Research Findings - Intramural Research

Chemistry and Drug Metabolism, CPTRB

Methadone Maintenance and Breastfeeding in the Neonatal Period
Lactation among methadone-maintained women is frequently challenged due to lack of knowledge and guidelines regarding this practice. In methadone-maintained breastfeeding women and a matched group of formula-feeding women, IRP scientists evaluated breast milk methadone concentrations and concentrations of methadone in maternal and infant plasma in both groups. Eight methadone-maintained (dose range 50-105 mg/day), lactating women provided blood and breast milk on days 1, 2, 3, 4, 14 and 30 after delivery at expected trough and peak times. Paired foremilk and hindmilk specimens were obtained at each sampling time. Eight matched formula-feeding subjects had blood drawn the same days. Infant blood for both groups was obtained on day 14. Urine toxicology between 36 weeks gestation and 30 days post-partum confirmed that subjects were not using illicit substances in the perinatal period. Concentrations of methadone in breast milk were low (range 21.0-462.0 ng/mL) and not related to maternal dose. There was a significant increase in methadone concentrations in breast milk over time for all six sampling times. Maternal methadone plasma concentrations were not different between groups and unrelated to maternal dose. Infant methadone plasma concentrations were low in all specimens (range 2.2 - 8.1 ng/mL). Infants in both groups had neurobehavioral assessments on days 3, 14 and 30; there were no significant effects of breastfeeding on neurobehavioral outcomes. Fewer infants in the breastfed group required pharmacotherapy for neonatal abstinence syndrome, but this was not a statistically significant finding. Results contribute to the recommendation of breastfeeding for methadone-maintained women. Jansson, L.M., Choo, R.E., Velez, M., Harrow, C., Schroeder, J., Shakleya, D.M. and Huestis, M.A. Pediatrics, 121, pp. 106-114, 2008.

Validation and Application of a Novel Method for the Determination of Buprenorphine, Norbuprenorphine and Their Glucuronide Conjugates in Human Meconium
A novel liquid chromatography tandem mass spectrometry (LCMSMS) method for buprenorphine, norbuprenorphine, and glucuronidated conjugates' quantification in meconium was developed and fully validated. Maternal self-report, the most common mechanism for identifying drug-exposed neonates is less reliable than biological monitoring of maternal and infant specimens. Meconium, the highly complex neonatal fecal material, is easy and non-invasive to collect and has higher sensitivity and specificity than urine to detect in utero drug exposure. Controlled administration of illicit drugs during pregnancy is unethical and unsafe, and administration of licit medications is recommended only as needed. Buprenorphine administration to pregnant opiate addicts to reduce illicit drug use and craving provides an important opportunity to study disposition of this drug in the maternal-fetal dyad. It is unknown whether buprenorphine dose is correlated to buprenorphine and/or metabolite concentrations in meconium, and if meconium concentrations predict neonatal outcomes. This research has not been possible due to the lack of a validated, quantitative chromatographic method for measuring buprenorphine in this neonatal matrix. This method will enable the question of whether drug doses predict meconium drug concentrations, and whether drug concentrations correlate with onset, magnitude and duration of neonatal abstinence syndrome and other outcomes. These findings could improve clinical care in this vulnerable population. Development of this new biomarker assay for buprenorphine and metabolites in meconium is a critical step for conducting this research. This sensitive and specific method will monitor in utero buprenorphine exposure and determine if correlations exist between buprenorphine exposure and neonatal outcomes. Kacinko, S.L., Shakleya, D.M. and Huestis, M.A. Analytical Chemistry, 80, pp. 246-252, 2008.

