Research Findings - Basic Neurosciences Research
Human White Blood Cells Synthesize Morphine
It has been reported that human plasma actually contains low concentrations of morphine. To date, however, the origin and biological substrates and pathways involved in the endogenous synthesis of morphine have been less clear. Now, researchers from the Neuroscience Research Institute at the State University of New York-College at Old Westbury have uncovered one such a pathway. This group, led by Dr. George Stefano, has shown that one likely source for plasma morphine is in the immune system's white blood cells (WBC). Specifically, Dr. Stefano and his colleagues recently reported that WBC express CYP2D6, an enzyme capable of synthesizing morphine from several substances including codeine. They further demonstrated that morphine can be synthesized from L-DOPA, the precursor for the neurotransmitter dopamine-- an unexpected finding. The research team went on to show that WBC, in fact, can release morphine. These results demonstrate that the biosynthetic pathways for the neurotransmitter dopamine and morphine are linked. Furthermore, these results raise additional questions about the role of these two chemical messengers in modulating the immune system. Zhu, W., Cadet, P., Baggerman, G., Mantione, K.J., and Stefano,G. Human White Blood Cells Synthesize Morphine: CYP2D6 Modulation. Journal of Immunology, 175, pp. 7357-7362, 2005.
Endocannabinoids Act on Inhibitory Interneurons and Modulate an Intrinsic Population Rhythm of Hippocampal Neurons
Theta rhythms are behaviorally relevant electrical oscillations, intrinsic to neuronal populations, particularly the hippocampus. These hippocampal theta waves are controlled by endocannabinoids (eCB) that act on inhibitory neurons. When a so-called principal neuron discharges an action potential, its release of eCB sends a feedback signal to the inhibitory interneurons, which in turn affects the discharge pattern and intensity of the principal neurons that they innervate. This phenomenon is seen wherever endocannabinoids affect theta rhythm. Often, glutamatergic and/or cholinergic inputs, shaped by these inhibitory postsynaptic potentials (IPSPs) drive the intra-hippocampal theta oscillations. Dr. Alger's recent work shows that the IPSPs controlling theta rhythm appear to be controlled by eCBs. Specifically, Dr. Alger has shown that if all the glutamate receptors are blocked, the cholinergically-induced theta rhythm IPSPs in the hippocampus can be transiently interrupted by action potential-induced, eCB release. Because these actions are rapid, the intrinsic neuronal network may constitute the fundamental mechanism for temporal coding and decoding in the hippocampus. Disruption of theta rhythms might be one mechanism by which cannabinoid drugs may cause cognitive dysfunction. Reich, C.G., Karson, M.A., Karnup, S.V., Jones, L.M. and Alger, B.E. Regulation of IPSP Theta Rhythm by Muscarinic Receptors and Endocannabinoids in Hippocampus. Journal of Neurophysiology, 94(6), pp. 4290-4299, 2005.
Pentapeptides and the Kappa Receptor
The rationale for the use of conformationally-restricted peptides has been that they reduce the number of multiple conformations available to flexible endogenous opioid peptides, such as dynorphin A, and allow a better understanding of those conformation(s) that may be contributing to the binding of a peptide to a receptor, and that may be involved in signal transduction or interruption of signal. Short cyclized peptides of four or five amino acids have been used with considerable success to better define the mu and delta receptor binding requirements; examples of these are the peptides prepared by Dr. Henry Mosberg and his associates: JOM-13 (delta receptor-specific), and JOM-6 (mu receptor-specific). Results previously obtained by Dr. Mosberg have suggested that pentapeptides cyclized by a 2,5 bridge produce enhanced kappa opioid receptor (KOR) affinity, and that a D-cysteine in the fifth position (rather than D-penicillamine), and a phenylalanine (Phe) in the third position was a requirement for kappa activity. These researchers have now prepared a series of pentapeptides with enhanced kappa receptor binding (Ki less than 5 nM), having a C-terminal cysteine, with a 2,5 cyclization consisting of disulfide, methylene, or ethylene dithioether linkage, and with a C-terminal amide structure. Modeling of the compound (Tyr-c[D-Cys-Phe-Phe-D-Cys]NH2) with the highest kappa affinity (Ki of 1.6 nM) bound to a model of the KOR, based on the rhodopsin crystal structure and previous modeling of the mu receptor, has indicated several important structural features, including hydrogen bonding between KOR anionic residues and the tyrosine nitrogen and phenolic oxygen in position one, interaction of Phe 3 with aromatic receptor residues in helix seven, accommodation of the disulfide bridge in a binding pocket between extracellular loop 2 and the extracellular ends of helices five and six. Phe 4 interacted with the side chains of residues in extracellular loop 2. Of considerable significance is the finding that this compound binds effectively at all three opioid receptor types, mu, delta, and kappa, with picomolar affinity at the mu receptor. The next step in this process will be to modify the Phe 3 side chain, such as introducing a basic substituent to interact with the neighboring negative charges of aspartate 204 and 206 in extracellular loop 2, in an effort to maximize or improve the kappa selectivity. The point of these findings is that cyclic pentapeptides can be designed, by careful amino acid selection and use of modeling, which can be accommodated by all three opioid receptor binding sites, and that they need not necessarily be longer peptides such as dynorphin A, in order to achieve reasonable kappa receptor binding. Further specific modifications of these pentapeptides may result in enhanced kappa receptor specificity.
