Research Findings - Basic Neuroscience Research
Toll-Like Receptor-3 Activation in Human Neuronal Cells Induces Interferon-λ Expression
Interferon lambda (IFN-λ) belongs to the type III family of IFNs which when activated exerts antiviral, antitumor and immunoregulatory activities. Toll-like receptors (TLRs) are a family of receptors that mediate innate immune responses to stimuli from pathogens and endogenous signals. It has been shown that agonists of TLR-3 can induce IFN-λ production in macrophages or plasmacytoid dendritic cells. Little is known about IFN-λ expression in human neuronal cells and whether IFN-λ expression in human neuron relies on the TLR ligand activation. Human neuronal cells expressed endogenous IFN-λ1, but not IFNλ2/3, however upon activation of TLR-3 in neuronal cells all three structurally related cytokines were significantly induced. Activation of TLR-3 also exhibited antiviral activity against pseudotyped HIV-1 infection of the neuronal cells. Human neuronal cells also expressed functional IFN-λ receptors which when activated inhibited pseudotyped HIV-1 infection and induced APOBEC 3G/3F, anti-HIV-1 cellular factors. This paper provides compelling evidence that there is intracellular expression and regulation of IFN-λ in human neuronal cells, which may have a major role in the innate neuronal protection against viral infections in the CNS. Zhou L, Wang X, Wang YJ, Zhou Y, Hu S, Ye L, Hou W, Li H, Ho WZ. Activation of Toll-Like receptor-3 induces interferon-λ expression in human neuronal cells. Neuroscience. 2009 Mar 17;159(2):629-637.
Chronic Cocaine Enhances Stress-Induced Potentiation of Excitatory (Glutamatergic) Neurotransmission in Dopamine Neurons within the VTA
Current concepts suggest that stress-induced release of neuromodulators such as corticotropin-releasing factor (CRF) can drive drug-dependent behaviors. Furthermore, prior drug exposure can enhance behavioral and neurochemical responses to stress. Previously, it had been shown that CRF can enhance NMDA-type glutamatergic transmission. This paper now shows that after repeated cocaine exposure, the magnitude and duration of the CRF-induced potentiation of NMDA receptor-mediated neurotransmission is significantly increased within ventral tegmental area (VTA) dopamine neurons, a key locus of drug- and stress-induced neuroadaptation. Furthermore, although CRF itself does not enhance AMPA-type glutamatergic transmission, CRF did enhance AMPA receptor-mediated transmission in mice that were exposed to chronic cocaine. Importantly, pharmacological experiments revealed that CRF receptor 1 and protein kinase A pathways were newly recruited after repeated cocaine for the enhancement of CRF-induced NMDAR potentiation and the appearance of AMPAR potentiation. Thus, enhanced CRF-induced potentiation of excitatory synaptic transmission onto VTA dopamine neurons after cocaine preexposure is likely to produce an abnormal increase in dopamine release during stressful events and could augment activation of addictive behaviors in response to stress. Hahn J, Hopf FW, Bonci A. Chronic cocaine enhances corticotropin-releasing factor-dependent potentiation of excitatory transmission in ventral tegmental area dopamine neurons. J Neurosci. 2009 May 20;29(20):6535-6544.
Impaired Synaptic Plasticity Following Chronic Cocaine
Dr. Peter Kalivas and colleagues have been providing novel evidence that chronic drug abuse results in future deficits in the ability to generate synaptic plasticity (for review, see Kalivas PW. Nat Rev Neurosci. 2009 Aug;10(8):561-572). In the present study, his group sought to determine whether withdrawal from repeated cocaine administration can alter the capacity of a subsequent cocaine injection to elicit morphological, biochemical, and physiological plasticity. Three weeks after termination of chronic daily cocaine or saline administration, the researchers filled neurons in the nucleus accumbens with the lipophilic dye, DiI. They observed a shift to larger diameter spines in the daily cocaine-pretreated rats. During the first 2 hours after an acute cocaine challenge, a bidirectional change in spine head diameter and increase in spine density was measured in the daily cocaine-pretreated animals. In contrast, no change in spine diameter or density was elicited by a cocaine challenge in daily saline animals during the first 2 hours after injection. However, spine density was elevated at 6 hours after a cocaine challenge in daily saline-pretreated animals. The time-dependent profile of proteins in the postsynaptic density subfraction of neurons elicited by a cocaine challenge in daily cocaine-pretreated subjects indicated that the changes in spine diameter and density were associated with a deteriorating actin cytoskeleton and a reduction in glutamate signaling-related proteins. Correspondingly, the amplitude of field potentials in accumbens evoked by stimulating prefrontal cortex was reduced for up to 6 hours after acute cocaine in daily cocaine-withdrawn animals. These data indicate that daily cocaine pretreatment dysregulates dendritic spine plasticity elicited by a subsequent cocaine injection. In humans, the altered drug experience caused by previous drug abuse may underlie diminishing behavioral control and contribute to a progressively deteriorating disorder. Shen H, Toda S, Moussawi K, Bouknight A, Zahm DS, Kalivas PW. Altered dendritic spine plasticity in cocaine-withdrawn rats. J Neurosci. 2009 Mar 4;29(9):2876-2884.
