Research Findings - Basic Neuroscience Research
Activation of the Kappa Opioid Receptor in the Dorsal Raphe Nucleus, and Involving Serotonergic but not Dopaminergic Transmission, Mediates the Aversive Effects of Stress and Reinstates Drug Seeking
Although stress has profound effects on motivated behavior including drug seeking, the underlying mechanisms responsible are incompletely understood. Previous studies have supported a role for the dynorphin/kappa opioid receptor (KOR) system in mediating the pro-addictive effects of stress-induced relapse. However, the brain region(s) and circuitry underlying KOR action are unresolved. This study elucidates a functional neural pathway in mouse brain that encodes the aversive effects of stress and mediates stress-induced reinstatement of cocaine conditioned place preference (CPP). Activation of the dynorphin/kappa opioid receptor (KOR) system by either repeated stress or agonist produces conditioned place aversion (CPA). Because KOR inhibition of dopamine release in the mesolimbic pathway has been proposed to mediate the dysphoria underlying this response, Dr. Chavkin and his colleagues tested dopamine-deficient mice in this study and found that KOR agonist in these mice still produced CPA. However, inactivation of serotonergic KORs by injection of the KOR antagonist norBNI into the dorsal raphe nucleus (DRN), blocked aversive responses to the KOR agonist U50,488 and blocked stress-induced reinstatement of CPP. Additionally, KOR knockout (KO) mice did not develop CPA to U50,488; however, lentiviral re-expression of KOR in the DRN of KOR KO mice restored place aversion. DRN serotonergic neurons project broadly throughout the brain, but the inactivation of KOR in the nucleus accumbens (NAc) coupled with viral re-expression of KOR in the DRN of KOR KO mice demonstrated that aversion was encoded by a DRN to NAc projection. These results suggest that the adverse effects of stress may converge on the serotonergic system and offers an approach to controlling stress-induced dysphoria and relapse. Land BB, Bruchas MR, Schattauer S, et al. Activation of the kappa opioid receptor in the dorsal raphe nucleus mediates the aversive effects of stress and reinstates drug seeking. Proc Natl Acad Sci U S A. 2009 Nov 10;106(45):19168-19173.
Dopamine Enables In Vivo Synaptic Plasticity Associated with the Addictive Drug Nicotine
Addictive drugs induce a dopamine signal that contributes to the initiation of addiction, and the dopamine signal influences drug-associated memories that perpetuate drug use. The addiction process shares many commonalities with the synaptic plasticity mechanisms normally attributed to learning and memory. Environmental stimuli repeatedly linked to addictive drugs motivate continued drug use. This paper, employing in vivo recording techniques, shows for the first time that in freely moving and behaving mice, physiologically relevant concentrations of the addictive drug, nicotine, directly cause in vivo hippocampal synaptic potentiation of the kind that underlies learning and memory. This nicotine-induced long-term synaptic plasticity required a local hippocampal dopamine signal. Disrupting general dopamine signaling prevented the nicotine-induced synaptic plasticity and conditioned place preference. These results suggest that dopaminergic signaling serves as a functional label of salient events by enabling and scaling synaptic plasticity that underlies drug-induced associative memory. Tang J, Dani JA. Dopamine enables in vivo synaptic plasticity associated with the addictive drug nicotine. Neuron. 2009 Sep 10;63(5):673-682.
Selective Blockade of the Hydrolysis of the Endocannabinoid, 2-AG, but not of Anandamide (AEA), Enhances Retrograde Endocannabinoid Signaling and the Duration of Endocannabinoid-mediated Synaptic Transmission
Endocannabinoid (eCB) signaling mediates depolarization-induced suppression of excitation (DSE) and inhibition (DSI), two prominent forms of retrograde synaptic transmission (synaptic depression). N-Arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), two known eCBs, are degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. Selective blockade of FAAH and MAGL is critical for determining the roles of the eCBs in DSE/DSI and understanding how their action is regulated. 4-Nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) is a recently developed, highly selective, and potent MAGL inhibitor that increases 2-AG but not AEA concentrations in mouse brain. Here, NIDA supported investigators report that JZL184 prolongs DSE in Purkinje neurons in cerebellar slices and DSI in CA1 pyramidal neurons in hippocampal slices. In contrast, neither the selective FAAH inhibitor cyclohexylcarbamic acid 3'-carbomoylbiphenyl-3-yl ester (URB597) nor FAAH knockout has a significant effect on DSE/DSI. These results indicate that the degradation of 2-AG by MAGL is the rate-limiting step that determines the time course of DSE/DSI and that JZL184 is a useful tool for the study of 2-AG-mediated signaling. Pan B, Wang W, Long JZ, Sun D, Hillard CJ, Cravatt BF, Liu QS. Blockade of 2-arachidonoylglycerol hydrolysis by selective monoacylglycerol lipase inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) enhances retrograde endocannabinoid signaling. J Pharmacol Exp Ther. 2009 Nov;331(2):591-597.