Przydzial, M.J., Pogozheva, I.D., Ho, J.C., Bosse, K.E., Sawyer, E., Traynor, J.R., and Mosberg, H.I. Design of High Affinity Cyclic Pentapeptide Ligands for _-opioid Receptors.
The Journal of Peptide Research, 66(5), pp. 255- 262, 2005.
Parallel Synthesis and Biological Evaluation of Different Sizes of Bicyclo[2,3]-Leu-Enkephalin Analogues
Parallel synthesis of peptides and peptidomimetics has been an important approach to search for biologically active ligands. A novel systematic synthesis of different size bicyclic dipeptide mimetics was developed on solid-phase supports. By taking advantage of the enantioselective synthesis of unsaturated amino acids and their N-methylated derivatives, the hemiaminal problem was prevented in the pathway to thiazolidine formation. The bicyclic dipeptide was generated on the solid-phase support in three steps by an unconventional method. By inserting this bicyclic scaffold into the synthesis of a larger bioactive peptide, 11 different sizes of bicyclo[2,3]-Leu-enkephalin analogues were synthesized in a fast and efficient way. Modeling studies show that a reversed turn structure at positions 2-3 was favored when an L- and L-bicyclic scaffold was used, and that an extended conformation at the N-terminal was favored when a D- and L-bicyclic scaffold was inserted. Binding affinities and bioassay studies show ligands with micromolar binding affinities and antagonist bioactivities for the [6,5]- and [7,5]-bicyclo-Leu-enkephalin analogues. Dr. Hruby and colleagues demonstrated that [6,5]-and [7,5]-bicyclic scaffold inserted peptide analogues can be generated in parallel synthesis on solid-phase support in 12 steps based on N-Fmoc chemistry using Wang resin. The amino acid precursors were synthesized from the previously developed methods followed by modification of their functional groups. It appears this is the first example of a parallel synthesis of [6,5]- and [7,5]-bicyclic dipeptide mimetics inserted into peptide analogues directly using -aldehyde amino acid derivatives and cysteine derivatives. Eleven analogues were synthesized, and their structure-biological activity relationship was examined. Some of the [6,5]- and [7,5]-bicyclo[2,3]-Leu-enkephalin analogues showed micromolar binding affinities and potent antagonist activities. The receptor selectivity decreased as the ring flexibility increased. Modeling studies indicated that the D- and L-bicyclic scaffold provided an extended backbone conformation similar to the DPDPE X-ray crystal structure at the corresponding positions. Interestingly, however, these analogues are antagonists whereas DPDPE is an agonist. Apparently the highly restricted bicyclic scaffold does not allow the ligand--receptor complex to form a bioactive agonist conformation. Gu, X., Ying , J., Min, B. , Cain, J.P., Davis, P., Willey, P., Navratilova , E., Yamamura , H.I., Porreca , F. and Hruby, V.J. Parallel Synthesis and Biological Evaluation of Different Sizes of bicyclo[2,3]-Leu-enkephalin Analogues. Peptide Science, 80(2-3), pp. 151-163, 2005.
Prenatal Cocaine Exposure and Brain Development
It has been demonstrated that exposure to cocaine increases cell death (i.e., apoptosis) in the fetal brain. To examine the molecular mechanisms of this effect, Dr. Michael Lidow and his group conducted studies in a mouse model of prenatal cocaine exposure. These studies demonstrate that maternal cocaine use is capable of interfering with a range of apoptosis-related genes in the cells of the fetal cerebral wall making these brain cells more sensitive to death-inducing signals. However, this increase in potential for apoptosis is likely to result in actual cell death only when and where the affected cells are subjected to a death-promoting local environment, with the distribution and harshness of such tissue environment depending not only on the cocaine exposure itself but also on fetal developmental stage, maternal health, nutritional status, etc. Consequently, the severity and scope of structural and functional impacts of the pro-apoptotic gene alterations in the offspring of cocaine-abusing mothers should be prone to significant variability due to idiosyncratic use of cocaine and a great diversity in the quality of prenatal care. Furthermore, apoptosis represents just one of several potentially negative influences of cocaine exposure on fetal corticogenesis, with proliferation, migration, and differentiation of cortical cells also being affected by this drug. Novikova, S.I., He, F., Bai, J., Badan, I., Lidow, I.A. and Lidow, M.S. Cocaine-induced Changes in the Expression of Apoptosis-related Genes in the Fetal Mouse Cerebral Wall. Neurotoxicology and Teratology, 27, pp. 3-14, 2005.
