enough of this cofactor. In addition, when combined with fluorescent-based methods for monitoring activity, the ChR2- NpHR system allows simultaneous real-time control and tracking of neuronal activity on both ends. The authors demonstrated that, when genetically targeted to specific types of neuron in the worm C. elegans, ChR2-NpHR system is able to rapidly and reversibly activate or silence a particular neuronal subtype with light, and simultaneously observe the effect on behavior, in this particular case, swimming behavior. The spectacular beauty of the technology is that the ChR2 and the NpHR are totally compatible with and complementary to each other in all measures examined, including temporal resolution (m sc), spatial specificity, and separation of absorption spectrum. That is, the spectral bandwidths for activation of ChR2 and NpHR is such that coexpression of both proteins in the same neuron allows different wavelengths of light - blue and yellow, respectively, to be used to selectively activate or inactivate the same cell. In essence, the ChR2- NpHR system provides a two-knob remote control for increasing or decreasing the activity of specific neurons using different colors of light. Also, there is an additional advantage; the spectral bandwidth remaining is sufficient enough to be used for monitoring neural activity optically using a fluorescent indicator of intracellular calcium. In conclusion, the technology is a stunning technical breakthrough. It allows targeting genes in specific neurons, is safe (nontoxic), fully reversible, has rapid onset and offset, allows bidirectional control, and offers experimenters remote control. The technology is unprecedented, immensely powerful and offers, for the first time, neuroscientists the opportunity to directly interrogate neural circuits dynamics and observe their effects on behavior in real time. The impact of the technical breakthrough is obvious, and will be far reaching beyond neurological and psychiatric disorders and the technology promises to revolutionize neuroscience research. The work has been featured in the same issue of Nature, Web Focus, Technical Breakthroughs in Neuroscience. Zhang, F., Wang, L.-P., Brauner, M., Liewald, J.F., Kay, K., Watzke, N., Wood, P.G., Bamber, E., Nagel, G., Gottschalk, A., and Deisseroth, K. Multimodal Fast Optical Interrogation of Neural Circuitry. Nature, 446(7136), pp. 633-639, 2007.

Dicarba Analogues of the Cyclic Enkephalin Peptides H-Tyr-c[D-Cys-Gly-Phe-D(or L)-Cys]NH2 Retain High Opioid Activity

Dicarba analogues of the cyclic opioid peptides might have potential as lead compounds for analgesic drug discovery. Dicarba analogues of the cyclic opioid peptides H-Tyr-c[D-Cys-Gly-Phe-D(or L)-Cys]NH2 were synthesized on solid phase by substituting allylglycines for the cysteines and cyclization by ring-closing metathesis between the side chains of the allylglycine residues. Mixtures of cis and trans isomers of the resulting olefinic peptides were obtained, and catalytic hydrogenation yielded the saturated -CH2-CH2- bridged peptides. The dicarba analogues retained high mu- and delta-agonist potencies. Remarkably, the trans isomer of H-Tyr-c[D-Allylgly-Gly-Phe-L-Allylgly]NH2 was a mu-agonist/delta-agonist with subnanomolar potency at both receptors. These chimeric peptides may potentially serve as lead compounds for analgesic drug discovery. Berezowska, I., Chung, N.N., Lemieux, C., Wilkes, B.C., and Schiller, P.W. Dicarba Analogues of the Cyclic Enkephalin Peptides H-Tyr-c[D-Cys-Gly-Phe-D(or L)-Cys]NH2 Retain High Opioid Activity. Journal of Medicinal Chemistry, 50, pp. 1414-1417, 2007.

Role of Endocannabinoids in Alcohol Consumption and Intoxication: Studies of Mice Lacking Fatty Acid Amide Hydrolase

