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NIDA Home > Publications > Director's Reports > February, 2008 Index    

Director's Report to the National Advisory Council on Drug Abuse - February, 2008

Research Findings - Basic Neuroscience Research

Inhalant-Induced Epigenetic Changes Produce Drug Tolerance

Epigenetic changes are heritable or stable changes in gene expression potential that are not encoded by the DNA sequence itself. The role of epigenetic regulation in illicit drug responses is an emerging but still understudied area of scientific discovery. Dr. Nigel Atkinson and co-workers exploit the genetically powerful fruit fly model system to investigate the molecular basis of inhalant tolerance. Briefly, Dr. Atkinson showed that a single exposure to inhalant can lead to epigenetic changes in the chromatin (the DNA/protein complex in the nucleus of a cell) near the "slowpoke" potassium channel gene, leading to altered expression of the slowpoke gene and reduced sensitivity (tolerance) to additional inhalant exposures. These studies are based on the observation that animals become tolerant to sedation by organic solvents, which can be abused as inhalants, and this reduced sensitivity to inhalants requires increased expression of the slowpoke potassium channel which in turn alters neuronal function. What is the molecular mechanism behind this observation? Dr. Atkinson and co-workers found that a single exposure to an inhalant led to epigenetic changes in regulatory regions of the slowpoke gene. Specifically they observed that the pattern of acetylation of the DNA-binding protein histone H4 was altered across the slowpoke gene, which likely led to a more open localized chromatin structure and subsequent increased expression of the slowpoke gene. Exposure of the animals to a pharmacological inhibitor of histone deacetylases, the class of enzymes responsible for the H4 histone acetylation, also led to similar epigenetic and gene expression changes as well as tolerance of the animals to the inhalant. Interestingly, Dr. Atkinson and colleagues found DNA elements within the slowpoke promoter that could be bound by the CREB transcription factor. A number of labs have shown that the CREB transcription factor is important in the responses of organisms to illicit substances, as well as in other neuroplastic processes such as learning and memory. Using a genetic trick to "turn off" CREB, the researchers found that the epigenetic and expression changes to slowpoke, and the development of behavioral tolerance no longer occurred. This indicates that the CREB transcription factor is required for these processes. Overall this work clearly shows that exposure to a drug of abuse can alter future sensitivity to the drug via epigenetic regulatory mechanisms. It also provides insight into the precise mechanisms by which exposure to an inhalant can lead to epigenetic and gene expression changes of a single gene, resulting in altered neuronal function and altered behavioral responses of an animal to future inhalant exposure. Although this work was done using an inhalant, similar mechanisms may well be utilized for responses to other drugs of abuse. Small molecules that alter the activity of epigenetic regulatory proteins are already in clinical trials to treat some forms of cancer, so it is possible that a deeper understanding of the role of epigenetics in response to drug exposure could lead to the development of "epigenetic therapies" to treat addiction and other neuropsychiatric diseases. Wang, Y., Kishnan, H.R., Ghezzi, A., Yin, J.C.P., and Atkinson, N.S. Drug-Induced Epigenetic Changes Produce Drug Tolerance. PLoS Biology 5, pp. 2342-2353, 2007.

Molecular Adaptations Underlying Susceptibility and Resistance to Social Defeat in Brain Reward Regions