Optical Fluorescent Tool Monitors Dopamine Release From Single Presynaptic Terminals
Investigators at Columbia University have designed optical tracers of monoaminergic neuro-transmitters, or false fluorescent neurotransmitters, which are taken up by secretory vesicles (VMAT2) and released after stimulation as if they were native transmitters. This has allowed the researchers to examine transmitter accumulation and exocytosis from single presynaptic terminals in the striatum and to make observations relevant to stimulation-dependent synaptic plasticity. They found a relatively low percentage of vesicles fusing to the plasma membrane with each stimulus, and that the release of transmitter differed across separate terminals. That is, some terminals were more active than others; additionally, transmitter release from each terminal also depended on the frequency of stimulation. Further, when the presynaptic autoreceptors on neurons were blocked, release was inhibited from two-thirds of the terminals, which were the terminals that were least active. Thus, activity-dependent terminal heterogeneity is associated with receptor-mediated responses and underscores the presence of frequency-dependent coding that may determine how particular synapses are activated during important functions such as decision-making and learning. Gubernator NG, Zhang H, Staal RGW, Mosharov EV, Pereira DB, Yue M, Balsanek V, Vadola PA, Mukerjee B, Edwards RH, Sulzer D, Sames D. Fluorescent false neurotransmitters visualize dopamine release from individual presynaptic terminals. Science. 2009;324:1441-1444.
Genome-wide Analysis of Chromatin Regulation by Cocaine Reveals a Role for Sirtuins
Long lasting changes in gene expression lead to changes in the reward systems of the brain. Normally investigators measure steady state mRNA levels to assess gene expression. However, advances in chromatin immunoprecipitation technology have enabled analysis of histone modifications which regulate chromatin structure at specific genomic locations. Chromatin structure is highly predictive for gene activation or repression. In initial experiments, Dr. Nestler and co-workers characterized genome wide levels of histone acetylation and methylation from the nucleus accumbens (NAc) brain region of rats treated chronically with cocaine. These studies revealed that many genes previously known to be up regulated by cocaine exposure also have additional acetylation of histone H3 and H4. Dr. Nestler then looked genome wide to see where the cocaine-induced transcription factors deltaFosB and CREB bound in the NAc of cocaine treated animals. Cross comparison of these data sets identified many genes that had not previously been implicated in response to cocaine, including the Sirtuin genes (Sirt1 and Sirt2) which function as NAD-dependent histone deacetylases. These genes function in many biological processes, including aging; however their role in the nervous system is poorly understood. Dr. Nestler used pharmacological activators and inhibitors of sirtuins to look at their function in cocaine responses. Interestingly, activation of sirtuins dramatically enhanced the rewarding effect of cocaine, while inhibition of sirtuins had the opposite effect. Thus genome wide chromatin immunoprecipitation strategies enabled Dr. Nestler and co-workers to identify a number of new genes likely to be in chronic cocaine responses. Two of these, Sirt1 and Sirt2, were functionally validated using pharmacological agents. Pharmaco-logical modulation of sirtuin function may be a fruitful future avenue to explore in the development of therapeutic agents to treat cocaine addiction. Renthal W, Kumar A, Xiao G, Wilkinson M, Covington HE, Maze I, Sikder D, Kodadek TJ, Stack A, Kabbaj M, and EJ Nestler. Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins. Neuron. 2009; 62:335-348.