Dr. Cravatt and colleagues have recently reported that endocannabinoid signaling plays an important role not only in the regulation of drug intake, but also in regulation of ethanol intake. Fatty acid amide hydrolase (FAAH) is a key membrane protein for metabolism of endocannabinoids, including anandamide, and blockade of FAAH increases the level of anandamide in the brain. To determine if FAAH regulates ethanol consumption, the PI studied mutant mice with deletion of the FAAH gene. Null mutant mice showed higher preference for alcohol and voluntarily consumed more alcohol than wild-type littermates. There was no significant difference in consumption of sweet or bitter solutions. To determine the specificity of FAAH for ethanol intake, the PI studied additional ethanol-related behaviors. There were no differences between null mutant and wild-type mice in severity of ethanol-induced acute withdrawal, conditioned taste aversion to alcohol, conditioned place preference, or sensitivity to the hypnotic effect of ethanol. However, null mutant mice showed shorter duration of loss of righting reflex induced by low doses of ethanol (3.2 and 3.4 g/kg) and faster recovery from motor in-coordination induced by ethanol. All three behavioral phenotypes (increased preference for ethanol, decreased sensitivity to ethanol-induced sedation, and faster recovery from ethanol-induced motor incoordination) seen in mutant mice were reproduced in wild-type mice by injection of a specific inhibitor of FAAH activity--URB597. These data suggest that increased endocannabinoid signaling increased ethanol consumption owing to decreased acute ethanol intoxication. Blednov, Y.A., Cravatt, B.F., Boehm II, S.L., Walker, D., and Harris, R.A. Role of Endocannabinoids in Alcohol Consumption and Intoxication: Studies of Mice Lacking Fatty Acid Amide Hydrolase. Neuropsychopharmacology, pp. 1-13, 2006, epub ahead of print.

Solubilized Neurotransmitter Assay

Membrane transporters such as the dopamine, serotonin, and norepinephrine transporters facilitate the uptake or reuptake of the corresponding neurotransmitters dopamine, serotonin, and norepinephrine across neuronal membranes in a process involving co-transport of sodium and chloride ions. Mammalian versions of these particular transporters are of interest to NIDA because of their affinity for cocaine and amphetamine. Because the limited expression of mammalian transporter quantities suitable for structural studies is a concern, researchers are also interested in over-expressing bacterial transporter homologs in hosts such as E. coli, for biophysical study of structure and function, and possible crystallization, which would yield a three dimensional picture of the transporter. Drs. Javitch and Quick have recently reported on the application of an existing assay technique, the scintillation proximity assay (SPA), to measure the tritiated tyrosine binding affinity and binding rate of bacterial tyrosine transporter Tyt1 solubilized in detergent. Using the SPA system, it was possible to show that Tyt1 has a high and specific affinity for tyrosine binding at pH 6.5-7.5, and operates with a stoichiometry of two sodium ions to one of tyrosine, with no dependence on chloride ion. The practical advantages of this system include obtaining binding affinity constants for tyrosine and sodium, comparison of results in different E. coli host expression systems and in different detergents, and optimization of purification techniques by comparison of results from different chromatographic fractions. In the case of newly isolated bacterial orphan transporters, this technique can be used to determine relative affinity toward a battery of commercially available tritiated amino acids. Quick, M. and Javitch, J.A., Monitoring the Function of Membrane Transport Proteins in Detergent-Solubilized Form. Proceedings of the National Academy of Sciences, 104(9), pp. 3603-3608, 2007.

Adolescent Anabolic-Androgen Steroids Exposure and its Consequences

Earlier work in hamsters has demonstrated that adolescent anabolic-androgen steroids (AAS) exposure facilitates offensive aggression, in part by altering the development and activity of anterior hypothalamic arginine vasopressin (AH-AVP). In a recent study Dr. Richard Melloni and his group first determined whether adolescent AAS exposure had lasting behavioral effects on aggression following suspension of AAS administration. Adolescent hamsters administered AAS were tested as adults for offensive aggression at 1, 4, 11, 18, or 25 days following AAS withdrawal. Data from this study showed that AAS-exposed hamsters were significantly more aggressive than age-matched controls through Day 12 of AAS withdrawal, however, these behavioral differences were no longer observable by Day 19 indicating the behavioral effects are not permanent. This study also examined whether the effects of AAS withdrawal on AH-AVP neural system were long-lasting or short-term. Using immuno-histochemical techniques, they measured AH-AVP afferent innervation. As seen with aggression, AH-AVP innervation was significantly higher in AAS-treated hamsters compared to controls through Day 12 of AAS withdrawal; however, these differences were no longer observable by Day 19 of withdrawal, at which point the neurobiology of AAS-treated hamsters reverted to that observed in controls, again as seen with the behavioral study. These findings suggest that adolescent AAS exposure has short-term, reversible effects on both aggression and AH-AVP, correlating AH-AVP with the aggressive/ non-aggressive behavioral phenotype during AAS withdrawal. Grimes, J.M., Ricci, L.A., and Melloni Jr, R.H. Plasticity in Anterior Hypothalamic Vasopressin Correlates with Aggression during Anabolic-androgenic Steroid Withdrawal in Hamsters. Behavioral Neuroscience, 120(1), pp. 115-124, 2006.