Potent Cannabinergic Indole Analogues as Brain Imaging Agents for the CB1 Cannabinoid Receptor and its X-Ray Crytallographic Structure
A series of novel aminoalkylindoles was synthesized in an effort to develop compounds that are potent agonists at the CB1 cannabinoid receptor and that are easily labeled with radioisotopes of iodine for biochemical and imaging studies. 2-Iodophenyl-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl]methanone (AM2233, 8) had a very high affinity for the rat CB1 receptor, with most of the affinity residing with the (R)-enantiomer. Radioiodinated 8, (R)-8, and (S)-8 were prepared by radioiododestannylation of the tributyltin analogues in high yields, radiochemical purities, and specific radioactivities. In a mouse hippocampal membrane preparation with [131I](R)-8 as radioligand, racemic 8 exhibited a Ki value of 0.2 nM compared with 1.6 nM for WIN55212-2. In autoradiographic experiments with mouse brain sections, the distribution of radioiodinated 8 was consistent with that of brain CB1 receptors. Again, very little specific binding was seen with the (S)-enantiomer [131I](S)-8 and none occurred with the (R)-enantiomer [131I](R)-8 in sections from CB1 receptor knockout mice. Radioiodinated 8 thus appears to be a suitable radioligand for studies of CB1 cannabinoid receptors. In addtion, for further gaining insights into structure activity relationship, X-ray crystallographic structure was determined. At present, there are only limited number of such successful studies on cannabinoid ligands and this is one of the successful elucidation of the three dimensional structure. Results of the single-crystal X-ray study of 8 were also reported in this publication. The solid-state structure outlines some important conformational features of the molecule. First, the N-methylpiperidinyl group exists in a chair conformation, with the N-methyl group being equatorial and in close proximity to the iodo substituent of the benzoyl ring. The carbonyl group lies in the plane with indole in an anti conformation with respect to the indole C2-C3 bond. Conversely, the 2-iodophenyl ring is gauche with its plane forming a 73 angle with the plane of the indole. Deng, H., Gifford, A.N., Zvonok, A.M., Cui, G., Li, X., Fan, P., Deschamps, J.R., Flippen-Anderson, J.L., Gatley S.J. and Makriyannis, A. Potent Cannabinergic Indole Analogues as Radioiodinatable Brain Imaging Agents for the CB1 Cannabinoid Receptor. Journal of Medicinal Chemistry, 48 (20), pp. 6386-6392, 2005.
Adamantyl Cannabinoids: A Novel Class of Cannabinergic Ligands
Structure activity relationship studies have established that the aliphatic side chain plays a pivotal role in determining the cannabinergic potency of tricyclic classical cannabinoids. Dr. Makriyannis and his coworkers have now synthesized a series of analogs in which a variety of adamantyl substituents were introduced at the C3 postion of delta-8-THC. A lead compound from PI's laboratory, (-)-3-(1-adamantyl)-delta-8-tetrahydrocannabinol (AM411), was found to have robust affinity and selectivity for the CB1 receptor as well as high in vivo potency. The X-ray crystal structure of AM411 was determined. Exploration of the side chain conformational space using molecular modeling approaches has allowed the investigators to develop cannabinoid side pharmacophore models for the CB1 and CB2 receptors. The results suggest that although a bulky group at the C3 position of classical cannabinoids could be tolerated by both CB1 and CB2 binding sites, the relative orientation of that group with respect to the tricyclic component can lead to receptor subtype selectivity. Lu, D., Meng, Z., Thakur,G.A., Fan, P., Steed, J., Tartal, C.L., Hurst, S.P., Reggio, P.H., Deschamps, J.R., Parrish, D.A., George, C., Jarbe, T.U.C., Lamb, R.J. and Makriyannis, A. Adamantyl Cannabinoids: A Novel Class of Cannabinergic Ligands. Journal of Medicinal Chemistry, 48, pp. 4576-4585, 2005.
Cannabinoids Reverse Capsaicin-Evoked Sensitization of Spinal Pain Fibers
Dr. Donald Simone and colleagues induced excitation and sensitization of wide-dynamic-range (WDR) and high-threshold (HT) spinal nociceptive neurons by intraplantar injection of 0.1% capsaicin (10 Ál) in rats. Low doses of the cannabinoid agonist, CP 55,940, applied intrathecally attenuated this capsaicin-evoked sensitization of WDR and HT spinal pain fibers. These same doses of capsaicin did not alter the responses of WDR and HT neurons in naïve rats. The effects of CP 55,940 on pain sensitization were blocked by pretreatment of the spinal cord with the CB1 receptor antagonist, SR141716A, indicating that the effect was CB1 receptor mediated. These studies demonstrate that cannabinoid application to the spinal cord prevents capsaicin-evoked central sensitization. Johanek, L.J. and Simone, D.A. Cannabinoid Agonist, CP55, 940, Prevents Capsaicin-induced Sensitization of Spinal Cord Dorsal Horn. Journal of Neurophysiology, 93(2), pp. 989-997, 2005.
Ketamine Produces a Lasting Disruption in Mouse Auditory Evoked Potentials
In humans, ketamine, an NMDA glutamate receptor antagonist, appears to produce persisting cognitive disruptions. In the present studies, auditory evoked potentials (AEP) were measured in four mouse strains administered ketamine. The acute effects of ketamine on AEP varied among the strains. The C3H and FVB strains were selected for a chronic ketamine study, based on the difference in effects of acute ketamine on the amplitude of the N1 evoked potential component. Acute ketamine dose-dependently decreased N1 amplitude in the C3H mice and did not affect any AEP component of FVB mice. The N1 is a cortically-generated evoked potential component. Both strains exhibited similar acute ketamine metabolism. In the chronic study, immediately after the final daily injection of ketamine, neither strain displayed alterations in AEP measures, suggesting adaptation of the previously vulnerable C3H mice. A week after the last exposure to ketamine, there was a decrement of the N1 amplitude in both the C3H (acutely sensitive) and FVB (acutely insensitive) strains, indicating a lasting deficit in information processing, even, in the FVB mice, in the absence of acute changes. Since decreased N1 amplitude is a reliable characteristic of patients with schizophrenia, the present observations in mice are consistent with the idea that NMDA hypo-function may be involved in the cognitive deficits of schizophrenia, as well as those of ketamine abusers. Maxwell, C.R., Liang, Y., Ehrlichman, R.S., Trief, D.F., Majumdar, S., Kanes, S.J., Karp, J. and Siegel, S.J. Ketamine Produces Lasting Disruptions in Encoding of Sensory Stimuli. Journal of Pharmacology and Experimental Therapeutics (epub), 2005.