Genetically Determined Interaction Between the Dopamine Transporter and the D2 Receptor on Prefronto-Striatal Activity and Volume in Humans
Dopamine modulation of neuronal activity during memory tasks identifies a nonlinear inverted-U shaped function. Both the dopamine transporter (DAT) and dopamine D(2) receptors (encoded by DRD(2)) critically regulate dopamine signaling in the striatum and in prefrontal cortex during memory. Moreover, in vitro studies have demonstrated that DAT and D(2) proteins reciprocally regulate each other presynaptically. Therefore, Dr. Sadee and his colleagues have evaluated the genetic interaction between a DRD(2) polymorphism (rs1076560) causing reduced presynaptic D(2) receptor expression and the DAT 3'-VNTR variant (affecting DAT expression) in a large sample of healthy subjects undergoing blood oxygenation level-dependent (BOLD)-functional magnetic resonance imaging (MRI) during memory tasks and structural MRI. They found a significant DRD(2)/DAT interaction in prefrontal cortex and striatum BOLD activity during both working memory and encoding of recognition memory. The differential effect on BOLD activity of the DAT variant was mostly manifest in the context of the DRD(2) allele associated with lower presynaptic expression. Similar results were also evident for gray matter volume in caudate. These interactions describe a nonlinear relationship between compound genotypes and brain activity or gray matter volume. Complementary data from striatal protein extracts from wild-type and D(2) knock-out animals (D2R(-/-)) show that DAT and D(2) proteins interact in vivo. Together, these data demonstrate that the interaction between genetic variants in DRD(2) and DAT critically modulates the nonlinear relationship between dopamine and neuronal activity during memory processing. Bertolino A, Fazio L, Di Giorgio A, Blasi G, Romano R, Taurisano P, Caforio G, et al., Genetically determined interaction between the dopamine transporter and the D2 receptor on prefronto-striatal activity and volume in humans. J Neurosci. 2009 Jan 28;29(4):1224-1234.
Mammalian Par3 Regulates Notch Signaling During Cortical Neurogenesis
Proper formation of the cerebral cortex depends on the orderly production of a large number of neurons during embryonic development. Radial glial cells spanning the primitive stages of developing cortex are a major population of neuronal progenitor cells during cortical development, in addition to their well-characterized role in guiding radial migration of newly born neurons generated in the ventricular zone. Radial glial cells divide to generate either a neuron or a new glial cell. The precise control of radial glial cell division in the developing cortex is likely a major factor in controlling the number of neurons in the mature cerebral cortex. While the asymmetric cell division of radial glial progenitors produces neurons as well as allowing self-renewal, little is known about the mechanism that generates asymmetry in daughter cell fate specification. A group of NIDA researchers at the Memorial Slone Kettering Cancer Center, led by Songhai Shi, found that mammalian partition defective protein 3 (mPar3), a key cell polarity determinant, exhibits dynamic distribution in radial glial progenitors. While it is enriched at the lateral membrane domain in the ventricular endfeet during interphase, mPar3 becomes dispersed and shows asymmetric localization as cell cycle progresses. Either removal or ectopic expression of mPar3 prevents radial glial progenitors from dividing asymmetrically yet generates different outcomes in daughter cell fate specification. Furthermore, the expression level of mPar3 affects Notch signaling, and manipulations of Notch signaling or the expression of Numb, a mediator of Notch, suppress mPar3 regulation of radial glial cell division and daughter cell fate specification. These results reveal a critical molecular pathway underlying asymmetric cell division of radial glial progenitors in the mammalian neocortex. These studies add to our understanding of normal and abnormal cortical development and may yield insights into developmental causes of pathological conditions such as seen in mental health disorders or mental retardation. Bultje RS, Castaneda-Castellanos DR, Jan LY, Jan YN, Kriegstein AR, Shi SH. Mammalian Par3 regulates progenitor cell asymmetric division via notch signaling in the developing neocortex. Neuron. 2009 July 30; 63:189-202.