Chronic Morphine Exposure Causes Pronounced and Altered Virus Replication

Two recent reports delineate some important results following exposure to a "trivirulent" SIV in combination with morphine dependence. This combination results in increased mortality and increased neurologic disorders were more prevalent with opiate-dependence. In a corresponding paper, increased genetic evolution of the SIV was associated with morphine-dependence. This may indicate that opiates could exacerbate viral strain evolution in humans and provide an understanding of some of the bases for different HIV viruses being present in drug abusers. Kumar, R., Orsoni, S., Norman, L., Verma, A.S., Tirado, G., Giavedoni, L.D., Staprans, S., Miller, G.M., Buch, S.J. and Kumar, A. Chronic Morphine Exposure Causes Pronounced Virus Replication in Cerebral Compartment and Accelerated Onset of AIDS in SIV/SHIV-infection, Virology, 354, pp. 192-206, 2006.

Synthesis of Salvinorin A Analogues as Opioid Receptor Probes

The main active constituent isolated from the leaves of Salvia divinorin is salvinorin A, a neoclerodane diterpene. This compound was found to be a potent and selective kappa opioid receptor agonist. Interestingly the pharmacology of Salvinorin A appeared to be different from other kappa agonists. Research has shown that salvinorin A decreases dopamine levels in the caudate putamen of mice and that this effect is blocked by the kappa opioid antagonist nor-binaltorphimine. Another study has shown that salvinorin A dose dependently increases immobility in the forced swim test, indicating that this compound has depressive like effects. Recently authors have described the synthesis of several analogues of salvinorin A that were found to be opioid receptor ligands. In this paper they have reported the semi-synthesis of neoclerdane diterpenes and the structure-affinity relationships at opioid receptors. This work will allow the further development of novel opioid receptor ligands that may be important for the treatment of substance abuse. Tidgewll, K., Harding, W.W., Lozama, A., Cobb, H., Shah, K., Kannan, P., Dersch, C.M., Parrish, D., Deschamps, J.R., Rothman, R.B., and Prisinzano, T.E. Synthesis of Salvinorin A Analogues as Opioid Receptor Probes. Journal of Natural Products, 69, pp. 914-918, 2006.

Novel Non-Opioid Analgesics Developed

Improgan has been found to have analgesic properties in animal models. Improgan is chemically related to histamine, yet does not produce analgesia via an action at the histamine receptor, or by a direct action at any other receptors known to be involved with pain modulation (e.g. opioid receptors). NIDA grantee Lindsay Hough (Albany Medical College, Albany, NY) and his colleagues have now produced a series of improgan congeners, where five compounds showed analgesic activity with potencies greater than that of improgan. One compound, VUF5420, produced maximal analgesia in rats after doses that produced no motor impairment or other obvious toxicity. VUF5420 analgesia was unaffected by the administration of the opioid antagonist naltrexone, but was inhibited by the CB1 cannabinoid antagonist SR141716A. However, VUF5420 had a very low affinity for the CB1-receptor in vitro, indicating that its analgesic action in vivo was not the result of a direct action on the CB1 receptor. Thus, VUF5420 may have clinical use as a non-opioid analgesic, which may indirectly activate cannabinoid pain-relieving mechanisms. Hough, L.B., de Esch, I.J.P., Janssen, E., Phillips, J., Svokos, K., Kern, B., Trachler, J., Abood, M.E., Leurs, R., and Nalwalk, J.W. Neuropharmacology, 51, pp. 447-456, 2006.