Methamphetamine Damage to Dopamine Nerve Endings May Be Due to Activation of Microglia
In the September 2005 NIDA Director's Report, several lines of evidence were reported from Donald M. Kuhn's lab indicating that microglia, activated by methamphetamine (METH), may contribute to METH's characteristic neurotoxicity to dopamine-containing neuron terminals. While microglia are the primary immune defense cells in the brain, usually safeguarding and supporting neuronal functions, excessive microglial activation can cause harm to neurons. Now, this group has supplied additional evidence in support of this hypothesis. Dizocilpine (MK-801) and dextromethorphan (DXM) were tested for the ability to block microglial activation in an in vivo (mouse) METH model of neurotoxicity. The METH-induced activation of striatal microglia was reduced significantly by doses of MK-801 and DXM that also protected against DA nerve terminal damage. The researchers also determined that MK-801 and DXM can directly block activation of mouse microglial cells in culture. Since the specific action or property of METH that leads to microglial activation in vivo is not yet known (they hypothesize that it may be mediated by dopamine quinone formation), lipopolysaccharide (LPS) and the neurotoxic HIV protein Tat72, which are prototypical microglial activators, and which cause dopamine neuronal toxicity after direct infusion into brain, were used as model compounds. Two independent measures of microglial activation were assessed - enhanced expression of cellular cyclooxygenase-2 and increased secretion of tumor necrosis factor-_. It was observed that MK-801 and DXM prevented both LPS- and Tat72-induced activation of microglia in a concentration-dependent manner. The present results indicate that the ability of MK-801 and DXM to protect against METH neurotoxicity is related to their common property as blockers of microglial activation. Several associated lines of investigation indicate that microglia are active participants in METH neurotoxicity and are not solely responding to neuronal damage. Thomas, D.M. and Kuhn, D.M. MK-801 and Dextromethorphan Block Microglial Activation and Protect Against Methamphetamine-induced Neurotoxicity. Brain Research, 1050, pp. 190-198, 2005.
Inhibitors of MEK Block Reconsolidation of Cocaine-Associated Memory
Memory for drug-paired cues resists extinction and contributes to relapse; however, the molecular mechanisms underlying these associations are not understood. Now researchers have shown that cocaine-conditioned place preference (CPP) in rats activates extracellular signal-regulated kinase 1/2 (ERK), two downstream transcription factors, ets-like gene-1 (Elk-1) and cAMP response element binding protein (CREB), and Fos, in the nucleus accumbens core (AcbC) but not shell. Intra-AcbC infusions of U0126, an inhibitor of the ERK kinase mitogen-activated protein kinase (MEK), immediately after CPP retrieval, prevent both the activation of ERK, Elk-1, CREB, and Fos and preference for the cocaine-associated chamber. When tested again 24 hr or 14 days after intra-AcbC infusions of U0126 or another MEK inhibitor, PD98059, CPP retrieval and concomitant protein activation were significantly attenuated. Thus, inhibition of the ERK signaling pathway in the AcbC not only blocked rats' preference for a drug-paired environment (CPP retrieval), but when the inhibitor was infused just after the recall had occurred, the rats had impairments in memory for the drug-paired cues that lasted at least two weeks afterward (reconsolidation paradigm). Together, these findings indicate the necessity of the AcbC ERK signaling pathway for drug-paired contextual cue memories and suggest that these strong memories can become susceptible to disruption by therapeutic agents. This experiment has translational relevance because the MEK inhibitors were given after the rats had already learned the cocaine-place association; this is analogous to treatments in people given after, not before, they are abusing drugs. Miller, C.A. and Marshall, J.F. Molecular Substrates for Retrieval and Reconsolidation of Cocaine-associated Contextual Memory. Neuron, 47(6), pp. 873-884, 2005.
A Role for the Distal Carboxyl Tails in Generating the Novel Pharmacology and G Protein Activation Profile of mu and delta Opioid Receptor Hetero-oligomers
Opioid receptor pharmacology in vivo has predicted a greater number of receptor subtypes than is explained by the profiles of the three cloned opioid receptors, and the functional dependence of the receptors on each other shown in gene-deleted animal models remains unexplained. Dr. Susan George has shown that one potential mechanism for such findings is the generation of novel signaling complexes by receptor hetero-oligomerization. In the present study, they show that deltorphin-II is a fully functional agonist of the mu-delta heteromer, which induced desensitization and inhibited adenylyl cyclase through a pertussis toxin-insensitive G protein. Activation of the mu-delta receptor heteromer resulted in preferential activation of Galpha(z), illustrated by incorporation of GTPgamma(35)S, whereas activation of the individually expressed mu and delta receptors preferentially activated Galpha(i). The unique pharmacology of the mu-delta heteromer was dependent on the reciprocal involvement of the distal carboxyl tails of both receptors, so that truncation of the distal mu receptor carboxyl tail modified the delta-selective ligand-binding pocket, and truncation of the delta receptor distal carboxyl tail modified the mu-selective binding pocket. The distal carboxyl tails of both receptors also had a significant role in receptor interaction, as evidenced by the reduced ability to co-immunoprecipitate when the carboxyl tails were truncated. The interaction between mu and delta receptors occurred constitutively when the receptors were co-expressed, but did not occur when receptor expression was temporally separated, indicating that the hetero-oligomers were generated by a co-translational mechanism.