Why do some individuals manage to cope with stress while others develop psychopathologies such as post-traumatic stress disorder (PTSD) and depression in response to adverse circumstances? Both genetic and environmental factors contribute to resiliency of individuals to stress. By examining the response of genetically identical animals to stress, environmental and genetic factors can be separated. Yet, if genetically identical animals respond differently to the same environmental stimuli what molecular mechanism can account for the different response? In a recent paper in Cell, Dr. Eric Nestler and his group examined the different responses of genetically identical C57BL/6J mice to social defeat. In the social defeat paradigm a smaller mouse is forced to intrude on the territory of a larger mouse of a more aggressive strain. The larger mouse responds aggressively to the intrusion of the smaller mouse, resulting in subsequent subordination by the smaller intruder mouse. Repeated exposure of C57Bl/6J mice to social defeat produces long lasting reductions in social interactions. Dr. Nestler and his colleagues report that nearly 50% of the chronically defeated C57BL/6J showed similar amounts of social interaction to non-defeated C57BL/6J mice. Susceptible mice showed increased sensitivity to low doses of cocaine, weight loss, and abnormalities in circadian regulation of body temperature, increased anxiety and increased corticosterone reactivity in response to stress. Increased levels of brain derived neurotropic factor (BDNF) and enzymatic activity of BDNF signaling molecules [(phosphorylated akt thymoma viral oncogene (Akt), glycogen synthase kinase 3_ (Gsk-3_)], and extracellular signal regulated kinase (ERK1/2) in the Nucleus Accumbens (NAC) was observed in susceptible mice but not in resilient mice. The source of the increase in BDNF in the NAC comes from BDNF synthesized by ventral tegmental area (VTA) dopamine neurons that project and transport BDNF to the NAC. While upregulation of BDNF explains susceptibility to social defeat, the result does not explain the molecular mechanisms of resilience. To identify the molecular mechanism of resilence, Dr. Nestler and his colleagues conducted a gene expression profiling experiment of transcripts expressed in the VTA from susceptible and resilient mice. Gene expression profiling showed that increased expression histone deacetylase-2 (Hdac2) and adenylyl cyclase 7 (Adcy7), and galanin (Gal) in the VTA is associated with susceptibility while increase expression of three voltage-gated potassium (K+) channels (Kcnf1, Kcnh3, and Kcnq3) in the VTA was only observed in resilient animals. As expected decreased excitability was observed in VTA dopamine neurons in resilient mice and susceptibility in susceptible mice could be reversed by increasing the expression of a potassium channel with a viral vector. The experimentally increased potassium channel resulted in decreased expression of BDNF protein in the NAC. To further test the hypothesis that the release of BNDF from VTA neurons is responsible for susceptibility, Nestler and his colleagues used a "knockin" mouse that has either a val/val or met/met variant in the BDNF gene. Met/met mice, displaying impaired BDNF secretion without affecting neuronal firing, were resilient while val/val knockin mice were highly susceptible to social avoidance following social defeat. These finding are highly relevant to the human condition because levels of BDNF were increased by 40% in the post-mortem brains of depressed patients. These results suggest that individuals releasing BDNF from VTA dopamine neurons in response to adversity are susceptible to stress. These results provide a novel insight into the development of novel therapeutic agents to promote resilience. It is anticipated that further research will explain the mechanisms by which differential gene expression in the VTA is observed in susceptible and resilient animals. Krishnan, V., Han, M.H., Graham, D.L., Berton, O., Renthal, W., Russo, S.J., Laplant, Q., Graham, A., Lutter, M., Lagace, D.C., Ghose, S., Reister, R., Tannous, P., Green, T.A., Neve, R.L., Chakravarty, S., Kumar, A., Eisch, A.J., Self, D.W., Lee, F.S., Tamminga, C.A., Cooper, D.C., Gershenfeld, H.K., and Nestler, E.J. Cell 131(2), pp. 391-404, 2007.

Histone Deacetylase 5 Epigenetically Controls Behavioral Adaptations to Chronic Emotional Stimuli

Although genetic mechanisms play an important role in vulnerability to addiction and psychiatric disorder, recent evidence suggest that epigenetic mechanisms such as enzymatic modification of histones or DNA methylation also play an important role in addiction and psychiatric illness. Epigenetic mechanisms permit long term changes in gene expression in response to environmental conditions without mutation or a change in the sequence of a gene. A recent paper in Neuron by Dr. Nestler and his colleagues focuses on the role of histone deacetylases (HDAC) that represses transcription by removing acetyl groups from histones in mediating responses to stress or cocaine. Previous work showed that systemic injections of HDAC inhibitors enhanced the rewarding effects of cocaine. Dr. Nestler and his colleagues show that the site of action of the systemic injection of the HDAC inhibitors on increased reward is the Nucleus Accumbens (NAC). HDAC5 and HDAC3 were found to have the highest level of expression in the NAC but their expression was not altered by acute or chronic cocaine treatment. Repeated injection of cocaine, however, was associated with increased phosphorylation of HDAC5 and transport of HDAC5 into the cytosol. The effect was specific to HDAC5. Nuclear export of HDAC5 in neurons of the NAC provides a mechanism by which genes induced by chronic cocaine can be regulated epigenetically. Over expression of HDAC5 in the NAC using viral vectors attenuated the response to cocaine and required the presence of the histone deacetylase domain in HDAC5. Knockout of the HDAC5 increased the rewarding properties of cocaine in mice following chronic but not acute cocaine treatment. Expressing HDAC5 in the nucleus accumbens restores normal sensitivity to the rewarding properties of cocaine. Gene expression profiling of HDAC5 knockout mice chronically treated with cocaine suggests that the enhanced sensititivity to the rewarding properties of cocaine is associated with increased expression and acetylation of the promoters of RapGEF6, Gnb4, Suv39H1, wnt5a and the NK1 receptor (NK1R). Chronic stress also resulted in decreasing HDAC5 activity by decreasing HDAC5 transcription, a mechanism distinct from the one through which cocaine decreases activity. Dr. Nestler and his colleagues suggest that chronic cocaine causes the phosphorylation of HDAC5 leading to the nuclear export of HDAC5 from the nucleus to the cytoplasm. This blocks the action of HDAC5 in the nucleus and increases histone acetylation and transcription of HDAC5 target genes. These results suggest that HDAC5 contributes to the behavioral transition between short-term physiological and long-term pathological responses to emotional stimuli and drug exposure. Renthal, W., Maze, I., Krishnan, V., Covington, H.E. 3rd, Xiao, G., Kumar, A., Russo, S.J., Graham, A., Tsankova, N., Kippin, T.E., Kerstetter, K.A., Neve, R.L., Haggarty, S.J,. McKinsey, T.A., Bassel-Duby, R., Olson, E.N., and Nestler, E.J. Neuron 56(3), pp. 517-529, 2007.