Persistent Changes in Brain Function after Repeated Ketamine
The N-methyl-D-aspartate (NMDA) receptor antagonist ketamine is a pediatric and veterinary anesthetic that is also abused at lower doses (as "Special K," etc.). Ketamine elicits and exacerbates psychotic symptoms resembling schizophrenia, including auditory hallucinations, blunted affect, and withdrawal. In some cases, past ketamine abuse is associated with a persisting psychosis, and it is not possible to determine whether a pre-existing psychosis was made florid by the drug, or the drug caused a new psychotic illness. Steven Siegel and his colleagues assessed the effects of chronic administration of a sub-anesthetic dose of ketamine on contextual fear conditioning, detection of pitch deviants, and auditory gating, in mice; deficits in these tests serve as models of schizophrenia. After four weeks of daily ketamine injections, mice exhibited decreased freezing in the fear conditioning paradigm (48 hr after cessation of treatment, which was the final test). Gating of the P80 component of auditory evoked potentials was significantly altered, as the four weeks of daily ketamine caused a significant decrease in S1 amplitude (3 and 5 weeks (last test) after cessation of treatment). S1 P20 latency was significantly increased as a result of ketamine treatment (3 and 5 weeks after cessation of treatment). Although there was no effect of ketamine on detection of pitch deviants, these results indicate that repeated ketamine impairs fear memory and has lasting effects on encoding of sensory stimuli. Such persisting effects add support to the belief that repeated ketamine abuse could have long-term effects on brain function and information processing, similar to schizophrenia. Though NMDA antagonists have been shown to kill neurons in animal models, the neural bases of these latest observations remain to be determined, and are presently being investigated. Amann LC, Halene TB, Ehrlichman RS, Luminais SN, Ma N, Abel T, Siegel SJ. Chronic ketamine impairs fear conditioning and produces long-lasting reductions in auditory evoked potentials. Neurobiol Dis. 2009 August;35(2):311-317.
D-Cycloserine Facilitates Extinction of Naloxone-Induced Conditioned Place Aversion in Morphine-Dependent Rats
The well-documented observation that cues paired with drug administration trigger relapse to drug seeking by inducing conditioned drug craving and withdrawal was extended to examine whether cues were also operative in conditioned morphine-withdrawal aversion. As drug cues hinder abstinence in addicts, therapies that reduce responsiveness to drug cues might facilitate rehabilita-tion. Drs. Myers and Carlezon used morphine-dependent rats and trained them to associate an environ-ment with naloxone-precipitated withdrawal. Subsequently, they received extinction training in which they were confined in the previously naloxone-paired environment in the absence of acute withdrawal. Some rats were given the N-methyl-D-aspartate (NMDA) receptor partial agonist D-cycloserine (DCS) before extinction training. Previously, this treatment has been shown to facilitate the extinction of conditioned fear responses and cocaine-conditioned place preferences. They found that morphine withdrawal-induced conditioned place aversion persisted in the absence of extinction training and that the administration of DCS before extinction training facilitated its extinction. Thus, D-cycloserine facilitated extinction of morphine withdrawal-associated place aversion, in a manner similar to the effect of DCS on extinction of conditioned fear, raising the possibility of common neural mechanisms. This work extends our understanding of drug cue responsivity and provides a rationale for the development of extinction-based treatments for addiction. Myers KM, Carlezon WA, Jr. D-cycloserine facilitates extinction of naloxone-induced conditioned place aversion in morphine-dependent rats. Biol Psychiatry 2010; Jan 1; 67(1):85-87.
Calcium Signaling Cascade Links Dopamine D1-D2 Receptor Heteromer to Striatal BDNF Production and Neuronal Growth
Although the perturbation of either the dopaminergic system or brain-derived neurotrophic factor (BDNF) levels has been linked to important neurological and neuropsychiatric disorders, there is no known signaling pathway linking these two major players. Dr. Susan George and her research team at the University of Toronto and the Centre for Addiction and Mental Health, Toronto, ON, Canada reported that the exclusive stimulation of the dopamine D1-D2 receptor heteromer, which they identified in striatal neurons and adult rat brain by using confocal FRET, led to the activation of a signaling cascade that links dopamine signaling to BDNF production and neuronal growth through a cascade of four steps: (i) mobilization of intracellular calcium through Gq, phospholipase C, and inositol trisphosphate, (ii) rapid activation of cytosolic and nuclear calcium/calmodulin-dependent kinase IIalpha, (iii) increased BDNF expression, and (iv) accelerated morphological maturation and differentiation of striatal neurons, marked by increased microtubule-associated protein 2 production. These effects, although robust in striatal neurons from D5(-/-) mice, were absent in neurons from D1(-/-) mice. They also demonstrated that this signaling cascade was activated in adult rat brain, although with regional specificity, being largely limited to the nucleus accumbens. This dopaminergic pathway regulating neuronal growth and maturation through BDNF may have considerable significance in disorders such as drug addiction, schizophrenia, and depression. Hasbi A, Fan T, Alijaniaram M, et al. Proc Natl Acad Sci U S A. 2009 Dec 15;106(50):21377-82. Epub 2009 Nov 30.