Inhibition of Cdk5 in the Nucleus Accumbens Enhances the Locomotor-Activating and Incentive-Motivational Effects of Cocaine

Genes provide instructions for carrying out cellular processes by telling the cell whether a protein or enzymes that carries out cellular processes is to be made. The instructions are carried out by first "transcribing" the DNA sequence in the gene into a messenger RNA transcript (mRNA) in the cell nucleus. The mRNA is then transported to the ribosomes in the cytoplasm where the mRNA is translated into proteins consisting of strings of amino acids. The process of transcription is regulated by proteins called transcriptional factors. One transcriptional factor called delta FOSB is upregulated in the nucleus accumbens by addictive drugs when administered chronically. The accumbens is a subcortical region involved in reward. One of the genes turned on by delta FOSB is cyclin dependent kinase-5 (cdk-5). Dr. Bibb and his colleagues examined the role of cdk-5 in the nucleus accumbens on cocaine-induced locomotor sensitization, responding for reward-associated stimuli (conditioned reinforcement), and cocaine self-administration. Injections of cdk-5 blockers into the nucleus accumbens augmented the increased sensitivity of rats to the locomotor effects of cocaine following repeated administration of cocaine. In addition, responding to a conditioned reward, in which a stimulus such as light is paired with cocaine injection, is greatly enhanced when rats received intra-accumbens injections of cdk-5 blockers. Dr. Bibb and his colleagues also show that rats will work harder to self-administer cocaine following injections in the nucleus accumbens of cdk-5 blockers. These results suggest that cdk-5 acts as negative feedback regulator to dampen the psychomotor and incentive-motivational effects of cocaine. Taylor, J.R., Lynch, W.J., Sanchez, H., Olausson, P., Nestler, E.J., and Bibb, J.A. Inhibition of Cdk5 in the Nucleus Accumbens Enhances the Locomotor-Activating and Incentive-Motivational Effects of Cocaine. Proceedings of the National Academy of Sciences 104, pp. 4147-4152, 2007.

IRS2-Akt Pathway in Midbrain Dopamine Neurons Regulates Behavioral and Cellular Responses to Opiates

Chronic administration of morphine is associated with tolerance or decrease in the ability to produce euphoria. During long term abstinence this process is reversed. Eric Nestler and his colleague show in a recent issue of Nature Neuroscience that the development of tolerance to the rewarding properties parallels the decreased size of Ventral Tegmental Area (VTA) dopamine neurons. The size of the VTA dopamine neurons returns to normal after tolerance to the euphoric properties of morphine is reversed by abstinence. Because knockouts of brain derived neurotrophic factor (BDNF) abolishes the rewarding effects of morphine and infusions of BDNF into the VTA block the decrease in size, Dr. Nestler and his colleagues hypothesized that one of the intracellular messengers activated by BDNF mediates the reduction in size. The BDNF uses three intracellular messengers to convey the BDNF signal from the cell surface to inside the cell. These are ras-erk pathway; the phopholipase C-gamma pathway, and the IRS2-Akt. Dr. Nestler and his colleagues report that the IRS-Akt pathway is downregulated following chronic morphine and is responsible for the decreased size of VTA dopamine neurons and the development of tolerance to morphine reward. The decrease in the amount of IRS parallels the decrease in size of the VTA neurons and the development of tolerance. The size of the VTA dopamine neurons and morphine reward as measured by conditioned place preference were decreased when infected with a virus expressing a mutant IRS protein that blocks the activation of the IRS-Akt pathway. The effect on size of the dopamine neurons was not due to the viral infection because normal viruses not expressing the mutant IRS protein did not have any effect on size. Furthermore, the decrease in size of the VTA dopamine neurons following chronic morphine could be reversed by infecting VTA dopamine neurons with a virus that expresses the normal IRS protein. The effect of the mutant IRS protein on cell size was selective because ERK and PLC-gamma mutants had no effect on cell size. Increased expression of the normal IRS protein in dopamine neurons is also associated with increased locomotor activity and reward in response to morphine. These results suggest that activation of the IRS protein is necessary and sufficient for regulating dopamine cell morphology and morphine reward. An understanding of the role of the IRS-Akt pathway may help in the development of therapeutics that can delay or reverse the transition to opiate addiction. Russo, S.J., Bolanos, C.A., Theobald, D.E., DeCarolis, N.A., Renthal, W., Kumar, A., Winstanley, C.A., Renthal, N.E., Wiley, M.D., Self, D.W., Russell, D.S., Neve, R.L., Eisch, A.J., and Nestler, E.J. IRS2-Akt Pthway in Midbrain Dopamine Neurons Regulates Behavioral and Cellular Responses to Opiates. Nature Neuroscience, 10, pp. 93-99, 2007.