Hemopressin is an Inverse Agonist of CB1 Cannabinoid Receptors

Hemopressin, a nine amino acid peptide derived from the _-chain of hemoglobin, was originally isolated from rat brain. It had been previously shown to inhibit hyperalgesia by an opioid-independent mechanism. In this study, the researchers report that hemopressin exhibits antinociceptive properties through interaction with the cannabinoid receptor, CB1. The antinociceptive properties were observed using a variety of modes of administering the peptide including oral administration. When the researchers looked at the effect of hemopressin on CB1 agonist activity, they found evidence that it blocks downstream signaling events suggesting that hemopressin is acting as an inverse agonist. Furthermore, hemopressin was selective for CB1 over the related receptor CB2 and was able to displace a known chemical antagonist for CB1 with sub-nanomolar affinity. Taken together, these properties make hemopressin a strong candidate for development of a new pain therapeutic. Heimann, A.S., Gomes, I., Dale, C.S., Pagano, R.L., Gupta, A., de Souza, L.L., Luchessi, A.D., Castro, L.M., Giorgi, R., Rioli, Ferro, E.S. and Devi, L.A. PNAS 104, pp. 20588-20593, December 18, 2007.

Tris-Azaaromatic Quaternary Ammonium Salts: Novel Templates as Antagonists at Nicotinic Receptors Mediating Nicotine-Evoked Dopamine Release

A series of tris-azaaromatic quaternary ammonium salts has been synthesized and evaluated for their ability to inhibit neuronal nicotinic acetylcholine receptors (nAChRs), which mediate nicotine-evoked [3H]dopamine release from superfused rat striatal slices, and for their ability to inhibit [3H]nicotine and [3H]methyl-lycaconitine binding to whole rat brain membranes. The 3-picolinium compound 1,3,5-tri-{5-[1-(3-picolinium)]-pent-1-ynyl}benzene tribromide (tPy3PiB), 3b, exhibited high potency and selectivity for nAChR subtypes mediating nicotine-evoked [3H]dopamine release with an IC50 of 0.2 nM and Imax of 67%. The IC50 values obtained for the mono-, bis-, and trispicolinium analogues, NDDPiI, bPiDDB, and tPy3PiB were 30 nM, 2.0 nM, and 0.2 nM, respectively, with Imax values of 62%, 68%, and 67%, respectively. These results clearly indicate that introduction of additional 3-picolinium head groups into the NDDPiI structure greatly augments inhibitory potency and may be attributed to an increase in the number of ionic interactions with putative negatively charged binding sites on nAChR proteins mediating nicotine-evoked DA release. However, one must also consider the potential contributions of the central planar aromatic moiety and triple bond in the linker units with respect to the improved potency of tPy3PiB compared with bPiDDB and NDDPiI. In summary, the novel tris-quaternary ammonium compounds described in the current study represent new leads in our search for subtype-selective nAChR antagonists as treatments for nicotine addiction. Zheng, G., Sumithran, S.P., Deaciuc, A.G., Dwoskin, L.P. and Crooks, P.A. tris-Azaaromatic Quaternary Ammonium Salts: Novel Templates as Antagonists at Nicotinic Receptors Mediating Nicotine-Evoked Dopamine Release. Bioorganic & Medicinal Chemistry Letters, 17, pp. 6701-6706, 2007.

Quantitative Analysis of Naltrexone and 6ss-Naltrexol in Plasma by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

To improve the analysis of naltrexone and its primary metabolite 6ss-naltrexol, a sensitive and specific method for the analysis of subnanogram per milliliter concentrations of these analytes in human, rat and rabbit plasma was developed utilizing liquid chromatography coupled to electrospray ionization (ESI) tandem mass spectrometry (MS-MS). Plasma samples were extracted utilizing a liquid-liquid extraction technique. This method was compared to an existing gas chromatography (GC)-MS method by analyzing plasma samples collected from a clinical study. Specificity determined from comparing blank plasma fortified with internal standard and analyte at the lower limit of quantitation (LLOQ) from six different human, rat, and rabbit sources demonstrated sufficient signal-to-noise to set the LLOQ for both analytes and by less than 10, 10, and 9% at higher concentrations for human, rat and rabbit plasma, respectively. No loss of analyte was observed after 24 hours of room temperature storage in human, rat, and rabbit plasma of three cycles of freezing and thawing of human plasma prior to extraction. Human plasma samples were stable for at least five days when stored frozen at -20_ C, and for at least two days when stored at room temperature. The GC-MS and LC-MS-MS methods correlated in the measured plasma concentrations of both naltrexone and 6ss-naltrexol. This method has been validated and subsequently used in the determination of the pharmacokinetics of Depotrex in rabbits. In rabbits, the parent compound shows dose dependent pharmacokinetics as seen in human, but rabbits have much lower conjugated metabolite, 6ss-nalrexol, than that seen in humans. Slawson, M.H., Chen, M., Moody, D.E., Comer, S.D., Nuwayser, E.S., Fang, W.B. and Foltz, R.L. Quantitative Analysis of Naltrexone and 6_-Naltrexol in Human, Rat, and Rabbit Plasma by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry with Application to the Pharmacokinetics of Depotrex(R) Rabbits. Journal of Analytical Toxicology, 31, pp. 453-461, 2007.