Striatal Specific Rhes Mediates Mutant-Huntingtin Cytotoxicity in HD
Huntington's disease is an inherited neurodegenerative disease that typically appears in middle age that is characterized by jerky movements (chorea), abnormal posturing, sleep disturbance, seizure, and dementia. In addition, anxiety, depression, and the worsening of substance abuse are frequently seen in these patients. Wood Guthrie, the famous American folksinger, who wrote "This Land is Your Land" suffered from Huntington's disease. The disease is caused by a polyglutamine repeat in the huntingtin protein. While the nature of the genetic mutation has been known for 15 years, scientists have been puzzled by the fact that neurodegeneration happens only in the striatum of the brain despite the fact that the mutation is expressed everywhere in the body. In a recent paper in Science, Dr. Solomon Snyder, funded by NIDA, has solved this puzzle. The Snyder lab shows that the selective cytotoxicity is caused by Rhes, a small GTP binding protein that is specifically expressed in the striatum. The Rhes protein binds more readily to the normal Huntingtin protein than the mutant huntingtin protein. Meanwhile the mutant Hungtintin protein but not the normal protein forms aggregates or clumps. When the mutant Huntingtin protein is expressed together with Rhes there is an increase in the amount of soluble mutant protein and a decrease in the number of aggregates that is associated with increased cytotoxicity. Depletion of the Rhes protein in cell lines decreased the cytotoxicity when the mutant huntingtin protein is expressed. The Snyder lab then showed that SUMOylation, the covalent attachment of the small ubiquitin-like modifier (SUMO) at lysine at residues 15 and 91 of Huntingtin protein is responsible for the increased solubility of the mutant huntingtin protein and cytotoxcity. Altering the Lys-residues to arginine and depleting the SUMOylation enzyme SUMO1 prevented cytotoxcity. Although, Dr. Snyder and his colleagues show that the SUMOylation of the huntingtin protein is not dependent on the GTPase activity of Rhes, the effect of Rhes on SUMOylation is dependent on the attachment of Rhes through farnesylation. Thus, farnesylation inhibitor Lonafarnib (SCH66336) is being tested in phase II clinical trials for progeria and may be useful for the prevention and treatment of Huntington's disease. Subramaniam S, Sixt KM, Barrow R, Snyder SH. Rhes, a striatal specific protein, mediates mutant-huntingtin cytotoxicity. Science. 2009 Jun 5;324(5932):1327-1330.
Carbon Nanotube Membranes for Active Transdermal Drug Delivery of Nicotine and Other Compounds
Cigarette smoking continues to be the leading cause of premature death and illness in every industrialized country in the world, and in the U.S. alone leads to more than 400,000 deaths each year. Quitting smoking leads to immediate health benefits, but is difficult because people become addicted to nicotine, the active ingredient in tobacco. The nicotine patch is a widely recognized over-the-counter treatment device for use in smoking cessation that delivers a constant dose of nicotine through the skin to help relieve the symptoms associated with withdrawal. Success rates for smokers using the patch to quit, while better than those who try to quit by cessation alone, have been less than optimal (<20%). Some have proposed that this may be due to the constant steady-state pattern of nicotine delivered by the patch does not match the intra-day peaks and valleys of nicotine blood levels associated with smoking each cigarette through the day. Dr. Bruce Hinds and his colleagues at the University of Kentucky have developed a novel skin patch device for delivering nicotine based on an active layer of aligned carbon nanotubes (CNT) approximately 1.5-7 nanometers in diameter crossing through a solid polymer film. The openings of the CNTs are modified chemically so that they can be opened or closed at any time by applying or removing a small electric current, respectively. Dr. Hinds has shown that in the open state, small molecules are actively pumped across the membrane five times faster than simple diffusion. In other words, the CNT patch is a programmable system that can be controlled by the physician or the patient to mimic the rapid attainment of high nicotine plasma levels similar to those associated with smoking a cigarette, and then closed to allow a slow return to normal. The usefulness of the CNT patch is not limited to nicotine; many other skin absorbable compounds could be used as well. For example, Dr. Hinds has now demonstrated current modulated transport of the alpha-adrenergic agonist clonidine in therapeutically useful doses through the CNT patch on human skin. He and his colleagues have proposed that opioid withdrawal symptoms could be relieved in a similar manner by the use of clonidine in the CNT patch. Presently, such treatment requires multiple injections per day over the 3-5 day opioid withdrawal syndrome in sick and often uncooperative patients. The CNT patch device represents a major step forward in developing a programmable, transdermal drug delivery system that can be useful to treat a variety of syndromes and that can be tailored to an individual patient's needs in a manner that will improve therapy and likely increase patient compliance. Majumder M, Stinchcomb A, Hinds BF. Towards mimicking natural protein channels with aligned carbon nanotube membrans for active drug delivery. Life Sci 2009 Apr 18 [E-pub ahead of print].