Disruption of the Circadian Regulator, CLOCK, Induces Mania-like Behavior in Mice

Individuals with bipolar disorder often have disturbances in their circadian rhythms (sleep, wake and activity cycles). Furthermore, a specific genetic polymorphism in the human circadian regulator Clock is associated with a propensity for manic episodes. To functionally test the relationship between Clock and mania, Dr. McClung and coworkers characterized the behaviors of mice with a mutation in the Clock gene. They found that compared to wild type mice, Clock mutant mice had an enhanced preference for rewarding stimuli such as cocaine or sucrose, reduced levels of anxiety, and reduced levels of depression-like behavior. In fact the behaviors exhibited by the Clock mutant mice were very similar to the behaviors exhibited by bipolar patients in a manic state. Furthermore, the mania-like behavior of the Clock mutant mice could be reduced by lithium, a common treatment for human mania. This further argues that the suite of behaviors exhibited by the Clock mutant animals models the manic phase of human bipolar disorder. Clock is expressed in a number of brain regions, but where is it required to regulate mania-like behaviors? Several lines of evidence link mania with the ventral tegmental area (VTA). Dr. McClung and colleagues used a viral gene transfer method to restore Clock function specifically to the VTA. The animals with restored Clock function in the VTA had levels of anxiety and activity similar to "normal" wild type animals, indicating that these behaviors are regulated by Clock function in the VTA. Dr. McClung's work has led to the development of a robust mouse genetic model for the manic phase of bipolar disorder. This mouse model will be extremely useful for future genetic and therapeutic investigations into this psychiatric disorder as well as the links between this disorder and drug addiction. Roybal, K., Theobold, D., Graham, A., DiNieri, J.A., Russo, S.J., Krishnan, V., Chakravarty, S., Peevey, J., Oehrlein, N., Birnbaum, S., Vitaterna, M.H., Orsulak, P., Takahashi, J.S., Nestler, E.J., Carlezon, W.A. Jr, and McClung, C.A. Proceedings of the National Academy of Sciences, 104, pp. 6406-6411, 2007.

Aberrant GABA Neuronal Migration Caused by Imbalance of Dopamine Receptors in Embryonic Brain

During brain development newly generated neurons migrate to the cortical walls to form layers, as well as to the limbic areas to form nuclei. GABA neurons, generated in the basal forebrain migrate into the neocortical areas during development and play a role in modulating neuronal proliferation and migration, thereby generating functional neural circuits. A team led by Pradeep Bhide at Harvard University and Massachusetts General Hospital reports that since GABA neurons express dopamine receptors D1 and D2, and since the embryonic basal forebrain is enriched in dopamine, GABA neuron migration is heavily influenced by the activation of dopamine receptors. Specifically, using ex vivo live cortical slices and in vivo tracing, the researchers show that increased dopamine concentration and activation of the D1 receptor promotes GABA neuron migration from the medial and caudal ganglionic eminences to the cerebral cortex, but increased dopamine concentration and activation of D2 receptor decreases this migration. This finding is then confirmed using slice preparations from D1 or D2 receptor knock-out mouse embryos. They show that D1 receptor electroporation into cells of the basal forebrain and pharmacological activation of these receptors promotes the migration of the electroporated cells to the cerebral cortex. Finally, to identify the cellular mechanisms involved, the team shows that dopamine receptor activation mobilizes striatal neuronal cytoskeleton in a manner consistent with the effects on neuronal migration. These data suggest that impairing the physiological balance between D1 and D2 receptors can alter GABA neuron migration from the basal forebrain to the cerebral cortex, with resulting consequences in widely distributed abnormalities in the embryonic brain. This observation provides insights into how the exposure of developing brains to certain drugs of abuse, such as cocaine and amphetamine that activate dopamine receptors, results in the malformation of the brain. Crandall, J.E., McCarthy, D.M., Araki, K.Y., Sims, J.R., Ren, J.-Q., and Bhide, P.G. Dopamine Receptor Activation Modulates GABA Neuron Migration from the Basal Forebrain to the Cerebral Cortex. Journal of Neuroscience, 27, pp. 3813-3822, 2007.