Cannabinoids and Pain

The CB2 receptor is of current interest in pain research because several studies have reported CB2 agonist efficacy in various animal models of nociceptive and neuropathic pain. One feature of this receptor is its high level of expression in the immune system and in peripheral organs, which suggests that CB2-selective ligands may not produce the side effects of catalepsy, sedation, and psychotropic action attributed to CB1 ligands, which affect the central nervous system. CB2 messenger RNA has also recently been reported in rat dorsal root ganglion neurons, and lumbar spinal cord, although the location of the receptor on neurons or microglia cells is controversial. At the supraspinal level, low levels of messenger RNA have been reported in various regions of animal brain, but not well correlated with CB2 protein localization. To date, studies of CB2 agonists having antinociceptive properties which NIDA has supported include "classical" tetrahydrocannabinols (HU210), a deoxy-delta-8-THC (JWH133), and cannabimimetic indoles (HU-308, AM 1241, and JWH 015). In a recent study, Dr. Alexandros Makriyannis and collaborators have reported on the preparation and preliminary properties of a new series of benzo(c)chromenones known as cannabilactones. These have been shown to bind preferentially to the CB2 versus the CB1 receptor in tissue binding assays, with one case of 500 fold selectivity for the CB2 receptor. They were shown to be partial agonists in terms of GTPgammaS stimulation, and they also decreased the level of forskolin-stimulated cAMP, indicative of agonist behavior. They did not affect ambulation in rats at ip dose injections of 3 mg/kg, as opposed to impaired ambulation shown by WIN 55,212-2 at the same dose. The 9-hydroxy and 9-methoxycannabi-lactones exhibited peripheral antinociception in the radiant tail-flick test in rats, which was blocked for the 9-hydroxy compound by injection of a CB2, but not a CB1 antagonist. Preliminary structural information from X-ray crystallography and modeling suggests that the presence of the 6-keto group rather than the 6-dimethyl substitution (found in classical THC) produces a planar three-ring structure, reduces affinity for the CB1 receptor, and modifies the interaction of the CB2 receptor with the cannabilactones at positions 3 and 9. It should be noted that the above CB2 selectivity found in animal tissue was much smaller when using the human CB2 receptor (82 % identity with the animal CB2 receptor) expressed in HEK cells. Additional structural modification and structure- activity relationship information will be needed to evaluate these cannabilactones as potential human therapeutics. Khanolkar, A.D., Lu. D. Ibrahim, M. Duclos, R.I., Thakur, G.A., Malan, P., Porreca, R. Veerappan, V., Tian, X., George, C., Parrish, D.A., Papahatjis, D.P., and Makriyannis, A. Cannabilactones: A Novel Class of CB2 Selective Agonists with Peripheral Analgesic Activity. Journal of Medicinal Chemistry, 50, pp. 6493-6500, 2007.

Antagonist to Improgan Discovered

Improgan is a highly effective non-opioid analgesic when injected into the CNS. Improgan has been extensively studied; however neither the improgan receptor, nor a pharmacological antagonist of improgan has been previously described. NIDA grantee Dr. Lindsay Hough (Albany Medical College) and colleagues have recently discovered that 4(5)-((4-iodobenzyl)thiomethyl)-1H-imidazole (CC12), produced a dose dependent attenuation of improgan analgesia in rats, while having no effect when given alone. Radioligand binding, receptor autoradiography, and electrophysiology experiments showed that CC12's effects are not explained by activity at 25 sites known to be relevant to analgesia, including sites where cannabinoids, opioids and histamine produce analgesia. Dr. Hough appears to have discovered a novel brain mechanism involved in pain and analgesia, which may provide a new target for the development of centrally acting analgesics. Hough, L.B., Nalwalk, J.W., Phillips, J.G., Kern, B., Shan, Z., Wentland, M.P., de Esch, I.J.P., Janssen, E., Barr, T. and Stadel, R. CC12, a High-Affinity Ligand for [3H]cimetidine Binding, is an Improgan Antagonist. Neuropharmacology, 52, pp. 1244-1255, 2007.