Homeostatic Synapse-Driven Membrane Plasticity in Nucleus Accumbens Neurons
Stable brain function relies on homeostatic maintenance of the functional output of individual neurons. In general, neurons function by converting synaptic input to output as action potential firing. To determine homeostatic mechanisms that balance this input-output/synapse-membrane interaction, Dr. Yan Dong of Washington State University focused on nucleus accumbens (NAc) neurons and demonstrated a novel form of synapse-to-membrane homeostatic regulation, homeostatic synapse-driven membrane plasticity (hSMP). Through hSMP, NAc neurons adjusted their membrane excitability to functionally compensate for basal shifts in excitatory synaptic input. Furthermore, hSMP was triggered by synaptic NMDA receptors (NMDARs) and expressed by the modification of SK-type Ca(2+)-activated potassium channels. Moreover, hSMP in NAc neurons was abolished in rats during a short- (2 d) or long- (21 d) term withdrawal from repeated intraperitoneal injections of cocaine. These results suggest that hSMP is a novel form of synapse-to-membrane homeostatic plasticity and dysregulation of hSMP may contribute to cocaine-induced cellular alterations in the NAc. Ishikawa M, Mu P, Moyer JT, et al. Homeostatic synapse-driven membrane plasticity in nucleus accumbens neurons. J Neurosci. 2009 May 6;29(18):5820-5831.
A Mechanisms by Which HIV-1 Evades the Immune System
HIV evades the defenses of the immune system leading eventually to the destruction of CD4+ T cells. Consequently, the host becomes susceptible to opportunistic infections. A paper in the October 2009 issue of the Journal of Virology by Professor Michael Gale and his colleagues at the University of Washington now provides an answer on how HIV evades destruction by the immune system of the host. Professor Gale and his colleagues show that HIV suppresses innate antiviral signaling and immune defenses within infected cells. Innate immunity differs from cellular immunity. In innate immunity signals within the cell fight infection; in cellular and adaptive immunity white cells such T cells, B cells, macrophages, neutrophils attack the foreign invader. Pathogen recognition receptors located on the cell surface of the host recognize viral proteins to trigger an intracellular messenger cascade that induces interferons and interferon induced pro-inflammatory cytokines. Induction of interferons is dependent upon the activation of IRF-3, IRF-7, and IRF-9. Professor Gale and his colleagues show that CD4+ T cells in vitro that normally clear paramyxovirus fail to clear paramyxovirus or HIV when infected with HIV. The inability to clear paramyxovirus or HIV infection is associated with depletion of the IRF-3 protein. Ectopic over expression of IRF-3 suppressed HIV infection. The depletion of IRF-3 is dependent on HIV replication and appears to be mediated by the Vpr protein of HIV. In vivo, Professor Gale and his colleagues show that IRF-3 levels are reduced in CD4+ T cells of patients with acute HIV-1 infection but not in long-term nonprogressors. These results suggest that suppression of IRF-3 is an important mechanism by which HIV-1 evades host defenses and suggest how the host might be more susceptible to viral coinfections such as hepatitis C. Progression of liver diseases occurs much more rapidly in HCV/HIV coinfected individuals. It is of interest to note that a gene variant in IRF-3/IL-28 is associated with inability to clear HCV infection. Thus, understanding how these two pathogens evades IRF-3 and innate immune system is central to understanding the pathogenesis of these diseases. Doehle BP, Hladik F, McNevin JP, McElrath MJ, Gale M Jr. Human immunodeficiency virus type 1 mediates global disruption of innate antiviral signaling and immune defenses within infected cells. J Virol. 2009 Oct;83(20):10395-10405.
Differential Inheritance of Mother Centrioles Determines Fates of Daughter Radial Glia
Abnormal cortical development underlies many pathological conditions seen in mental disorders or mental retardation. Microcephaly, a cortical developmental disorder associated with maternal alcoholism, is characterized by a small head with small brain and may be caused by a disturbance in the rapid growing of neural cells during cortical development, when a large number of neural cells are generated by the progenitor cells of radial glia at the ventricular zone (VZ). It has been known that asymmetric divisions of radial glia progenitors produce self-renewing radial glia and differentiating cells simultaneously in the VZ. Whereas differentiating cells leave the VZ to constitute the future neocortex, renewing radial glia progenitors stay in the VZ for subsequent divisions. The differential behavior of progenitors and their differentiating progeny is essential for neocortical development; however, the mechanisms that ensure these behavioural differences are unclear. A group of NIDA funded researchers, led by Song-Hai Shi, at Memorial Sloan Kettering Cancer Centre show that asymmetric centrosome inheritance regulates the differential behavior of renewing progenitors and their differentiating progeny in the embryonic mouse neocortex. Centrosome duplication in dividing radial glia progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glia progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results indicate that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glia progenitors in the developing mammalian neocortex. Irregular gene expression or mutations of genes that encode centrosomal components, or disturbances in the expression and regulation of such genes in substance abuse, may therefore play roles in abnormal cortical development. Wang X, Tsai JW, Imai JH, Lian WN, Vallee RB, Shi SH. Asymmetric centrosome inheritance maintains neural progenitors in the neocortex. Nature. 2009; 461: 947-955.