Activation of the cAMP/PKA/DARPP-32 Signaling Pathway is Required for Morphine Psychomotor Stimulation but not for Morphine Reward

In this study, using a mouse model the researchers show that, in key brain regions, acute administration of morphine resulted in an increase in the state of phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) at one site of phosphorylation, Thr34, without affecting phosphorylation at Thr75. In the striatum, activation of dopamine D1 receptors leading to phosphorylation of Thr34 leads to a cascade of molecular events ultimately potentiating responses produced by activation of the cAMP cascade. These researchers examined the possible involvement of DARPP-32 in the acute motor stimulant effect of morphine, and in morphine behavioral sensitization. The study shows that morphine's psychomotor effects are mediated through DARPP-32 and specifically through the phosphorylation of Thr34, but not Thr75, which are known to have opposing effects on DARPP-32 activity. To address the question of the possible role of DARPP-32 in the rewarding properties of morphine, they used the conditioned place preference (CPP) paradigm. The data clearly show that morphine-induced place preference is present in mice lacking the DARPP-32 gene, providing strong evidence that DARPP-32 is not involved in morphine reward. This is in contrast to similar studies conducted with cocaine, suggesting a different mechanism for morphine reward. These results demonstrate that dopamine D1 receptor-mediated activation of the cAMP/DARPP-32 cascade in striatal neurons is involved in the psychomotor action, but not in the rewarding properties, of morphine. Borgkvist, A., Usiello, A., Greengard, P., and Fisone, G. Activation of the cAMP/PKA/DARPP-32 Signaling Pathway is Required for Morphine Psychomotor Stimulation but not for Morphine Reward. Neuropsychopharmacology, pp. 1-9, 2007.

Association of the Neuronal Nicotinic Receptor B2 Subunit Gene (CHRNB2) with Subjective Responses to Alcohol and Nicotine

Nicotine and alcohol dependence are highly co-morbid disorders and may share overlapping genetic components. Dr. Ehringer and her colleagues examined two neuronal nicotinic receptor subunit genes (CHRNA4 and CHRNB2) that code for the alpha4 and beta2 subunits. The alpha4 beta2 subunits are the most frequently encountered nicotinic receptor subtype in the brain, and are promising candidates for genetic studies with nicotine and alcohol dependence. So far, strong evidence supports a role for alpha4 in dependence, but there is no evidence for involvement of beta2. In this report, six single nucleotide polymorphisms (SNPs) in the alpha4 gene and two SNPs in the beta 2 gene were examined individually and by haplotype analysis for association with nicotine and alcohol phenotypes (use), including subjective measures of response to each drug in the period shortly after initiation to capture critical steps in early development of addictive behaviors. The results provided stronger support for the beta 2 subunit in early subjective (negative physical responses to tobacco) responses to alcohol and nicotine with the SNP rs2072658. This SNP is located 42 bp upstream of the transcription initiation site, and may be important for regulating expression of the gene. This study highlights the potential importance of careful definition of phenotypes when studying complex disorders such as addiction. This study also provides the first evidence for association between the CHRNB2 gene and nicotine- and alcohol-related phenotypes, and suggests that polymorphisms in the CHRNB2 gene may be important in mediating early responses to nicotine and alcohol. Ehringer, M.A., Clegg, H.V., Collins, A.C., Corley, R.P., Crowley, T., Hewitt, J.K., Hopfer, C.J., Krauter, K., Lessem, J., Rhee, S.H., Schlaepfer, I., Smolen, A., Stallings, M.C., Young, S.E., and Zeiger, J.S. Association of the Neuronal Nicotinic Receptor _2 Subunit Gene (CHRNB2) with Subjective Responses to Alcohol and Nicotine. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics, 2007. (epub ahead of print).