High Specific Activity (+)-Amphetamine and (+)-Methamphetamine
In general, the (S)-(+)-enantiomers of amphetamine and methamphetamine have been demonstrated to have about five times greater psychostimulant activity than their respective (R)-forms. In a recent study of the effects of (S)- and (R)- methamphetamine in humans, the pharmacokinetic parameters for the enantiomers administered separately were found to be similar, but the elimination half-life was longer for (R)-methamphetamine, it did not increase the systolic pressure like (S)-methamphet-amine. Interestingly, (R)-methamphetamine was psychoactive producing intoxication and drug-liking ratings similar to those for (S)-methamphetamine at the same dose. However, despite the longer half- life of (R)-methamphetamine, its effects were dissipated twice as fast as those of (S)-methamphetamine. Investigation of amphetamine binding sites has been hindered by the lack of the separate antipodes, (S)- and (R)-, of amphetamine and methamphetamine with high specific activity. Thus, it has been reported that, using (+)-tritiated amphetamine (specific activity 15.7 Ci/mmol), binding site with apparent affinity constants of 96 and 279 nM were detected in hypo-thalamic membrane preparations from rat brain, but has been challenged as artefactual, resulting from inadequate filtration technique. Data obtained using both filtration and centrifugation techniques in a follow up investigation were consistent with the presence of a binding site for (S)-amphetamine, but could neither confirm nor exclude the presence of a second binding site. As part of a NIDA supported program to provide useful biochemical tools to researchers, the PI and coworkers have synthesized higher specific activity (>30 Ci/mmol) (S)-amphetamine and (S)-meth-amphetamine by reductive dechlorination of (S)-(3',5'-dichlorophenyl-2-propylazide and (S)-2',6'-dichloro-methamphetamine, respectively, while the latter was readily obtained by resolution of racemic 2',6'-dichloromethamphetamine using (+)-dibenzoyltartaric acid, the analogous amphetamine resisted all efforts to resolve it. Hence, the required chiral precursor was prepared by stereospecific total synthesis following methodology that had been previously been developed in authors' laboratories. Lamb PB, McElhinny CJ, Sninski T, Purdom H, Carroll FI, Lewin AH. High specific activity (+)-amphetamine and (+)-methamphetamine, J. Label Compd. Radiopharm. (E-pub) July 15, 2009.
Morphine Enhances Tat-induced Activation in Murine Microglia
Increasing evidence suggests that opiates accelerate the pathogenesis and progression of acquired immunodeficiency syndrome (AIDS), as well as the incidence of human immunodeficiency virus (HIV) encephalitis (HIVE), a condition characterized by inflammation, leukocyte infiltration, and microglial activation in the brain. The mechanisms, by which the HIV-1 transactivating protein Tat and opioids exacerbate microglial activation, however, are not fully understood. In the current study, researchers explored the effects of morphine and HIV-1 Tat(1-72) on the activation of mouse BV-2 microglial cells and primary mouse microglia. Both morphine and Tat exposure caused up-regulation of the chemokine receptor CCR5, an effect blocked by the opioid receptor antagonist naltrexone. Morphine in combination with Tat also induced morphological changes in the BV-2 microglia from a quiescent to an activated morphology, with a dramatic increase in the expression of the microglial activation marker CD11b, as compared with cells exposed to either agent alone. In addition, the mRNA expression of inducible nitric oxide synthase (iNOS), CD40 ligand, Interferon-gamma-inducible protein 10 (IP-10), and the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, and IL-6, which were elevated with Tat alone, were dramatically enhanced with Tat in the presence of morphine. In summary, these findings shed light on the cooperative effects of morphine and HIV-1 Tat on both microglial activation and HIV coreceptor up-regulation, effects that could result in exacerbated neuropathogenesis. Bokhari SM, Yao H, Bethel-Brown C, Fuwang P, Williams R, Dhillon NK, Hegde R, Kumar A, Buch SJ. Morphine enhances Tat-induced activation in murine microglia. Journal of Neurovirology. 2009 May 22; 15:219-228.