MDMA, HMMA, MDA, and HMA Plasma Pharmacokinetics in Humans Following Controlled MDMA Administration
An extended pharmacokinetic analysis of MDMA or Ecstasy and three metabolites, HMMA, MDA and HMA, characterizing Cmax, Tmax, AUC()(), detection windows, t1/2, Vd/F, CL/F, and metabolite ratios for up to 143 h after oral MDMA dosing in young adults was performed. The aim of the study was to document whether non-linearity in MDMA pharmacokinetics occurred at recreational doses, and to characterize MDMA pharmacokinetics in African-Americans and women for the first time. Seventeen female and male participants received placebo, low (1.0 mg/kg), and high (1.6 mg/kg) oral MDMA doses in a double blind, randomized, balanced, within-subject design. Study strengths included metabolite measurements, concentrations after low and high doses, frequent and extended plasma sampling, and residence of participants on a closed research unit with 24-h monitoring to prevent self-administration of MDMA or other drugs. A fully validated 2D-GC/MS method simultaneously quantified MDMA and metabolites. Mean +/- SD maximum plasma concentrations (Cmax) were 162.9+/-39.8 and 171.9+/-79.5 ng/mL for MDMA and HMMA, respectively, after low and 291.8+/-76.5 and 173.5+/-66.3 ng/mL after high MDMA doses, demonstrating non-linear MDMA pharmacokinetics. Mean MDMA volume of distribution was constant for low and high doses; clearance was significantly higher after the low dose. MDMA primarily affects the serotonergic system, acting as an indirect monoaminergic agonist; however, the mechanism(s) by which MDMA causes toxicity are not fully understood. Non-linearity in MDMA dose-concentration relationships and wide variability between subjects at typical recreational doses could contribute to observed MDMA toxicity. Preliminary data on gender differences in drug elimination also were noted. Kolbrich, E.A., Goodwin, R.S., Gorelick, D.A., Hayes, R.J., Stein, E.A., and Huestis, M.A. Presentation, 2008. American Academy of Forensic Sciences (AAFS) Annual Meeting, Washington, DC, February 18-22, 2008.

Office of the Scientific Director

Longitudinal ECG Changes in Cocaine Users During Extended Abstinence
Cocaine causes acute changes in the human electrocardiogram (ECG), such as lengthened QTc interval, which can be a marker for cardiac arrhythmias, but the effects of extended abstinence after chronic cocaine use are not well understood. The authors are not aware of any prospective, longitudinal study addressing this issue. This study recorded weekly ECGs from 25 physically healthy, adult, chronic cocaine users during up to 3 months of monitored abstinence on a closed research ward. The first (baseline) ECG was recorded a mean of 20.5 hours after last cocaine use. The greater the total amount of cocaine used and amount used per day in the 2 weeks prior to ward admission, the longer the baseline QTc interval, consistent with an effect of cocaine use. There was a significant shortening of QTc interval during the first week of abstinence, with no further significant changes thereafter. There were no significant changes in other ECG parameters. These findings suggest that cocaine-associated QTc prolongation (and possibly risk for cardiac arrhythmia) returns toward normal during the first week of cocaine abstinence. Levin K.H., Copersino M.L., Epstein D.H., Boyd S.J., and Gorelick D.A. Drug and Alcohol Dependence, 95, pp. 160-163, 2008.

Molecular Neuropsychiatry Branch

Differential Neurochemical Consequences of an Escalating Dose-binge Regimen Followed by Single-day Multiple-dose Methamphetamine Challenges
Chronic intake of methamphetamine (METH) causes tolerance to its behavioral and subjective effects. To better mimic human patterns of drug abuse, the present study used a rodent model that took into account various facets of human drug administration and measured METH-induced effects on brain monoamine levels. Adult male Sprague-Dawley rats were injected with METH or saline according to an escalating dose schedule for 2 weeks. This was followed by a challenge regimen of either saline or one of two doses of METH (3 x 10 mg/kg every 2 h or 6 x 5 mg/kg given every hour, both given within a single day). Both challenge doses of METH caused significant degrees of depletion of dopamine in the striatum and norepinephrine and serotonin in the striatum, cortex, and hippocampus. Animals pre-treated with METH showed significant attenuation of METH-induced striatal dopamine depletion but not consistent attenuation of norepinephrine and serotonin depletion. Unexpectedly, METH pre-treated animals that received the 3 x 10 mg/kg challenge showed less increases in tympanic temperatures than saline pre-treated rats whereas METH pre-treated animals that received the 6 x 5 mg/kg METH challenge showed comparable increases in temperatures to saline pre-treated rats. Therefore, pre-treatment-induced partial protection against monoamine depletion is probably not because of attenuated METH-induced hyperthermia in those rats. Graham, D.L., Noailles, P.A. and Cadet, J.L. J. Neurochem., 2008, Epub ahead of print.