EphA and Ephrin-A: Signaling Pair Teams Up Functional Cortical Columns
The Eph proteins play essential roles in many aspects of brain development, from neuronal differentiation, axonal branching, pathfinding, to establishing columnar maps. The cerebral cortex is a laminated sheet of neurons composed of the arrays of intersecting radial columns. During development, excitatory projection neurons originating from the proliferative units at the ventricular surface of the embryonic cerebral vesicles migrate along elongated radial glial fibers to form a cellular infrastructure of radial (vertical) ontogenetic columns in the overlaying cortical plate. However, a subpopulation of these clonally related neurons also undergoes a short lateral shift and transfers from their parental to the neighboring radial glial fibres, and intermixes with neurons originating from neighboring proliferative units. This columnar organization acts as the primary information processing unit in the cortex. The molecular mechanisms, role and significance of this lateral dispersion for cortical development are not understood. Two groups of NIDA researchers, one led by Pat Levitt of University of Southern California, and the other led by Pasko Rakic at Yale University, jointly show that an Eph receptor A (EphA) and ephrin A (Efna) signalling-dependent shift in the allocation of clonally related neurons is essential for the proper assembly of cortical columns. As has been seen in the ocular dominance columns, these researchers found that EphA receptors and Efna exhibit highly regulated, stereotypic spatial and temporal expression pattern in the neocortex. In addition, in contrast to the relatively uniform labeling of the developing cortical plate by various molecular markers and retrograde tracers in wild-type mice, they found alternating labeling of columnar compartments in Efna knockout mice that are caused by impaired lateral dispersion of migrating neurons rather than by altered cell production or death. Furthermore, in utero electroporation of EphA- and Efna-expressing plasmid showed that lateral dispersion depends on the expression levels of EphAs and Efnas during neuronal migration. This mechanism seems to be essential for the proper intermixing of neuronal types in the cortical columns, which, when disrupted, may contribute to neuropsychiatric disorders associated with abnormal columnar organization. Torii M, Hashimoto-Torii K, Levitt P, Rakic P. Integration of neuronal clones in the radial cortical columns by EphA and ephrin-A signaling. Nature. 2009;461:524-528.
Retinoic Acid from the Surface to the Cortex: How Meninges Regulate Brain Development
The factors that control the timing and continued progression of cortical neurogenesis have remained largely obscure. Samuel Pleasure and his colleagues at UCSF, present evidence that all-trans retinoic acid (atRA) released from the meninges is involved in the decision of neuroepithelial cells to generate IPCs and neurons. His group demonstrates that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. Using Foxc1 mutant mice with defects in forebrain meningeal formation, they show that Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. They further demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10- and Raldh2- expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants, and Rdh10 mutants had a cortical phenotype similar to the Foxc1 null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal derived cue required for successful corticogenesis. Since Vitamin A derivative signaling plays essential roles during brain development and in mesolimbic dopaminergic pathway formation and plasticity, this newly discovered role of RA provides important insights for addiction research. Siegenthaler JA, Ashique AM, Zarbalis K, Patterson KP, Hecht JH, Kane MA, Folias AE, Choe Y, May SR, Kume T, Napoli JL, Peterson AS, Pleasure SJ. Cell. 2009;139:597-609.
CHRNA5 Nonsynonymous Polymorphism Associated with Nicotine Dependence in African-Americans
Genome-wide association studies of nicotine dependence have consistently shown a top association signal on chromosome 15q24-25, and include a nonsynonymous single nucleotide polymorphism (SNP) at rs16969968 within exon 5 of the alpha5 cholinergic nicotinic receptor subunit gene. The risk allele causes a functional amino acid change from aspartic acid at position 398 to asparagine. Additionally, this risk allele has been associated with lung cancer, chronic pulmonary obstructive disorder, and peripheral artery disease in European-descent samples. Dr. Saccone and her colleagues have studied this region in African descent to confirm its role in this population, and to leverage the contrasting genetic architecture of these two populations to help pin-point the functional source(s) of the disease associations. They report that the most significant SNP in the African-American sample is rs16969968 (P=0.0147; OR, 2.04; 1.15-3.62). They also confirm that the most significant SNP of the 76 tested in the chr 15q24-25 region in the combined African-American and European-American sample was rs16969968 (P=4.49 x 10-8; OR, 1.42; 95% CI, 1.25-1.61). This replication in African-Americans provides crucial information because of the different linkage disequilibrium structure and population history from the European-American samples. The rs16969968 SNP is rare in African-Americans, with a frequency of ~5% compared with a frequency of ~35% in European-Americans. Additionally, another SNP (rs555018) in the region is associated with brain mRNA levels of CHRNA5 in European-descent samples, but this result is not significant. When a rs16969668 is included as a covariate in the analysis with rs555018, a protective effect is seen in European-Americans, but not in African-Americans. These data suggest that mRNA levels of CHRNA5 may be influenced by additional variants in the region, and that these two SNPs may be responsible for two biological mechanisms affecting nicotine dependence risk. Saccone NL, Wang JC, Breslau N, et al. The CHRNA5-CHRNA3-CHRNB4 Nicotinic Receptor Subunit Gene Cluster Affects Risk for Nicotine Dependence in African-Americans and European-Americans. Cancer Res. 2009 Sept 69:6848-6856.