Multiple ADH Genes Modulate Risk for Drug Dependence in both African- and European-Americans

Cocaine dependence (CD) and opioid dependence (OD) are commonly co-morbid with alcohol dependence, and studies indicate that there are common genetic risk factors for these addictive disorders. Dr. Gelernter and his colleagues recently studied the relationship among seven alcohol dehydrogenase (ADH) genes in CD and OD. ADH genes have been shown to be important risk factors for alcohol dependence, and since CD and OD are commonly co-morbid with alcohol dependence, the hypothesis was that ADH genes might also contribute to CD and OD. Sixteen markers within the ADH gene cluster and 38 ancestry-informative markers were genotyped in a sample of 718 individuals. Analysis showed that the ADH5 and ADH6 genotypes, and diplotypes (pairs of haplotypes that comprise genetic data from an individual) from ADH1A, ADH1B, ADH1C and ADH7 were associated with CD and OD in both African- and European Americans. This is the first report to link alcohol risk loci with CD and OD, and provides new insights into explaining the high rate of co-morbidity between these addictive disorders. Luo, X., Kranzler, H.R., Zuo, L., Wang, S., Schork, N.J., and Gelernter, J. Multiple ADH Genes Modulate Risk for Drug Dependence in both African- and European-Americans. Human Molecular Genetics, 16, pp. 380-390, 2007.

DAT-Ubiquitin Conjugates are Required for Protein Kinase C-Induced Internalization of DAT

The amount of DAT at the cell surface is determined by its relative rates of internalization and recycling. Activation of protein kinase C (PKC) increases the rate of endocytosis of DAT. PKC activation also results in the ubiquitination of DAT. Dr. Sorkin's group investigated whether ubiquitination of DAT and DAT endocytosis were linked and by mutating the ubiquitination sites of the DAT, found that three sites in the amino terminus of the DAT were critical for internalization. They also conducted an RNA interference screen and discovered that ubiquitin ligase, Nedd4-2 (neural precursor cell expressed, developmentally downregulated 2-4) was essential for the internalization of DAT. These data proved the first evidence for a new model of DAT regulation, showing that DAT-ubiquitin conjugates may be the molecular interface of transporter interaction with endocytic machinery. Miranda, M., Dionne, K.R., Sorkina, T., and Sorkin, A. Three Ubiquitin Conjugation Sites in the Amino Terminus of the Dopamine Transporter Mediate Protein Kinase C-dependent Endocytosis of the Transporter. Molecular Biology of the Cell, 18, pp. 313-323, 2007. Sorkina, T. Miranda, M., Dionne, K.R., Hoover, B.R., Zahniser, N.R., and Sorkin, A. RNA Interference Screen Reveals an Essential Role of Nedd4-2 in Dopamine Transporter Ubiquitination and Endocytosis. The Journal of Neuroscience, 26, pp. 8195-8205, 2006.

Mechanisms Governing Secretory Granule Biogenesis and Exocytosis: Proteins of the Regulated Pathway Are Sequestered in Secretory Granules Differently from Proteins of the Constitutive Pathway

Secretory granules carrying fluorescent cargo proteins are widely used to study granule biogenesis, maturation, and regulated exocytosis. The investigators fused the soluble secretory protein peptidylglycine _-hydroxylating monooxygenase (PHM) to green fluorescent protein (GFP) to study granule formation. When expressed in AtT-20 or GH3 cells, the PHM-GFP fusion protein partitioned from endogenous hormone (adrenocorticotropic hormone, growth hormone) into separate secretory granule pools. Both exogenous and endogenous granule proteins were stored and released in response to secretagogue. Neither luminal acidification nor cholesterol-rich membrane microdomains play essential roles in soluble content protein segregation. Their data suggest that intrinsic biophysical properties of cargo proteins govern their differential sorting, with segregation occurring during the process of granule maturation. Proteins that can self-aggregate are likely to partition into separate granules, which can accommodate only a few thousand copies of any content protein; proteins that lack tertiary structure are more likely to distribute homogeneously into secretory granules. Therefore, a simple "self-aggregation default" theory may explain the little acknowledged, but commonly observed, tendency for both naturally occurring and exogenous content proteins to segregate from each other into distinct secretory granules. Sobota, J.A., Ferraro, F., Baeck, N., Eipper, B.A., and Mains, R.E. Not All Secretory Granules Are Created Equal: Partitioning of Soluble Content Proteins. Molecular Biology of the Cell, 17, pp. 5038-5052, 2006.