Studies of Drug Actions on Basic HIV Infection and Growth

Several different laboratories have recently published data on drug action on HIV proliferation in various mammalian systems. There are three papers recently published that describe different types of drug action (cannabinoid or opioid) on immune systems; the third paper describes the basic mechanism of action (describing enzyme systems conducting the message) through which these drugs exert their actions. (1) The first paper describes the inhibition of growth of HIV in cell cultures by a synthetic cannabinoid. NIDA researcher Guy Cabral and colleagues observed that this action was mediated primarily through the "peripheral" cannabinoid system. They also observed, unexpectedly, that the more "central" cannabinoid system, also present, exerted the opposite effect. This occurred not through the action of the agonist (the usual mode of "pro" action through a receptor), but via the antagonist. Rock, R.B., Gekker, G., Hu, S., Sheng, W.S., Cabral, G.A., Martin, B.R. and Peteron, P.K. WIN55,212-2-Mediated Inhibition of HIV-1 Expression in Microglial Cells: Involvement of Cannabinoid Receptors. Journal of Neuroimmune Pharmacology, 2, pp. 178-183, 2007. (2) The second study, reported by NIDA research Dr. S.L. Chang focuses on opioid actions. In a special rat model which expresses some HIV viral proteins, these circulating viral entities enhanced the expression of morphine receptor activity through which increased levels of HIV viral growth could occur, especially in humans. This would demonstrate that not only bacterial, but viral growth (including HIV) could be enhanced by opiates administered to humans. Chang, S.L., Beltran, J.A. and Swarup, S. Expression of the Mu Opioid Receptor in the Human Immunodeficiency Virus Type 1 Transgenic Rat Model. Journal of Virology, 81, pp. 8406-8411, 2007. (3) A CXCR4 receptor system resides on immune and neural supporting cells. This receptor plays a pivotal role in immune responses, the pathogenesis of infection such as HIV, and cellular trafficking. However, the signaling mechanisms regulating SDF-driven (the agent acting on the CXCR4 receptor) T cell migration are not well defined. Dr. Burt Sharp and colleagues have focused on understanding how the signal is transmitted from CXCR4 receptor to accomplish its actions: i.e. alter HIV actions and reproduction and have characterized the kinases involved (enzyme systems which are involved in many cellular functions), which are different from those primarily associated with HIV type actions. Shahabi, N.A., McAllen, K. and Sharp, B.M. Stromal Cell-Derived Factor 1-_ (SDF)-Induced Human T Cell Chemotaxis Becomes Phosphoinositide 3-Kinase (PI3K-Independent: Role of PKC-_. Journal of Leukocyte Biology, 83, 2008. (epub ahead of print)

Cell-specific Upregulation of 4 Nicotinic Receptor Subunits May Explain both Tolerance and Sensitization due to Chronic Nicotine Exposure

Chronic nicotine induces at least two behavioral phenomena: sensitization, both locomotor and cognitive, and tolerance. Additional studies have shown that chronic nicotine upregulates nicotinic receptor levels throughout the brain, and that these receptors are functional. The conundrum is that increased nicotinic receptor activity produced by upregulation of receptors is consistent with sensitization but not tolerance. Conversely, desensitization of nicotinic receptors which would result in reduced activity would be consistent with tolerance but not sensitization. This study developed mice that express a normal functioning, fluorescently labeled 4 nicotinic receptor subunit and examined the brain region and cell type expression of this subunit, and the functional consequences of its expression. Focusing on the midbrain and hippocampus, it was found that 4 receptors localize to the GABAergic neurons of the VTA and substantia nigra while 4 receptor expression is little changed in dopaminergic neurons. Consequently, increased inhibitory inputs would reduce dopaminergic transmission and produce a decreased response to nicotine that may underlie the development of nicotine tolerance. In the hippocampus, chronic nicotine increased 4 receptors on glutamatergic neurons of the medial perforant path, thereby increasing excitability and consistent with the development of sensitization to nicotine. These different mechanisms provide a possible explanation for the development of tolerance of dopaminergic transmission in midbrain and sensitization of synaptic transmission in forebrain due to chronic nicotine exposure. Nashmi, R., Xiao, C., Deshpande, P., McKinney, S., Grady, S.R., Whiteake, P., Huang, Q., McClure-Begley, T., Lindstrom, J.M., Labarca, C., Collins, A.C., Marks, M.J., Lester, H.A. Chronic Nicotine Cell Specifically Upregulates Functional alpha 4* Nicotinic Receptors: Basis for Both Tolerance in Midbrain and Enhanced Long-term Potentiation in Perforant Path. Journal of Neuroscience, 27, pp. 8202-8218, 2007.

Haloperidol, after a Neurotoxic Regimen of Methamphetamine, Kills GABAergic Neurons in the Substantia Nigra Pars Reticulata of Rats

Methamphetamine-induced psychoses are often managed with haloperidol, a typical antipsychotic (neuroleptic) drug. Neuroleptics are dopamine D2 receptor antagonists. Since the loss of dopaminergic axons due to chronic methamphetamine in rats results in persistent elevated glutamate in the substantia nigra, Bryan Yamamoto's group hypothesized that D2 blockade by haloperidol following methamphetamine neurotoxicity might further disinhibit (i.e., enhance) glutamate release in the substantia nigra pars reticulata by D2 antagonism on striatal GABAergic output neurons and on subthalamo-nigral glutamatergic terminals in the substantia nigra, the combination of which results in the disinhibition of glutamate release from subthalamo-nigral terminals. Indeed, the researchers found that haloperidol, after a neurotoxic regimen of methamphetamine, killed GABAergic neurons in the substantia nigra pars reticulata by glutamatergic excitotoxicity, as reflected by loss of GAD67 mRNA expression, a marker for GABAergic neurons, and DNA fragmentation, accompanied by enhanced extracellular glutamate, and blocked by the glutamate NMDA (N-methyl-D-aspartate) receptor antagonist dizocilpine. While it is not known if this GABAergic neurotoxicity occurs in humans, these findings suggest that the current therapeutic management of methamphetamine psychoses with haloperidol may be contraindicated because of a resultant GABAergic cell death in the substantia nigra pars reticulata, which may predispose some individuals to the development of hyperkinetic movement disorders and seizures. Atypical antipsychotics or benzodiazepines may be a better option. Hatzipetros, T., Raudensky, J.G., Soghomonian, J.J. and Yamamoto, B.K. Haloperidol Treatment after High-Dose Methamphetamine Administration Is Excitotoxic to GABA Cells In The Substantia Nigra Pars Reticulata. Journal of Neuroscience, 27(22), pp. 5895-902, 2007.