Humphrey Bogart and Betty Davis: Identification of Nicotine Response Genes in Zebrafish
Human genetic studies have recently been used with great success to identify genes associated with nicotine dependence. Animal genetic studies are an important complement to human studies since they can be used to 1) perform unbiased screens to identify genes and pathways associated with responses to drugs of abuse and 2) functionally characterize genes/variants identified in human or animal studies. Dr. Ekker and colleagues developed a nicotine behavioral response assay that allowed the identification of mutant zebrafish with altered behavioral responses to nicotine. Two independent mutant strains were identified and named Betty Davis and Humphrey Bogart (after celebrities that suffered from tobacco-related disease). The mutant animals were generated using a gene break transposon strategy which facilitated identification of the mutated genes responsible for the behavioral phenotypes. The Betty Davis mutant had a defect in the cct8 (chaperonin containing protein 8) gene. The human genome encodes an analogous version of cct8, so now the human cct8 gene can be tested for a possible role in human nicotine responses. The Humphrey Bogart mutant had a defect in the zebrafish GABA(B) receptor which binds to the neurotransmitter GABA. Interestingly, human gene variants in GABA(B) had previously been associated with nicotine dependence. This zebrafish work provides unbiased genetic confirmation of a role for the GABA(B) receptor in nicotine responses and suggests that at least a part of the nicotine behavioral response pathway is evolutionarily conserved. Furthermore the identification of the Humphrey Bogart and Betty Davis mutants in the genetically tractable zebrafish system will allow functional assessment of the role of these genes (and potentially the human variants of these genes) in nicotine responses. The development of nicotine behavioral response assays in zebrafish also could enable future inexpensive pharmacological screens to identify small molecules that could serve as the foundation for future nicotine dependence therapeutics. Petzold AM, Balciunas D, Sivasubbu S, et al. Nicotine response genetics in the zebrafish. Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18662-18667. Epub 2009 Oct 26.
Stress-Induced Potentiation of Cocaine Reward: A Role for CRF R1 and CREB
Studies in humans and animal models indicate that stress increases vulnerability to develop addiction, and potentiates drug taking and relapse in addicted individuals. Understanding the molecular mechanisms of this interaction could aid in the development of addiction treatment options. Exposure to both stress or drugs of abuse results in long-term adaptations in the brain that involve persistent alterations in gene expression or activation of transcription factors, such as the cAMP Response Element Binding (CREB) protein. Potential CREB target genes such as corticotropin-releasing factor (CRF), Brain derived neurotrophic factor (BDNF), and dynorphin are associated with initiation or reinstatement of drug reward and are also altered following stress. Dr. Blendy has shown that a single exposure to forced swim (FS) reinstates extinguished conditioned place preference (CPP) to cocaine and that CREB is necessary for this response. Chronic exposure to FS in advance of conditioning enhances cocaine CPP in wild-type mice, but this is blocked in CREB-deficient mice. As CREB can be activated by corticotropin releasing factor (CRF) receptor type 1 (CRF(R1)) binding, which mediates neuroendocrine and behavioral responses to stress as well as to drugs of abuse, contribution of CREB and CRF(R1) to the changes in cocaine reward elicited by previous exposure to stress was investigated. Pretreatment with the CRF(R1) antagonist, antalarmin, before FS exposure, blocks the stress-induced enhancement of cocaine CPP. Furthermore, FS-induced increase in phosphorylated CREB (pCREB), specifically in the lateral septum (LS) and nucleus accumbens (NAc) is also blocked by antalarmin. Thus, both CREB and CRF(R1) activation are necessary for stress-induced potentiation of drug reward. Kreibich AS, Briand L, Cleck JN, Ecke L, Rice KC, Blendy JA. Stress-induced potentiation of cocaine reward: a role for CRF R1 and CREB. Neuropsycho-pharmacogy. 2009 Nov;34(12):2609-2617.
Crystal Structure of FAAH with an Inhibitor Reveals Enzymatic Inactivation Mechanisms
Ben Cravatt and his collaborators recently reported the cocrystal X-ray structures of two isomeric R-ketooxazole inhibitors (1 (OL-135) and 2) bound to fatty acid amide hydrolase (FAAH), a key enzymatic regulator of endocannabinoid signaling. The active site catalytic Ser241 is covalently bound to the inhibitors' electrophilic carbonyl groups, providing the first structures of FAAH bound to an inhibitor as a deprotonated hemiketal mimicking the enzymatic tetrahedral intermediate. The work also offers a detailed view of the oxyanion hole and an exceptional "in-action" depiction of the unusual Ser-Ser-Lys catalytic triad. These structures capture the first picture of inhibitors that span the active site into the cytosolic port providing new insights that help to explain FAAH's interaction with substrate leaving groups and their role in modulating inhibitor potency and selectivity. The role for the activating central heterocycle is clearly defined and distinguished from that observed in prior applications with serine proteases, reconciling the large electronic effect of attached substituents found unique to this class of inhibitors with FAAH. Additional striking active site flexibility is seen upon binding of the inhibitors, providing insights into the existence of a now well-defined membrane access channel with the disappearance of a spatially independent portion of the acyl chain-binding pocket. Finally, comparison of the structures of OL-135 (1) and its isomer 2 indicates that they bind identically to FAAH, albeit with reversed orientations of the central activating heterocycle, revealing that the terminal 2-pyridyl substituent and the acyl chain phenyl group provide key anchoring interactions and confirming the distinguishing role of the activating oxazole. Mileni M, Garfunkle J, DeMartino JK, Cravatt BF, Boger DL, Stevens RC. Binding and inactivation mechanism of a humanized fatty acid amide hydrolase by α-Ketoheterocycle inhibitors revealed from cocrystal structures. J Am Chem Soc 2009 Jul 8;131(30):10497-10506.