Transcriptional Responses to Reinforcing Effects of Cocaine in the Rat Hippocampus and Cortex
The psychostimulant effects of cocaine are thought to result from its ability to block dopamine (DA) uptake and increase DA levels in ventral striatum. In addition, cocaine causes biochemical changes in the brain areas involved in learning and memory, including hippocampus and cortex, whose role in drug reinforcement is now being actively investigated. Thus, IRP researchers studied molecular events in the hippocampus and frontal cortex of rats treated with cocaine conditioned place preference (CPP) paradigm. After exposure to cocaine conditioning (cocaine paired), cocaine alone (cocaine non-paired) or saline rats were tested for place conditioning. Cocaine (10 mg/kg) caused increases in time spent in the drug-paired compartment. By using microarray analyses, the authors examined gene expression in the hippocampi and frontal cortices of cocaine-paired rats, cocaine non-paired and saline-treated controls. Their study revealed that 214 transcripts were differentially regulated in the hippocampi of cocaine-paired rats. These include genes that play roles in protein phosphorylation, RNA processing and protein synthesis, ubiquitin-dependent protein degradation and cytoskeleton organization. In contrast, 39 genes were differently expressed in the frontal cortex. These data support the possibility that molecular changes in the hippocampus might participate in the formation and maintenance of memory patterns induced by cocaine in the brain. Differences in the transcriptional responses in the hippocampus and cortex suggest the primary importance of the hippocampus for recent memory processing associated with cocaine-induced CPP. Krasnova, I.N., Li, S.M., Wood, W.H., McCoy, M.T., Prabhu, V.V., Becker, K.G., Katz, J.L. and Cadet, J.L. Genes Brain Behavior 7(2), pp. 193-202, 2008.

EEG of Chronic Marijuana Users During Abstinence: Relationship to Years of Marijuana Use, Cerebral Blood Flow and Thyroid Function
Marijuana abuse is associated with neurological changes including increases in frontal EEG alpha during abstinence. Research is needed to assess to what extent these EEG patterns are indicative of cerebral perfusion deficits. IRP scientists recorded the resting eyes closed EEG of 75 abstinent marijuana users and 33 control subjects. Fifty-six marijuana users used marijuana for less than eight years and 19 used for eight years or more. The EEG evaluation occurred within 72h of admission to an inpatient unit. Fifty-nine marijuana users remained abstinent for a month and were tested twice. Supplemental psychological and physiological data were also collected. Log alpha2 and beta2 power at posterior sites were significantly lower for the marijuana abusers that used eight years or more than the other marijuana abusers and the control subjects. These EEG changes continued for the month of abstinence. The marijuana users who used marijuana for more than eight years, also, had lower heart rates and thyroid function (T4) compared to the other marijuana users and the control subjects. Chronic marijuana use was also associated with reduced EEG power in alpha and beta bands at posterior sites. These reductions in EEG power appear to be related to cerebral perfusion deficits and/or thyroid function in marijuana abusers. These results suggest EEG, cerebral blood flow velocity, cardiovascular and thyroid function alterations in marijuana abusers with an extended period of use. These alterations reflect under arousal in these systems. Herning, R., Better, W. and Cadet, J.L. Clin. Neurophysiol. 119(2), pp. 321-331, 2008.