Cocaine During Adolescence Enhances Dopamine in Response to a Natural Reinforcer

One of the most important questions in addiction research is whether the developmental period during which one begins to use drugs is important in determining an individual's vulnerability to future drug use. Increased dopamine release may be predictive of a substance's reinforcing properties. The present study investigated whether cocaine pretreatment in either adolescence or adulthood altered the dopaminergic response to a naturally reinforcing substance in adulthood. Animals were treated with cocaine for a single ten-day period as adolescents or as adults, and were prepared for microdialysis, with cannulae implanted in the nucleus accumbens septi (NAcc). One week later, all animals were exposed to sucrose in their drinking water and dialysate samples were collected. Regardless of age all saline-pretreated rats had significant increases in sucrose-induced extracellular dopamine (DA) levels in the NAcc compared to baseline levels. Rats pretreated with cocaine as adults also had significant increases in DA levels after sucrose. Interestingly, sucrose intake significantly enhanced DA levels in cocaine-pretreated adolescent rats as compared to all other conditions. The results from the present study show that rats given cocaine during adolescence have an enhanced dopaminergic response relative to adults when exposed to a naturally reinforcing substance, suggesting a greater sensitivity to sucrose in the younger animals. Cocaine exposure during adolescence produces long-term functional changes in the mesolimbic pathway. Future studies need to ascertain the underlying mechanisms and their potential role in cocaine addiction. Catlow, B.J. and Kirstein, C.L. Cocaine During Adolescence Enhances Dopamine in Response to a Natural Reinforcer. Neurotoxicology and Teratology, 1, pp. 57-65, 2007.

Gingko Biloba Extract Protects Against Astrocyte-Mediated Neurotoxicity in Mouse Model of HIV Neuropathogenesis

One factor believed to play a significant role in HIV-associated neuropathogenesis is the secretion of the HIV-1 Tat protein from infected cells in the brain. However, the mechanisms underlying Tat neurotoxicity are still not completely understood, and few therapeutics have been developed to specifically target HIV infection in the brain. Recent development of an inducible brain-specific Tat transgenic mouse model has made it possible to define the mechanisms of Tat neurotoxicity and evaluate anti-neuroAIDS therapeutic candidates in the context of a whole organism, as well as to study these issues in combination with models of drug abuse. This study showed that administration of EGb 761, a standardized formulation of Ginkgo biloba extract, markedly protected Tat transgenic mice from Tat-induced developmental retardation, inflammation, death, astrocytosis, and neuron loss. EGb 761 directly down-regulated glial fibrillary acidic protein (GFAP) expression, a marker of astrocyte activation, at both protein and mRNA levels. This down-regulation was, at least partially mediated by EGb 761 alteration of the interactions of the AP1 and NF-kappaB transcription factors with the GFAP promoter. Most strikingly, the characteristic Tat-induced macrophage/microglia activation, central nervous system infiltration of T lymphocytes, and oxidative stress were significantly alleviated in GFAP-null/Tat transgenic mice. Taken together, these results provide the first evidence to support the potential for clinical use of EGb 761 to treat HIV-associated neurological diseases. Moreover, these findings suggest for the first time that GFAP activation is directly involved in Tat neurotoxicity, supporting the notion that astrocyte activation or astrocytosis may directly contribute to HIV-associated neurological disorders. Zou,W., Kim, B.O., Zhou, B.Y., Liu, Y., Messing, A. and He, J.J. Protection Against Human Immunodeficiency Virus Type 1 Tat Neurotoxicity by Ginkgo biloba Extract EGb 761 Involving Glial Fibrillary Acidic Protein. American Journal of Pathology, 171, pp. 1923-1935, 2007.

Cocaine Directly Induces Breakdown of Blood-Brain Barrier and Increased Chemotactic Signaling of Monocytes, Factors Involved in Exacerbation of NeuroAIDS Pathogenesis

One of the hallmark features underlying the pathogenesis of HIV encephalitis is the disruption of the blood-brain barrier (BBB). Cocaine, often abused by HIV-infected patients, has been suggested to worsen the HIV-associated dementia (HAD) via unknown mechanisms. The present study examined the effects of cocaine on BBB permeability using human brain microvascular endothelial cells (HBMECs), as well as on the chemokine CCL2 and its receptor CCR2, which play crucial roles in the recruitment of inflammatory cells into the central nervous system in HIV-associated neurological disease. Exposure of HBMECs to cocaine correlated with the breakdown of ZO-1 tight junction protein and reorganization of the cytoskeleton resulting in stress fiber formation. Furthermore, cocaine also modulated upregulation of the CCL2/CCR2 axis in monocytes. These findings support the idea that cocaine exposure may lead to accelerated progression of HIV-1 neuropathogenesis. Dhillon N.K., Peng, F., Bokhari, S., Callen, S., Shin, S.H., Zhu, X., Kim, K.J. and Buch, S.J. Cocaine-Mediated Alteration in Tight Junction Protein Expression and Modulation of CCL2/CCR2 Axis Across the Blood-Brain Barrier: Implications for HIV-Dementia. Journal of Neuroimmune Pharmacology, 2007, epub ahead of print.