Fan, T., Varghese, G., Nguyen, T., Tse, R., O'Dowd, B.F. and George, S.R.. A Role for the Distal Carboxyl Tails in Generating the Novel Pharmacology and G Protein Activation Profile of mu and delta Opioid Receptor hetero-oligomers. Journal of Biological Chemistry, 280(46), pp. 38478-38488, 2005. [Epub 2005 Sep 13]
Dopamine Receptor Oligomerization Visualized in Living Cells
G protein-coupled receptors occur as dimers within arrays of oligomers. Dr. George and her group visualized ensembles of dopamine receptor oligomers in living cells and evaluated the contributions of receptor conformation to the dynamics of oligomer association and dissociation, using a strategy of trafficking a receptor from one cellular compartment to another. They incorporated a nuclear localization sequence into the D1 dopamine receptor, which translocated from the cell surface to the nucleus. Receptor inverse agonists blocked this translocation, retaining the modified receptor, D1-nuclear localization signal (NLS), at the cell surface. D1 co-translocated with D1-NLS to the nucleus, indicating formation of homooligomers. Administration of a dopamine antagonist retained both receptors at the cell surface, and removal of the drug allowed translocation of both receptors to the nucleus. They found that differences in receptor conformation disrupted the oligomer that ligand-binding pocket occupancy by the inverse agonist induced a conformational change. They demonstrated robust heterooligomerization between the D2 dopamine receptor and the D1 receptor. The heterooligomers could not be disrupted by inverse agonists targeting either one of the receptor constituents. In sum, they describe a novel method showing that a homogeneous receptor conformation maintains the structural integrity of oligomers, whereas conformational heterogeneity disrupts it. O'Dowd, B.F., Ji, X., Alijaniaram, M., Rajaram, R.D., Kong, M.M., Rashid, A., Nguyen, T. and George, S.R. Dopamine Receptor Oligomerization Visualized in Living Cells. Journal of Biological Chemistry, 280(44), pp. 37225-37235, 2005. [Epub 2005 Aug 22]
Brain Dopamine Fluctuates After Cocaine Administration In Behaving Rats
Cocaine is known to alter the extracellular concentrations of dopamine in dopamine-rich areas of the brain. Both rapid (phasic) and slower (tonic) changes in its extracellular concentration contribute to its complex actions. However, most of the data we have on these fluctuations come from studies of microdialysates, which cannot provide temporal resolution on a real-time scale. Cyclic voltammetry overcomes this limitation, and provides continuous resolution at the millisecond scale. Dr. Wightman and his group showed dose-dependent changes in extracellular dopamine after repeated intravenous infusions of the drug into behaving rats. He found that cocaine modified dopamine release in two ways: dopamine concentration transients increase in frequency and in magnitude immediately upon infusion, and there was a gradual increase in the steady-state concentration of dopamine which occurred over 90 seconds, and likely reflected the ability of cocaine to inhibit dopamine uptake. Heien, M.L., Khan, A.S., Ariensen, J.L., Cheer, J.F., Phillips, P.E.M., Wassum, K.M. and Wightman, R.M., Real-time Measurement of Dopamine Fluctuations after Cocaine in the Brain of Behaving Rats. Proceedings of the National Academy of Sciences, 102, pp. 10023-10028, 2005.