D1-D2 Dopamine Receptor Heterooligomers with Unique Pharmacology Are Coupled to Rapid Activation of Gq/11 in the Striatum

Dr. Susan George and her research team at the University of Toronto discovered a heteromeric D1-D2 dopamine receptor signaling complex in brain that is coupled to Gq/11 and requires agonist binding to both receptors for G protein activation and intracellular calcium release. The D1 agonist SKF83959 was identified as a specific agonist for the heteromer that activated Gq/11 by functioning as a full agonist for the D1 receptor and a high-affinity partial agonist for a pertussis toxin-resistant D2 receptor within the complex. They provide evidence that the D1-D2 signaling complex can be more readily detected in mice at 8 months of age compared to 3 months of age, suggesting that calcium signaling through the D1-D2 dopamine receptor complex is relevant for function in the post-adolescent brain. Activation of Gq/11 through the heteromer increases levels of calcium/calmodulin-dependent protein kinase II_ (CAMKII_) in the nucleus accumbens, indicating a mechanism by which D1-D2 dopamine receptor complexes may contribute to synaptic plasticity. The formation of a distinct dopaminergic signaling unit by two receptors that signal through separate pathways is highly significant in that it provides a greater repertoire of signaling pathways by which dopamine can modulate neuronal function than would be possible by each of the five different dopamine receptor subtypes acting solely as separate units. Characterization of changes in this signaling unit with age and the functional consequences of signaling through the complex will increase our understanding of how D1-D2 heteromers contribute to neuronal function as well as the role this pathway may play in the etiology or pathophysiology of disorders in which altered dopamine signaling is implicated, such as schizophrenia and drug addiction. Rashid, A.J., So, C.H, Kong. M.M.C., Furtak, T., El-Ghundi, M., Cheng, R., O'Dowd, B.F., and George, S.R. D1-D2 Dopamine Receptor Heterooligomers with Unique Pharmacology Are Coupled to Rapid Activation of Gq/11 in the Striatum. Proceedings of the National Academy of Sciences, 104, pp. 654-659, 2007.

Neuronal Oxidative Stress Induced By HIV-1 and Alcohol Blocked By Cholesterol-Depleting Statin Drugs

In the brain, both HIV-1 and alcohol induce oxidative stress, which is considered a precursor for cytotoxic responses. Several reports have suggested that statins exert antioxidant as well as anti-inflammatory pleiotropic effects, besides their inherent cholesterol-depleting potentials. In this study, post-mitotically differentiated neurons were co-cultured with uninfected or HIV-1-infected monocytes, T cells, or their cellular supernatants in the presence or absence of physiological concentrations of alcohol. In neurons cultured with HIV-infected monocytes or T cells, or with supernatants from those cells, neuronal oxidative stress responses (8-isoprostane-F2-alpha, NOS activity, and Hsp70) were significantly increased compared to co-cultures using uninfected cells. Exposure to ethanol further elevated Hsp70 in both infected and uninfected cultures. The amount of total nitrates was significantly elevated in the co-culture system when both infected cells and alcohol were present. Surprisingly, pretreatment of postmitotic neurons with clinically available inhibitors of HMG-coenzyme A reductase (statins) inhibited HIV-1-induced release of stress/toxicity-associated parameters (i.e., Hsp70, isoprostanes, and total nitrates) from HIV-1-infected cells. The statins also blocked the enhanced oxidative stress due to HIV-1-infected cells and ethanol. The results of this study provide new insights into HIV-1 neuropathogenesis aimed at the development of future HIV-1 therapeutics to eradicate viral reservoirs from the brain. Acheampong, E., Parveen, Z., Mengistu, A., Ngoubilly, N., Wigdahl, B., Lossinsky, A.S., Pomerantz, R.J., and Mukhtar, M. Cholesterol-Depleting Statin Drugs Protect Postmitotically Differentiated Human Neurons against Ethanol- and Human Immunodeficiency Virus Type 1-Induced Oxidative Stress In Vitro. Journal of Virology, 81, pp. 1492-1501, 2007.