CYP2B6 Genotype Alters Abstinence Rates in a Bupropion Smoking Cessation Trial

Bupropion is a non-nicotine smoking cessation treatment medication. Although the efficacy of bupropion relative to placebo is firmly established, the majority of smokers relapse to smoking within 6 months after a quit attempt. It's known that CYP2B6 is the primary enzyme that metabolizes bupropion. Genetic variations in CYP2B6, such as the variant CYP2B6*6, can alter bupropion metabolism and may affect treatment outcome. To understand the pharmacogenetics of bupropion metabolism, Rachel Tyndale and her colleagues conducted a smoking cessation trial of bupropion versus placebo. Subjects were assessed for abstinence and other outcomes 10 weeks after start of treatment, and then again for a 6-month follow-up, and genotyped for CYP2B6. They showed that among smokers with *6 variants (n=147), bupropion treatment showed higher abstinence rates than placebo at the end of treatment (32.5% vs. 14.3%, p=0.01) and at the 6 month follow-up (31.2% vs. 12.9%, p=0.008). In contrast, bupropion was no more effective than placebo for smokers with the CYP2B6*1 gene variant (n=179), at the end of treatment (31% vs. 31.6%, p=.93) or at the 6-month follow up (22.0% vs. 21.5%, p=.94). Taken together, this study suggests that smokers with the CYP2B6*6 genotype may be especially 1.good candidates for bupropion treatment for smoking cessation. Lee, A.M, Jepson, C., Hoffmann, E., Epstein, L., Hawk, L.W., Lerman, C., and Tyndale, R.F. Biol. Psychiatry 62(6), pp. 635-641, 2007.

The Classical Complement Cascade Mediates CNS Synapse Elimination

During development synapses are initially formed and many inappropriate synapses are eliminated to establish the necessary circuitry for the proper function of the nervous system. Axons emanating from the retinas of both eyes initially connect to overlapping areas of the lateral geniculate nucleus, an area involved in processing visual information. Through a process of neuronal activity the overlap in the projections to the lateral geniculate nucleus is eliminated and the inputs from each eye become separated. This occurs during a specific time window, known as the critical period. If neuronal activity is blocked and only occurs after the critical period, synaptic refinement cannot take place. The process by which inappropriate synapses are eliminated has not been understood up until now. Work by the Barres lab at Stanford led by Dr. Beth Stevens shows that a molecule used to fight infection, C1q, in the complement cascade is required for synapse elimination. C1q is expressed in postnatal neurons in response to immature astrocytes and is localized to synapses throughout the postnatal CNS and retina. In the adult mouse brain C1q is not expressed. Thus, the expression of C1q coincides with the critical period for synapse elimination. The knockout of the C1q blocks synapse elimination and prevents segregation of the connections by the two retinas to the lateral geniculate nucleus. The mechanism by which synapse elimination takes place appears to be mediated by the classical complement cascade because knockout out of C3 produces the same effect on synapse elimination and segregation of synaptic input from the two retinas. C3 is the next molecule in complement cascade activated by C1q. While C1q is normally down regulated in the adult nervous system, C1q is upregulated in the retina in a mouse model of glaucoma, a neurodegenerative disease. These results suggest that the complement cascade plays an important role in synapse elimination during normal development and that complement-mediated synapse elimination may become reactivated in neurodegenerative disease. Stevens, B., Allen, N.J., Vazquez, L.E., Howell, G.R., Christopherson, K.S., Nouri, N., Micheva, K.D., Mehalow, A., Huberman, A.D., Stafford, B., Sher, A., Litke, A.M., Lambris, J.D., Smith, S.J., John, S.W.M., and Barres, B.A. Cell, 131, pp. 1164-1178, 2007.