N-arachidonoyl Dopamine (NADA), A Putative Endocannabinoid and Endovanilloid, Is Biosynthesized Via Conjugation of Arachidonic Acid with Dopamine
N-arachidonoyl dopamine (NADA) is an endogenous ligand that activates the cannabinoid type 1 receptor and the transient receptor potential vanilloid type 1 channel. Two potential biosynthetic pathways for NADA have been proposed, though no conclusive evidence exists for either. The first is the direct conjugation of arachidonic acid with dopamine and the other is via metabolism of a putative N-arachidonoyl tyrosine (NA-tyrosine). In the present study, Walker and colleagues investigated these biosynthetic mechanisms and report that NADA synthesis requires TH in dopaminergic terminals; however, NA-tyrosine, which is identified here as an endogenous lipid, is not an intermediate. The investigators show that NADA biosynthesis primarily occurs through an enzyme-mediated conjugation of arachidonic acid with dopamine. While this conjugation likely involves a complex of enzymes, their data suggests a direct involvement of fatty acid amide hydrolase in NADA biosynthesis either as a rate-limiting enzyme that liberates arachidonic acid from AEA, or as a conjugation enzyme, or both. Hu SS, Bradshaw HB, Benton VM, et al. The biosynthesis of N-arachidonoyl dopamine (NADA), a putative endocannabinoid and endovanilloid, via conjugation of arachidonic acid with dopamine. Prostaglandins Leukot Essent Fatty Acids, 2009 Oct; 81(4):291-301.
Inhibitors of Endocannabinoid Metabolizing Enzymes Reduce Precipitated Withdrawal Responses in THC Depenedent Mice
Abstinence symptoms in cannabis-dependent individuals are believed to contribute to the maintenance of regular marijuana use. However, there are currently no medications approved by the FDA to treat cannabis related disorders. The only treatment currently shown consistently to alleviate cannabinoid withdrawal in both animals and humans is substitution therapy, using the psychoactive constituent of marijuana, δ9-tetrahydrocannabinol (THC). However new genetic and pharmacological tools are available to increase endocannabinoid levels by targeting fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the enzymes responsible for the degradation of the endogenous cannabinoids ligands anandamide (AEA) and 2-arachidonylglycerol (2-AG), respectively. In the present study the authors have investigated whether increasing endogenous cannabinoids levels through the use of FAAH (-/-) mice as well as the FAAH inhibitor URB597 or the MAGL inhibitor JZL184 significantly attenuated rimonabant precipitated withdrawal signs in the THC dependent mice. By contrast, FAAH (-/-) mice showed identical withdrawal responses as wild type mice under a variety of conditions, suggesting that the absence of this enzyme across the development of dependence and during rimonabant challenge does not affect withdrawal responses. Of importance, subchronic administration of URB 597 did not lead to cannabinoid dependence, and neither URB597 nor JZL184 impaired rotarod motor coordination. These results support the concept of targeting endocannabinoid metabolizing enzymes as a promising treatment for cannabis withdrawal. Schlosburg JE, Carlson BLA, Ramesh D, Long JZ, Cravatt BF, Lichtman AH, Abdullah RA. Inhibitors of endocannabinoid metabolizing enzymes reduce precipitated withdrawal responses in THC dependent mice. The AAPS Journal, 2009 Jun;11(2):342-352.
MOR Antagonist Design
Mu opioid receptor (MOR) antagonists are of pharmacological interest in the field of analgesia, and are utilized in drug addiction research. Important objectives in the design of such antagonists include reversible rather than irreversible binding to the MOR, metabolic stability, a non-peptide structure for cell permeability, and relative selectivity toward the MOR versus the other opioid receptors. A recent report from Dr. Yan Zhang and colleagues at the Virginia Commonwealth University demonstrates the use of receptor modeling and message/address concepts to target a ligand to the opioid receptors (the message), while varying individual ligand substituents to improve selectivity (the address). Homology models of the three opioid receptors (MOR, DOR, KOR) were generated based on the 3-D structure of bovine rhodopsin, including both transmembrane helical and loop regions. The receptor models were included in a phospholipid bilayer environment, and structures were optimized using molecular dynamics. Naltrexone was used as a model antagonist, and was docked into the transmembrane binding pocket of each receptor, using an electrostatic distance constraint between the positively charged protonated nitrogen of naltrexone and the negative charge of an aspartate (position 147 in the MOR) in helix three of each receptor. The receptor-ligand complexes were subjected to molecular dynamics to yield the conformation of lowest energy in each case. The major difference found between the MOR model versus the DOR or KOR was the proximity of extracellular loop aromatic amino acid residues (tyrosine 210, phenylalanine 221, tryptophan 318) near the naltrexone carbonyl at the C(6) position. This suggested that suitable modification of the ligand naltrexone in terms of hydrogen bond formation would enhance the selectivity of binding toward the MOR. A series of naltrexamine amides were then synthesized with these findings in mind. One series of compounds introduced a pyridyl group to interact with the aromatic binding region of the MOR, and in the second case, introduced an isoquinoline group for the same purpose. These series also provided a pyridine nitrogen atom to act as a possible hydrogen bond acceptor with the hydroxyl of Tyr 210 (extracellular loop 2) or isoquioline nitrogen with the hydroxyl of Tyr 318 (extracellular loop 3). From these series, two particular compounds were reported to be selective toward the MOR versus the DOR or KOR, and showed partial agonism in the GTPgammaS functional assay. They were reported to be competitive and full antagonists of morphine response in-vivo, using the tail immersion test in mice, with AD50 values of .45 mg/kg or 4.5 mg/kg, and with no agonism effects seen up to 100 mg/kg. This work illustrates the application of homology modeling to design antagonists emphasizing or maximizing receptor-selective interactions. Li G, Aschenbach LC, Chen J, et al. Design, synthesis, and biological evaluation of 6-alpha and 6-beta-N-heterocyclic substituted naltrexamine derivatives as mu opioid receptor selective antagonists. J Med Chem. 2009 Mar 12; 52(5):1416-1427.