Moody: a Novel Conserved Gene Required for Cocaine and Nicotine Sensitivity
Drosophila has been invaluable in illuminating many aspects of basic biology because of the power and rapidity of fruit fly genetics. Drosophila is particularly useful for identifying new and unexpected genes involved in a biological process, since no a priori assumptions are made about the identities of genes involved. This can be achieved for example when a chemical mutagen is used to disrupt the DNA of a large number of flies. The animals are then sorted or screened to identify the very rare animals that have a particularly interesting phenotype, in this case sensitivity to cocaine. The mutant gene is then mapped to smaller and smaller chromosomal regions until the single gene (in flies, 1 out of about 12,000) responsible for the mutant phenotype is identified. Alternatively, transposons, made of up of DNA can be made to hop into different genes, and in this manner disrupt the function of the normal genes. Each and every fly with a different gene disrupted by the transposon can be screened. The sequence of the transposon can be used to locate the disrupted gene that is associated with the altered phenotype. Many biological processes are evolutionarily conserved, so identification and characterization of genes involved in a phenomenon such as drug sensitivity in fruit flies may shed light on drug sensitivity in humans. Drs. Bainton, Heberlein, and co-workers used a genetic screen to identify Drosophila mutants with increased sensitivity to cocaine. Interestingly, the cocaine-sensitive "moody" mutant also had increased sensitivity to nicotine, but was resistant to the acute effects of alcohol. Molecular identification of "moody" revealed that it is an evolutionarily conserved G-protein coupled receptor (GPCR) of previously unknown function. Moody encodes two alternate splice forms: moody-alpha and moody-beta. Surprisingly, if either splice form is defective, the flies are more sensitive to cocaine. Antibody staining shows that moody protein is expressed in surface glial cells required for proper maintenance of the blood-brain barrier (BBB). Data are also presented that indicate that moody-alpha and moody-beta "are actively required to maintain the integrity of the blood-brain barrier in the adult fly." How does "moody" modulate sensitivity to cocaine? One possibility is that a minor BBB defect simply allows more drug to enter the CNS of the flies. Several lines of reasoning argue against this possibility, the most compelling of which is that "moody" mutants are sensitive to cocaine and nicotine, but resistant to alcohol which readily passes through the BBB. Another possibility is that minor defects in the BBB could increase permeability of the CNS to ions, hormones, or other small molecules and that these molecules in turn modify drug sensing CNS structures leading to increased sensitivity to particular drugs. The identification of a novel GPCR that mediates cocaine and nicotine sensitivity and also plays a role in blood-brain barrier maintenance suggests several new and previously unanticipated research avenues. Future research into the human version of "moody" and related genes may provide insight into why some people are more or less sensitive to particular drugs. Identification of pharmaceuticals that increase the levels of the human version of "moody" could potentially be of use in reducing the effects of cocaine and nicotine. Bainton, R.J., Tsai, L.T., Schwabe, T., DeSalvo, M., Gaul U. and Heberlein, U. Moody Encodes Two GPCRs that Regulate Cocaine Behaviors and Blood-brain Barrier Permeability in Drosophila. Cell. Vol. 123, pp. 145-156, 2005. Schwabe, T., Bainton, R.J., Fetter R.D., Heberlein, U. and Gaul, U.. GPCR Signaling is Required for Blood-brain Barrier Formation in Drosophila. Cell, 123, pp. 133-144, 2005.
Chromatin Remodeling Mediates Neuronal and Behavioral Changes Induced By Cocaine
Epigenetics is the study of heritable and long-term changes in gene function that occur without a change in the DNA sequence. Chromatin structure and function, including regulation of gene expression, is heavily regulated through a series of enzyme-mediated covalent modifications that target genomic DNA, histones, and other molecules. Histone deacetylases (HDACs) and histone acetylases remodel chromatin, a complex of histone proteins and DNA, in the vicinity of a particular gene by removing or adding acetyl groups to histones, respectively. Long term changes in gene expression are thought to underlie addiction. Researchers in the Nestler and Self laboratories investigated the role of histone modification near the promoters of genes that play a critical role in cocaine addiction. Acute, but not chronic cocaine exposure is known to induce expression of the cfos gene while chronic exposure leads to the induction of the delta Fos B gene. Using chromatin immunoprecipitation (ChIP) the Nestler and Self laboratories found that acute but not chronic cocaine exposure leads to increased acetylation of histone H4 at the cfos promoter, but had no significant effect on histone H3 acetylation. Conversely, this group found that chronic but not acute cocaine exposure leads to increased acetylation of histone H3 of the delta FosB promoter, but had no significant effect on histone H4 acetylation. In addition, chronic cocaine induces acetylation of histone H3 at the brain derived neurotrophic factor (BDNF) and cyclin-dependent kinase 5 (Cdk5) promoter. Using a ChIP assay, the Nestler and Self laboratories show directly that the deltaFosB protein directly regulates the expression of the Cdk5 gene and that BDNF is regulated by chronic cocaine through a different transcription factor. These data suggest that a histone code (acetylation of histones H4 or H3 near the promoters of particular genes) may in part specify which genes are modulated in response to acute or chronic cocaine administration. Taking these experiments one step further, the Nestler and Self laboratories showed that administration of the HDAC inhibitor trichostatin A, prior to cocaine administration enhances reward responses to cocaine by increasing H3 acetylation, while overexpression of the HDAC4 gene in the striatum decreases reward responses to cocaine by inhibiting H3 acetylation. Pharmaceuticals targeting histone modifying enzymes could therefore be potentially valuable therapeutic agents for treating addiction to cocaine or, possibly, other drugs of abuse. HDAC inhibitors are currently under investigation for the treatment of Huntington's Disease and other neurodegenerative diseases. Kumar, A., Choi, K.H., Renthal, W., Tsankova, N.M., Theobald, D.E., Truong, H.T., Russo, S.J., Laplant, Q., Sasaki, T.S., Whistler, K.N., Neve, R.L., Self, D.W. and Nestler, E.J. Chromatin Remodeling is a Key Mechanism Underlying Cocaine-induced Plasticity in Striatum. Neuron, 48, pp. 303-314, 2005.