Evidence for HIV Tat-Induced Dopamine System Dysfunction
Individuals infected with human immunodeficiency virus (HIV) may develop neuropsychological impairment, and a modest percentage may progress to HIV-associated dementia (HAD). Research using human and nonhuman, in vitro and in vivo models, demonstrates that subcortical dopamine (DA) systems may be particularly vulnerable to HIV-induced neuro-degeneration. In this study, Booze and colleagues examined how the HIV-1 protein Tat altered brain striatal DA transmission; her approach involved in vivo brain microdialysis in rats. The current study investigated Tat-induced neuronal dysfunction between 24-h and 48-h post-Tat administration, and demonstrates a reduction in evoked DA for the Tat-treated group relative to vehicle-treated group at 24 and 48 h. The Tat-induced reduction of DA overflow by 24 h suggests dysfunction of nerve terminals, and a compromised DA system in Tat-treated animals. Furthermore, the current study provides direct support for HIV-associated decline of DA function at a systemic level, helping to characterize the functional outcome of the relatively large amount of research on the molecular and behavioral levels of HIV-induced neurotoxicity. This initial study may provide additional characteristics of Tat-induced neuronal dysfunction to inform research on therapeutic intervention, and it provides a springboard for future in vivo research currently needed in the field. Ferris MJ, Frederick-Duus D, Fadel J, Mactutus CF, Booze RM. In vivo microdialysis in awake, freely moving rats demonstrates HIV-1 Tat-induced alterations in dopamine transmission. Synapse. 2009 Mar;63(3):181-185.
NOP Ligand Activity
The "orphan" receptor NOP (nociceptin opioid peptide) is found in the forebrain and spinal cord of animals and humans. Its distribution can be measured by the binding of its natural ligand, known as nociceptin, in radiolabeled form. As an endogenous ligand, nociceptin may affect a range of neuronal circuits and brain regions. When administered to animals by an icv route it can display a pronociceptive effect, and when given intrathecally, an antinociceptive effect. Synthetic ligands for the NOP have been discovered since nociceptin was identified, both peptides and heterocyclic non-peptides, having a range of activities from partial agonism to full antagonism. These determinations are based largely on cell membrane and tissue in-vitro binding, and on the degree of stimulation of radiolabeled GTPgammaS binding, and the degree of cellular calcium stimulation or inhibition measured in electrophysio-logical studies. Recently, Dr. Nurulain Zaveri of the Molecular Medicine Research Institute and collaborators at SRI International have been examining NOP ligands with "mixed" properties: NOP agonist/MOP (mu opioid receptor) agonist, and NOP antagonist/MOP agonist. For this work, a chronic neuropathic pain model in rats was used. This work has shown that compound SR 16435 (a ligand with high affinity for both the NOP and MOP receptors) exhibited anti-allodynic effects at 3 or 10 mg/kg by i.p. administration, and this effect was largely blocked by naloxone co-administration at 1 mg/kg. This suggests the effect on the MOP predominates in this case. In another example, the researchers found that NOP antagonists SR 16430 and SR 14148 (having partial MOP agonism) co-administered at 3 or 10 mg/kg with morphine at 3 or 10 mg/kg could significantly enhance the anti-allodynic effect of morphine. Further experiments are planned with additional ligands intended to optimize the balance between opioid (MOP) and non-opioid (NOP) effects on neuropathic pain. Khroyan TV, Polgar WE, Orduna J, Jiang F, Olsen, C, Toll, L, Zaveri NT. Activity of new NOP receptor ligands in a rat peripheral mononeuropathy model: potentiation of morphine anti-allodynic activity by NOP receptor antagonists. Eur J Pharmacol May 21 2009;610(1-3):49-54.
The Biosynthesis of N-arachidonoyl Dopamine (NADA), A Putative Endocannabinoid and Endovanilloid, 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 tyrosine hydroxylase 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, Chen JS, Huang SM, Minassi A, Bisogno T, Masuda K, Tan B, Roskoski R, Cravatt BF, Di Marzo V, Walker JM. 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, Jun 28, [E-pub ahead of print].