Serine Phosphorylation Controls PSD-95: A Mini-Switch for Learning and Memory

Long term potentiation (LTP) and long term depression (LTD) play major roles in the most basic neural mechanisms for learning and memory. In the hippocampal region of the brain, LTP is the long-lasting enhancement in communication between two neurons that results from stimulating them simultaneously. LTD has the opposite role in neuronal communication. The cellular and molecular biology of LTP and LTD are not fully understood. PSD-95 is a scaffold protein at the post synaptic density (PSD) of the neuron that regulates synaptic strength. Evidences suggests that synaptic (chemical) LTP promotes PSD-95 accumulation at PSD, increasing synaptic strength; and LTD induces dispersal of PSD-95, reducing synaptic strength. Therefore PSD-95 is functioning like a switch to turn communication on and off between neurons for memory and learning. How the synaptic activities results in PSD-95 switch being turned on and off is not known. A NIDA funded research team led by Dr. Yasunori Hayashi, researcher at MIT in collaboration with a research group at University of Bristol, UK, reports that synaptic activities regulate PSD-95 accumulation at the synaptic site through the phosphorylation and dephosphorylation of a specific serine residue on the protein. Thus, phosphorylation of ser-295 occurs in vivo, and it enhances the ability of PSD-95 to accumulate in the PSD, leading to the recruitment of AMPA receptors and the strengthening of synaptic transmission. On the other hand, over expression of a mutant, S295D-PSD-95, which mimics phosphorylation but cannot be "dephosphorylated", blocks the induction of LTD. They suggest that dephosphorylation of PSD-95 ser-295 is critical for LTD, presumably because this dephosphorylation is required for dispersal of PSD-95 from synaptic sites and subsequent mobilization and internalization of AMPA receptors. Using pharmacological blocking agents, the researchers further identified that the Rac1-JNK1 kinase pathway is the main mediator of the PSD-95 serine phosphorylation, and the activation of the PP1 and/or PP2A phosphatases are responsible for PSD-95 dephosphorylation. Kim, M.J., Futai, K., Jo, J., Hayashi, Y., Cho, K. and Sheng, M. Synaptic Accumulation of PSD-95 and Synaptic Function Regulated by Phosphorylation of Serine-295 of PSD-95. Neuron 56(3), pp. 488-502, 2007.

Navigating Neurons Get to Know Where and When in the Developing Cerebral Cortex through Lhx2

During the development of cerebral cortex, newly born neurons in the ventricular zone migrate towards the cortical area and form six cortical layers. How the navigating neurons know where they are during the migration and when to stop upon their destination have been important research topics. A NIDA supported research team at Mass General Hospital/Harvard University, led by Dr. Pradeep Bhide, previously reported that embryonic exposure to cocaine results in irregularity of neuronal migration in the developing cortex, indicating the consequences of drugs of abuse in causing brain disorders. In the most recent study, the team reveals a molecular mechanism that migrating neurons rely on for positioning. Thus, the projection neurons of the neocortex are produced in the pseudostratified ventricular epithelium (PVE) lining of the embryonic lateral ventricles. Over a 7 day period in mouse, these neurons arise in an overlapping layer VI-to-II sequence and in an anterolateral to posteromedial gradient [the transverse neurogenetic gradient (TNG)]. At any time in the 7 day neurogenetic interval, a given PVE cell must know what class of precursor cell or neuron to form next. How this information is encoded in the PVE has not been known. With comparative experiments in wild-type and double-transgenic mice, overexpressing the cell cycle inhibitor p27Kip1, they show that a gradient of expression of Lhx2, a LIM homeodomain transcription factor, together with a gradient in duration of the G1 phase of the cell cycle, are sufficient to specify a positional mapping system that informs the PVE cell what class of neuron to produce next. The researchers suggest that Lhx2 likely is representative of an entire class of transcription factors expressed along the TNG. This mapping system consisting of a combination of signals from two different sources is a novel perspective on the source of positional information for neuronal specification in the developing CNS. Bernhard, S.B., Nowakowski, R.S., Bhide, P.G., and Caviness, V.S. Navigating Neocortical Neurogenesis and Neuronal Specification: A Positional Information System Encoded by Neurogenetic Gradients. J. Neurosci. 27, pp. 10777-10784, 2007.

Alpha2-Chimaerin Is the Newly Discovered Player in Eph-Ephrin Signaling Pathways in Axonal Path-finding

Eph-ephrin, the ligand-receptor signaling pair, is a family of principal cell guidance system during vertebrate and invertebrate development. They play essential roles in determining body segmentation, axon guidance and fasciculation, cell migration, angiogenesis, and cancer. A well known phenotype observed in neurobiological research is that mice lacking EphA4 exhibit a pronounced locomotor defect that results in rabbit-like hopping of the hindlimbs, largely due to aberrant axonal path-finding and crossing in the spinal cord during CNS development. While many ephrins, as the receptors for Ephs, have been identified, little has been known on the downstream signaling events and players inside the cell as effectors for Eph-ephrin signaling. Dr. Peter Scheiffele, a NIDA researcher at Columbia University, reports that his team has recently identified an important player, alpha2-chimaerin, as the effector in EphA4-ephrin signaling. They observed that the RacGAP alpha2-chimaerin interacts with activated EphA4 and is required for ephrin-induced growth cone collapse in cortical neurons. Alpha2-chimaerin mutant mice exhibit the same rabbit-like hopping gait with synchronous hind limb movements that phenocopies mice lacking EphA4 kinase activity. Anatomical and functional analyses of corticospinal and spinal interneuron projections reveal that loss of alpha2-chimaerin results in impairment of EphA4 signaling in vivo. These findings identify alpha2-chimaerin as an indispensable effector for EphA4 in cortical and spinal motor circuits. Beg, A.A., Sommer, J.E., Martin, J.H., and Scheiffele, P. Alpha2-Chimaerin Is an Essential EphA4 Effector in the Assembly of Neuronal Locomotor Circuits. Neuron 55(5), pp. 768-778, 2007.


Research Findings

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