Activity-Dependent Subcellular Localization of NAC1
NAC1 is a protein that is upregulated in the brain's nucleus accumbens following chronic self-administration of cocaine. Overexpressing NAC1 in the nucleus accumbens antagonizes the actions of cocaine suggesting that NAC1 acts as a homeostatic protein. This protein belongs to the BTB/POZ family of proteins. Because of its nuclear localization in the cell and its ability to suppress transcription in a yeast assay of transcription, Dr. Mackler and his colleagues have previously suggested that NAC1 acts as a transcription factor. In a report in the European Journal of Neuroscience, Dr. Mackler and his colleagues suggest that neuronal activity causes the translocation of NAC1 from the nucleus of a nerve cell into the cytoplasm. Blockade of electrical activity by tetrodotoxin in cortical nerve cells prevented translocation of NAC1 from the nucleus to the cytoplasm. Depolarization by high potassium of both undifferentiated PC12 cells, a pheochromocytoma cell line, and Neuro2A cells also caused translocation of NAC1 from the nucleus to the cytoplasm. Dr. Mackler and his colleagues subsequently show that the translocation of NAC1 from the nucleus to the cytoplasm is mediated by protein kinase C phosphorylation at serine residue 245 in NAC1. Dr. Mackler suggests that the exclusion of NAC1 from the nucleus by activity may function to remove repression of transcription of target genes. Because BTB/POZ proteins function to degrade proteins through ubiquitination in cullin-E3 ligase complexe in the proteosomes, and the preliminary observation that NAC1 binds cullin3, Dr. Mackler and his colleagues suggest that NAC1 may also function in the cytoplasm to degrade specific proteins. Korutla, L., Champtiaux, N., Shen, H.W., Klugmann, M., Kalivas, P.W. and Mackler, SA. Activity-dependent Subcellular Localization of NAC1. European Journal of Neuroscience, 22(2), pp. 397-403, 2005.
Extracellular Signal-Regulated Kinases (ERK) and DARPP-32 Modulation by Drugs of Abuse
Pharmacological blockade of ERK kinases have been shown to block cocaine reward while the knockout of ERK1 increases morphine reward. The differences may be attributed to upregulation of ERK2 activity in the ERK1 knockout. In a recent paper Valjent et al. show that ERK2 activation by amphetamine requires activation of D1 dopamine receptors and NMDA receptors. The activation of ERK by drugs of abuse such as amphetamine cocaine, nicotine, morphine, and 9-tetrahydrocannabinol is absent in mice lacking DARPP-32. A point mutation in DARPP-32 at Thr-34 residue specifically involved in protein phosphatase-1 inhibition prevented ERK activation by drugs of abuse in the striatum but not in the prefrontal cortex. Phosphorylation of DARPP-32 at Thr-34 inhibits the protein-phosphatase-1 leading to increased phosphorylation of striatal enriched tyrosine phosphatase (STEP). This in turns prevents dephosphorylation of ERK. Blocking either phosphorylation of DARPP-32 on Thr34 or reducing ERK phosphorylation by 80 percent, prevent cocaine sensitization. These results suggest that DARPP-32 acts as a coincidence detector of glutamatergic and dopaminergic signals. Thus converging inputs from the prefrontal cortex and striatum triggered by environmental stimuli and internal rewarding stimuli are integrated by DARPP-32 to activate ERK producing changes in a specific subset of striatal neurons. Valjent, E., Pascoli, V., Svenningsson, P., Paul, S., Enslen, H., Corvol, J.C., Stipanovich, A., Caboche, J., Lombroso, P.J., Nairn, A.C., Greengard, P., Herve, D. and Girault, J.A. Regulation of a Protein Phosphatase Cascade Allows Convergent Dopamine and Glutamate Signals to Activate ERK in the Striatum. Proceedings of the National Academy of Sciences, USA, 102(2), pp. 491-496, 2005.
Distinct Cell Process Regulation Observed in Neuronal Migration
During brain development, neuronal proliferation, neuronal migration and axonal pathfinding are three major events to lay down basic structures for neural circuit formation, followed by axonal termination and synapse formation to complete the initial stage of brain development. Alteration in the formation of neuronal circuits is hypothesized to contribute to addiction. Secreted guidance cues such as netrins, ephrins, semaphorins, Slits, and the chemokine SDF are thought to guide axons to their target neurons. A fundamental question is whether the same guidance cues are used by migrating neurons to locate their final position as those used by axons to find their target neurons. In axonal pathfinding, axons turn by retracting an area of growth cone in the direction being repelled and extend the growth cone towards the area that the axon is turning, resulting in a reorienting of the axonal shaft. To test whether migrating neurons use the same process to find their final location, Dr. Rao and his collaborators observed the migration of neurons from the subventricular zone to the olfactory bulb in post-natal rats. They found that the process of guiding migrating neurons is different from the one used by axons. Instead of a single growth cone bending toward or away from a guidance cue, migrating neurons extend processes and retract multiple processes until the appropriated direction is discovered. Dr. Rao and his colleagues show that neurons from the subventicular zone turn away from the chemorepellent, Slit protein, through repeated rounds of process extension and retraction, instead of choosing a direction and reorienting the movement of the cell body into a given area of the extended process. A close observation reveals that Slit causes the cell body to preferentially extend its processes on the side away from this chemorepellent; however, it takes rounds of repeated process extension and retraction before the cell body completes a turn. Dr. Rao and his colleagues suggest that the distinct cellular events in neuronal migration reflect different cell signal transduction in interpreting the cues and in sensing the gradient of diffusing environmental cues. Ward, M.E., Jiang, H. and Rao Y. Regulated Formation and Selection of Neuronal Processes Underlie Directional Guidance of Neuronal Migration. Molecular and Cellular Neuroscience, 30(3), pp. 378-387, 2005.