Development and Plasticity Section, Cellular Neurobiology Research Branch

Postmortem Diagnosis and Toxicological Validation of Illicit Substance Use
The present study examines the diagnostic challenges of identifying ante-mortem illicit substance use in human postmortem cases. Substance use, assessed by clinical case history reviews, structured next-of-kin interviews, by general toxicology of blood, urine and/or brain, and by scalp hair testing, identified 33 cocaine, 29 cannabis, 10 phencyclidine and nine opioid cases. Case history identified 42% cocaine, 76% cannabis, 10% phencyclidine and 33% opioid cases. Next-of-kin interviews identified almost twice as many cocaine and cannabis cases as Medical Examiner (ME) case histories, and were crucial in establishing a detailed lifetime substance use history. Toxicology identified 91% cocaine, 68% cannabis, 80% phencyclidine and 100% opioid cases, with hair testing increasing detection for all drug classes. A cocaine or cannabis use history was corroborated by general toxicology with 50% and 32% sensitivity, respectively, and with 82% and 64% sensitivity by hair testing. Hair testing corroborated a positive general toxicology for cocaine and cannabis with 91% and 100% sensitivity, respectively. Case history corroborated hair toxicology with 38% sensitivity for cocaine and 79% sensitivity for cannabis, suggesting that both case history and general toxicology underestimated cocaine use. Identifying ante-mortem substance use in human postmortem cases are key considerations in case diagnosis and for characterization of disorder-specific changes in neurobiology. The sensitivity and specificity of substance use assessments increased when ME case history was supplemented with structured next-of-kin interviews to establish a detailed lifetime substance use history, while comprehensive toxicology, and hair testing in particular, increased detection of recent illicit substance use. Lehrmann, E., Afanador, Z.R., Deep-Soboslay, A., Gallegos, G., Darwin, W.D., Lowe, R.H., Barnes, A.J., Huestis, M.A., Cadet, J.L., Herman, M.M., Hyde, T.M., Kleinman, J.E., and Freed, W.J. Addiction Biology, 13(1), pp. 105-117, 2008.

An Immortalized Rat Ventral Mesencephalic Cell Line, RTC4, is Protective in a Rodent Model of Stroke
One therapeutic approach to stroke is the transplantation of cells capable of trophic support, reinnervation, and/or regeneration. Previously, IRP researchers have described the use of novel truncated isoforms of SV40 large T antigen to generate unique cell lines from several primary rodent tissue types. Here they describe the generation of two cell lines, RTC3 and RTC4, derived from primary mesencephalic tissue using a fragment of mutant T antigen, T155c (cDNA) expressed from the RSV promoter. Both lines expressed the glial markers vimentin and S100beta, but not the neuronal markers NeuN, MAP2, or beta-III-tubulin. A screen for secreted trophic factors revealed substantially elevated levels of platelet-derived growth factor (PDGF) in RTC4, but not RTC3 cells. When transplanted into rat cortex, RTC4 cells survived for at least 22 days and expressed PDGF. Because PDGF has been reported to reduce ischemic injury, the authors examined the protective functions of RTC4 cells in an animal model of stroke. RTC4 or RTC3 cells, or vehicle, were injected into rat cortex 15-20 min prior to a 60-min middle cerebral artery ligation. Forty-eight hours later, animals were sacrificed and the stroke volume was assessed by triphenyl-tetrazolium chloride (TTC) staining. Compared to vehicle or RTC3 cells, transplanted RTC4 cells significantly reduced stroke volume. Overall, the authors generated a cell line with glial properties that produces PDGF and reduces ischemic injury in a rat model of stroke. Harvey, B.K., Chen, G.J., Schoen, C.J., Lee, C.T., Howard, D.B., Dillon-Carter, O., Coggiano, M., Freed, W.J., Wang, Y., Hoffer, B.J., and Sanchez, J.F. Cell Transplantation, 16(5), pp. 483-491, 2007.

Gene Expression Profile of Neuronal Progenitor Cells Derived from hESCs: Activation of Chromosome 11p15.5 and Comparison to Human Dopaminergic Neurons
IRP scientists initiated differentiation of human embryonic stem cells (hESCs) into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription. Dopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS). Individual genes as well as regions of the genome which were activated were determined. A number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, the authors found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture. The present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons. Freed, W.J., Chen, J., Baeckman, C.M., Schwartz, C.M., Vazin, T., Cai, J., Spivak, C.E., Lupica, C.R., Rao, M.S., and Zeng, X. Public Library of Science One, 3(1), pp. e1422, 2008.