An Ultra-Short Dopamine Pathway Regulates Basal Ganglia Output
Substantia nigra pars reticulata (SNr) is a key basal ganglia output nucleus critical for movement control. Its GABA-containing projection neurons intermingle with nigral dopamine (DA) neuron dendrites. Here the authors show that SNr GABA neurons coexpress dopamine D(1) and D(5) receptor mRNAs and also mRNA for TRPC3 channels. Dopamine induced an inward current in these neurons and increased their firing frequency. These effects were mimicked by D(1)-like agonists, blocked by a D(1)-like antagonist. D(1)-like receptor blockade reduced SNr GABA neuron firing frequency and increased their firing irregularity. These D(1)-like effects were absent in D(1) or D(5) receptor knock-out mice and inhibited by intracellularly applied D(1) or D(5) receptor antibody. These D(1)-like effects were also inhibited when the tonically active TRPC3 channels were inhibited by intracellularly applied TRPC3 channel antibody. Furthermore, stimulation of DA neurons induced a direct inward current in SNr GABA neurons that was sensitive to D(1)-like blockade. Manipulation of DA neuron activity and DA release and inhibition of dopamine reuptake affected SNr GABA neuron activity in a D(1)-like receptor-dependent manner. Together, these findings indicate that dendritically released dopamine tonically excites SNr GABA neurons via D(1)-D(5) receptor coactivation that enhances constitutively active TRPC3 channels, forming an ultra-short substantia nigra pars compacta --> SNr dopamine pathway that regulates the firing intensity and pattern of these basal ganglia output neurons. Zhou FW, Jin Y, Matta SG, Xu M, Zhou FM. An ultra-short dopamine pathway regulates basal ganglia output. J. Neurosci. 2009 Aug 19;29(33):10424-10435.
Visualization of Chemokine Receptor Activation in Transgenic Mice Reveals Peripheral Activation of CCR2 Receptors in States of Neuropathic Pain
CCR2 chemokine receptor signaling has been implicated in the generation of diverse types of neuropathology, including neuropathic pain. For example, CCR2 knock-out mice are resistant to the establishment of neuropathic pain, and mice overexpressing its ligand, monocyte chemoattractant protein-1 (MCP1; also known as CCL2), show enhanced pain sensitivity. However, whether CCR2 receptor activation occurs in the central or peripheral nervous system in states of neuropathic pain has not been clear. NIDA supported investigators developed a novel method for visualizing CCR2 receptor activation in vivo by generating bitransgenic reporter mice in which the chemokine receptor CCR2 and its ligand MCP1 were labeled by the fluorescent proteins enhanced green fluorescent protein and monomeric red fluorescent protein-1, respectively. CCR2 receptor activation under conditions such as acute inflammation and experimental autoimmune encephalomyelitis could be faithfully visualized by using these mice. The investigators examined the status of CCR2 receptor activation in a demyelination injury model of neuropathic pain and found that MCP1-induced CCR2 receptor activation mainly occurred in the peripheral nervous system, including the injured peripheral nerve and dorsal root ganglia. These data explain the rapid antinociceptive effects of peripherally administered CCR2 antagonists under these circumstances, suggesting that CCR2 antagonists may ameliorate pain by inhibiting CCR2 receptor activation in the periphery. The method developed here for visualizing CCR2 receptor activation in vivo may be extended to G-protein-coupled receptors (GPCRs) in general and will be valuable for studying intercellular GPCR-mediated communication in vivo. Jung H, Bhangoo S, Banisadr G, Freitag C, Ren D, White FA, Miller RJ. Visualization of chemokine receptor activation in transgenic mice reveals peripheral activation of CCR2 receptors in states of neuropathic pain. J Neurosci. 2009 Jun 24;29(25):8051-8062.
Association of Nicotine Metabolite Ratio and CYP2A6 Genotype with Smoking Cessation Treatment in African-American Light Smokers
Cytochrome P450 2A6 (CYP2A6) is the main nicotine (NIC)-metabolizing enzyme in humans. Rachel Tyndale, Neal Benowitz and their collaborators investigated the relationships between CYP2A6 genotype, baseline plasma trans- 3'-hydroxycotinine/ cotinine (3HC/COT) (a phenotypic marker of CYP2A6 activity), and smoking behavior in African-American light smokers. Cigarette consumption, age of initiation, and dependence scores did not differ among 3HC/COT quartiles or CYP2A6 genotype groups. Slow metabolizers (SMs; both genetic and phenotypic) had significantly higher plasma NIC levels, suggesting that cigarette consumption was not reduced to adjust for slower rates of NIC metabolism. Individuals in the slowest 3HC/COT quartile had higher quitting rates with both placebo and NIC gum treatments (odds ratio 1.85, 95% confidence interval (CI) 1.08-3.16, P = 0.03). Similarly, the slowest CYP2A6 genotype group had higher quitting rates, although this trend did not reach significance (odds ratio 1.61, 95% CI 0.95-2.72, P = 0.08). The determination of the 3HC/COT ratio, and possibly CYP2A6 genotype, may be useful in the future for personalizing the choice of smoking cessation treatment in African-American light smokers. Ho MK, Mwenifumbo JC, Al Koudsi N, Okuyemi KS, Ahluwalia JS, Benowitz NL, Tyndale RF. Association of nicotine metabolite ratio and CYP2A6 genotype with smoking cessation treatment in African-American light smokers. Clin Pharmacol Ther. 2009 Jun;85:635-643.