Developmental Hypothalamus Affected by NMDA Receptor Mediated Changes of Gap Junctions between Differentiating Neurons
During brain development, the initial communications between neurons relies heavily on electric transmission through gap junctions. During the maturation of the brain, with onset of chemical transmission via chemical synaptic junctions, gap junctions between neurons are uncoupled, down regulated or mostly eliminated, except in some subset of neurons in specific areas, including at least glutaminergic neurons in the prefrontal cortex and nucleus accumbens. Using gap junction permeable dye, contrasted with un-permeable dye, an initial wave of gap junction coupling was observed at neonatal age in rat hypothalamus, and was found to decrease dramatically two weeks after birth. This decrease and uncoupling of gap junction was associated with the activation of NMDA receptor, whose activity is essential for memory, reward and addiction. The NMDA receptor initiates a signaling cascade that increases the phosphorylation of calcium-cyclic AMP response element binding protein (CREB) that eventually down regulates the expression of connexin 36, the component protein for neuronal gap junction. Inhibition of NMDA receptors with antagonists or suppressing CREB phosphorylation abolishes uncoupling of gap junction and downregulation of connexin 36. The researchers suggest that NMDA receptor activity contributes to the developmental uncoupling of gap junctions via CREB-dependant down regulation of connexin 36. This work may provide new insight on how developing brain can be altered as the consequence of being exposed to drugs of abuse, since both cocaine and amphetamine have recently been found to suppress connexin 36 expression in prefrontal cortex and nuceus accumbens. Arumugam, H., Liu, X., Colombo, P.J., Corriveau, R.A. and Belousov, A.B. NMDA Receptors Regulate Developmental Gap Junction Uncoupling Via CREB Signaling. Nature Neuroscience, 8(12), pp. 1720-1726, 2005.
Melanocortin-1 Receptor Gene Variants Affect Pain and m-Opioid Analgesia In Mice And Humans
Melanocortin-1 receptor (MCR1) was recently found to mediate k-opioid receptor analgesia, especially in female mice and human volunteers. This was an unexpected finding because MCR1 is thought to be primarily involved in skin/hair pigmentation and immunomodulation. Mogil and colleagues tested MCR1 mutant mice and humans of various hair color for sensitivity to pain and m-opioid analgesia. Mice were tested for their basal sensitivity on six different assays of acute and tonic nociception. Human volunteers were also tested for tolerance to acute pain. For both mice and humans, morphine and its derivative, morphine-6-glucuronide (M6G), were assessed for their effectiveness in reducing pain. In mice, mutants for MCR1 displayed reduced nociceptive responses to morphine and M6G and this effect was significant for both sexes. Studies in human volunteers with two or more variant alleles of the MCR1 gene in amino acids known to abolish MCR1 functionality, compared to volunteers with no or one variant, showed that baseline tolerance differed significantly between genotypes, with greater tolerance for pain in volunteers with the MCR1 variant. In addition, analgesic responses after M6G were greater in MCR1 variant subjects compared to controls. In contrast to the k-opioid receptor analgesia, the effect on the m-opioid receptor is not sex-dependent. This study eloquently demonstrates the power of direct mouse to human translation in genetic studies. Mogil and colleagues show a significant link between MCR1 gene variants, pain tolerance, and potential efficacy of analgesics acting on the m-opioid receptor. Larger human studies are needed to validate these findings in this important area of pain research. Mogil, J.S., Ritchie, J., Smith S.B., Strasburg, K., Kaplan, L., Wallace, M.R., Romberg, R.R., Bijl, H., Sarton, E.Y., Fillingim, R.B. and Dahan, A. Melanocortin-1 Receptor Gene Variants Affect Pain and Mu-opioid Analgesia in Mice and Humans. Journal of Medical Genetics, 42, pp. 583-587, 2005.
Identification and Functional Characterization of Brainstem Cannabinoid CB2 Receptors
There are two known receptor targets for cannabis, cannabinoid receptors 1 and 2 (CB1 and CB2). It has long been believed that CB1 was present in the central nervous system, while CB2 was used primarily by cells of the immune system and not present in the nervous system. NIDA researchers have found that CB2 is present and functional in neurons of some select brain regions, particularly the brain stem. They found evidence of CB2 receptors in neurons of the dorsal motor nucleus of the vagus, the nucleus ambiguus and the spinal trigeminal nucleus. The blood vessels and glia in these regions did not appear to express CB2 receptors. The potential significance of finding CB2 receptors in the brainstem is that therapeutics could be developed to affect the CB2 receptor selectively without the psychoactive effects that act through the CB1 receptor. Such therapeutics, if localized to the brainstem might be used to alleviate emesis and nausea. Van Sickle, M.D., Duncan, M., Kingsley, P.J., Mouihate, A., Urbani, P., Mackie K., Stella, N., Makriyannis, A., Piomelli, D., Davison, J.S., Marnett, L.J., Di Marzo, V., Pittman, Q.J., Patel, K.D. and Sharkey, K.A. Identification and Functional Characterization of Brainstem Cannabinoid CB2 Receptors. Science, 310(5746), pp. 329-332, 2005.