Cellular Pathobiology Section, Cellular Neurobiology Research Branch

An Update on the Development of Drugs for Neuropsychiatric Disorders: Focusing on the Sigma1 Receptor Ligand
The sigma1 receptor is an intracellular molecule that shares no homology with any mammalian proteins. Sigma1 receptors normally localize at the endoplasmic reticulum and regulate a variety of signal transductions including intracellular Ca2+ dynamics and neurotrophic factor signaling. In the brain, sigma1 receptors are known to regulate the activity of diverse ion channels via protein-protein interactions. Accumulated evidence strongly indicates that the activation/upregulation of sigma1 receptors promotes the neuronal differentiation as well as a robust antiapoptotic action. In animals, sigma1 receptor agonists exhibit an antidepressant-like action. Furthermore, the agonists enhanced neuronal survival even though they were administered several hours after a brain ischemia. Thus, primary clinical targets of sigma1 receptor ligands are proposed to include stroke, neurodegenerative disorders and depression. Ligands for the sigma1 receptor may constitute a new class of therapeutic drugs targeting an endoplasmic reticular protein. Hayashi T., and Su, T.P. Expert Opinion on Therapeutic Targets, 12(1), pp. 45-58, 2008.

Sigma-1 Receptor Chaperones at the ER-Mitochondrion Interface Regulate Ca(2+) Signaling and Cell Survival
Communication between the endoplasmic reticulum (ER) and mitochondrion is important for bioenergetics and cellular survival. The ER supplies Ca(2+) directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). IRP scientists found here that the ER protein sigma-1 receptor (Sig-1R), which is implicated in neuroprotection, carcinogenesis, and neuroplasticity, is a Ca(2+)-sensitive and ligand-operated receptor chaperone at MAM. Normally, Sig-1Rs form a complex at MAM with another chaperone, BiP. Upon ER Ca(2+) depletion or via ligand stimulation, Sig-1Rs dissociate from BiP, leading to a prolonged Ca(2+) signaling into mitochondria via IP3Rs. Sig-1Rs can translocate under chronic ER stress. Increasing Sig-1Rs in cells counteracts ER stress response, whereas decreasing them enhances apoptosis. These results reveal that the orchestrated ER chaperone machinery at MAM, by sensing ER Ca(2+) concentrations, regulates ER-mitochondrial interorganellar Ca(2+) signaling and cell survival. Hayashi T., and Su, T.P. Cell, 131(3), pp. 596-610, 2007.

Electrophysiology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

MPTP-Induced Deficits in Striatal Synaptic Plasticity are Prevented by Glial Cell Line-Derived Neurotrophic Factor Expressed Via an Adeno-Associated Viral Vector
This study determined the consequences of dopamine denervation of the striatum on synaptic plasticity and prevention of these changes with gene therapy using an adeno-associated viral vector (AAV) expressing glial cell line-derived neurotrophic factor (GDNF). C57BL6/J mice were injected with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP); long-term depression (LTD) or potentiation (LTP) were measured in vitro. Fast-scan cyclic voltammetry measured electrically released dopamine from a functionally relevant pool in these same striatal slices. After MPTP, dopamine release and uptake were greatly diminished, and LTP and LTD were blocked in the striatal slices. The loss of plasticity resulted directly from the loss of dopamine since its application rescued synaptic plasticity. Striatal GDNF expression via AAV, before MPTP, significantly protected against the loss of dopamine and prevented the blockade of corticostriatal LTP. These data demonstrate that dopamine plays a role in supporting several forms of striatal plasticity and that GDNF expression via AAV prevents the loss of dopamine and striatal plasticity caused by MPTP. The authors propose that impairment of striatal plasticity after dopamine denervation plays a role in the symptomology of Parkinson's disease and that AAV expression of neurotrophic factors represents a tenable approach to protecting against or slowing these neurobiological deficits. Chen, Y.H., Harvey, B.K., Hoffman, A.F., Wang, Y., Chiang, Y.H., and Lupica, C.R. Federation of American Societies for Experimental Biology, 22(1), pp. 261-275, 2008.