Greater Impulsivity Leads to Escalation of Cocaine Self-Administration in Rats
Impulsivity has emerged as a major risk factor in the vulnerability to several aspects of drug abuse. The relationship between impulsivity and stimulant abuse is especially robust. Stimulant abusers exhibit higher levels of impulsivity, relative to non-drug-using controls as assessed by self-report and behavioral laboratory measures. Impulsivity, as measured by the delay-discounting task, predicts the acquisition of cocaine self-administration and reinstatement of cocaine seeking in rats. This study extended the current literature to the escalation phase of drug self-administration. Female rats were initially screened for high or low impulsivity for food reinforcement using a delay-discounting procedure. They were training to self-administer cocaine and exposed to progressive ratio and fixed ratio testing as well as an extended access paradigm. The results indicated that high impulsivity rats escalated cocaine-reinforced responding during the extended access condition, but low impulsivity rats did not. High impulsivity rats also earned significantly more infusions than low impulsivity rats under the post-escalation short access fixed-ratio condition. This work suggests that individual differences in impulsivity predict escalation of cocaine self-administration in rats, which may have implication in prediction of binge-like patterns of cocaine intake in humans. Anker JJ, Perry JL, Gliddon LA, Carroll ME. Impulsivity predicts the escalation of cocaine self-administration in rats. Pharmacol Biochem Behav. 2009 Sept; 93(3):343-348.
Spinal Microglia Play a Role in Visceral Hyperalgesia and NK1R Up-Regulation in a Rat Model of Chronic Stress
Chronic stress has been known to be associated with visceral hyperalgesia and increased expression of spinal NK1 receptors (NK1Rs). Dr. Yaksh and his associates sought to identify the role of spinal microglia in this process by exposing rats to the chronic stress of a water avoidance procedure or a sham stress procedure in control animals. Rats were given an injection of minocycline, a p38 inhibitor (SB203580), or saline each day. Phosphorylation levels of kinase p38 (P-p38), the microglia marker OX42, NK1R, and IκBα were assessed by immunoblotting and/or immunostaining of spinal samples collected on the day following the last exposure to the water stress (day 11). The investigators found that protein levels of P-p38 and immunoreactivity were increased in the stressed rats, and these changes were co-localized with OX42-positive cells and neurons in the dorsal horn of the spinal cord. Furthermore, the investigators found that these increases were reversed by minocycline or SB203580 exposure. They also reported that the stress-induced increase in NK1R expression was blocked by minocycline, but not by the SB compound. The water stress-induced hyperalgesia was blocked by minocycline and the SB compound when administered intrathecally. Dr. Yaksh and his colleagues conclude that this is the first demonstration that stress-induced activation of spinal microglia has a key role in visceral hyperalgesia and associated spinal NK1R up-regulation. Radesi R, Svensson CI, Steinauer J, Pothoulakis C, Yaksh TL, Mayer EA. FEBS Let. 2009 Apr;136(4):1339-1348.
Significant Association of ANKK1 and Detection of a Functional Polymorphism with Nicotine Dependence in an African-American Sample
The dopaminergic system in the brain plays a critical role in nicotine addiction. Genetic variants in the dopaminergic system, including those in dopamine receptor genes, represent plausible candidates for the genetic study of nicotine dependence (ND). Dr. Li and colleagues investigated various polymorphisms in the dopamine D(2) receptor gene (DRD2) and its neighboring ankyrin repeats and kinase domain containing 1 gene (ANKK1) to determine whether they were associated with ND. They examined 16 single nucleotide polymorphisms (SNPs) at DRD2 and 7 SNPs at ANKK1 in their Mid-South Tobacco Family cohort, which consisted of 2037 participants representing two distinct American populations. Several SNPs (rs7131056, rs4274224, rs4648318, and rs6278) in DRD2, along with the Taq IA polymorphism (rs1800497) in ANKK1, revealed initial significant associations with ND in European Americans, but not after correction for multiple testing, indicating a weak association of DRD2 with ND. In contrast, associations for ANKK1 with ND in the African-American and pooled samples, specifically for SNP rs2734849, remained significant after correction. With a non-synonymous G to A transition, rs2734849 produces an amino-acid change (arginine to histidine) in C-terminal ankyrin repeat domain of ANKK1. Using the luciferase reporter assay, the research team further demonstrated that the variant alters expression level of NF-kappaB-regulated genes. Since DRD2 expression is regulated by transcription factor NF-kappaB, they suspect that rs2734849 may indirectly affect dopamine D(2) receptor density. Dr. Li and colleagues conclude that ANKK1 is associated with ND and polymorphism rs2734849 in ANKK1 represents a functional causative variant for ND in African-American smokers. Huang W, Payne TJ, Ma JZ, Beuten J, Dupont RT, Inohara N, Li MD. Significant association of ANKK1 and detection of a functional polymorphism with nicotine dependence in an African-American sample. Neuropsychopharmacology. 2009 Jan;34(2):319-330.