The Endocannabinoid Anandamide Inhibits the Function of Alpha4beta2 Nicotinic Acetylcholine Receptors
The effects of the endocannabinoid anandamide (arachidonylethanolamide, AEA) on the function of alpha4beta2 nicotinic acetylcholine receptors (nAChR) stably expressed in SH-EP1 cells were investigated using the whole-cell patch-clamp technique. In the concentration range of 200 nM to 2 microM, AEA significantly reduced the maximal amplitudes and increased the desensitization of acetylcholine (ACh)-induced currents. The effects of AEA could be neither replicated by the exogenous cannabinoid Delta(9)-tetrahydrocannabinol (1 microM) nor reversed by the selective CB1 receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (SR-141716A) (1 microM). The actions of AEA were apparent when applied extracellularly but not during intracellular dialysis. Furthermore, the effects of AEA ACh currents were not altered by the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. The onset and washout of the AEA effects required several minutes (10-30 min), but the latter was significantly decreased in the presence of lipid-free bovine serum albumin (BSA). Moreover, BSA alone increased peak ACh current amplitudes and diminished desensitization rates in naive cells, suggesting a tonic modulation of alpha4beta2 nAChR function by an endogenous AEA-like lipid. Further analysis of AEA effects on alpha4beta2 nAChR-mediated currents, using a two-stage desensitization model, indicated that the first forward rate constant leading to desensitization, k(1), increased nearly 30-fold as a linear function of the AEA concentration. In contrast, the observation that the other three rate constants were unaltered by AEA suggested that AEA raised the energy of the activated state. These results indicate that AEA directly inhibits the function of alpha4beta2 nAChRs in a CB1 receptor-independent manner. Spivak, C.E., Lupica, C.R., and Oz, M. Molecular Pharmacology, 72(4), pp. 1024-1032, 2007.

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Volatile Anesthetics and Endogenous Cannabinoid Anandamide have Additive and Independent Inhibitory Effects on Alpha(7)-Nicotinic Acetylcholine Receptor-Mediated Responses In Xenopus Oocytes
In earlier studies, the volatile anesthetics and the endogenous cannabinoid anandamide have been shown to inhibit the function of alpha(7)-nicotinic acetylcholine receptors. In the present study, interactions between the effects of volatile anesthetics and anandamide on the function of alpha(7)-nicotinic acetylcholine receptors expressed in Xenopus oocytes were investigated using the two-electrode voltage-clamp technique. Anandamide and volatile anesthetics isoflurane and halothane inhibited currents evoked with acetylcholine (100 muM) in a reversible and concentration-dependent manner. Coapplication of anandamide and volatile anesthetics caused a significantly greater inhibition of alpha(7)-nicotinic acetylcholine receptor function than anandamide or volatile anesthetics alone. Analyses of oocytes by matrix-assisted laser desorption/ionization mass spectroscopy indicated that volatile anesthetics did not alter the lipid profile of oocytes. Results of studies with chimeric alpha(7)-nicotinic acetylcholine-5-HT(3) receptors comprised of the N-terminal domain of the alpha(7)-nicotinic acetylcholine receptor and the transmembrane and carboxyl-terminal domains of 5-HT(3) receptors suggest that while isoflurane inhibition of the alpha(7)-nicotinic acetylcholine receptor is likely to involve the N-terminal region of the receptor, the site of action for anandamide involves transmembrane and carboxyl-terminal domains of the receptors. These data indicate that endocannabinoids and isoflurane have additive inhibitory effects on alpha(7)-nicotinic acetylcholine receptor function through allosteric binding sites located on the distinct regions of the receptor. Jackson, S.N., Singhal, S.K., Woods, A.S., Morales, M., Shippenberg, T., Zhang, L., and Oz. M. European Journal of Pharmacology, 582(1-3), pp. 42-51, 2008.

A Snapshot of Tissue Glycerolipids
The lipid membrane is the portal to the cell and its first line of defense against the outside world. Its plasticity, diversity and powers of accommodation in a myriad of environments, mirrored by the varied make up of the cells it protects, are unparalleled. Glycerophospholipids are one of its major components. In cell membranes the extracellular layer is mainly made up of positively charged glycolipids, while the intracellular one's main components are negatively charged. Advances in mass spectrometry have allowed the direct probing of tissues, and thus a direct approach to probing membranes make up was developed. Until recently most studies have focused on proteins. An overview of the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) for the direct analysis of phospholipids in various tissue is presented. Molecular ions corresponding to phosphatidlycholines, sphingomyelin, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols and sulfatides were mapped. Woods, A.S., Wang, H.Y., and Jackson, S.N. Current Pharmaceutical Design, 13(32), pp. 3344-3356, 2007.

A Stargardt Disease-3 Mutation in the Mouse Elovl4 Gene Causes Retinal Deficiency of C32-C36 Acyl Phosphatidylcholines
Stargardt disease-3 (STGD3) is a juvenile dominant macular degeneration caused by mutations in elongase of very long chain fatty acid-4. All identified mutations produce a truncated protein which lacks a motif for protein retention in endoplasmic reticulum, the site of fatty acid synthesis. In these studies of Stgd3-knockin mice carrying a human pathogenic mutation, IRP scientists examined two potential pathogenic mechanisms: truncated protein-induced cellular stress and lipid product deficiency. Analysis of mutant retinas detected no cellular stress but demonstrated selective deficiency of C32-C36 acyl phosphatidylcholines. The authors conclude that this deficit leads to the human STGD3 pathology. McMahon, A., Jackson, S.N., Woods, A.S., and Kedzierski, W. FEBS Letters, 581(28), pp. 5459-5463, 2007.

Adenosine Receptor Heteromers and Their Integrative Role in Striatal Function
By analyzing the functional role of adenosine receptor heteromers, IRP investigators review a series of new concepts that should modify our classical views of neurotransmission in the central nervous system (CNS). Neurotransmitter receptors cannot be considered as single functional units anymore. Heteromerization of neurotransmitter receptors confers functional entities that possess different biochemical characteristics with respect to the individual components of the heteromer. Some of these characteristics can be used as a "biochemical fingerprint" to identify neurotransmitter receptor heteromers in the CNS. This is exemplified by changes in binding characteristics that are dependent on coactivation of the receptor units of different adenosine receptor heteromers. Neurotransmitter receptor heteromers can act as "processors" of computations that modulate cell signaling, sometimes critically involved in the control of pre- and postsynaptic neurotransmission. For instance, the adenosine A1-A2A receptor heteromer acts as a concentration-dependent switch that controls striatal glutamatergic neurotransmission. Neurotransmitter receptor heteromers play a particularly important integrative role in the "local module" (the minimal portion of one or more neurons and/or one or more glial cells that operates as an independent integrative unit), where they act as processors mediating computations that convey information from diverse volume-transmitted signals. For instance, the adenosine A2A-dopamine D2 receptor heteromers work as integrators of two different neurotransmitters in the striatal spine module. Ferre, S., Ciruela, F., Quiroz, C., Lujan, R., Popoli, P., Cunha, R.A., Agnati, L.F., Fuxe, K., Woods, A.S., Lluis, C., and Franco, R. Scientific World Journal, 7, pp. 74-85, 2007.

Anatomy and Cell Biology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Hippocampal Interneurons Co-Express Transcripts Encoding the Alpha7 nicotinic Receptor Subunit and the Cannabinoid Receptor 1
The notion of functional interactions between the alpha7 nicotinic acetylcholine (alpha7 nACh) and the cannabinoid systems is emerging from recent in vitro and in vivo studies. Both the alpha7 nACh receptor and the cannabinoid receptor 1 (CB1) are highly expressed in the hippocampus. To begin addressing possible anatomical interactions between the alpha7 nACh and the cannabinoid systems in the rat hippocampus, IRP scientists investigated the distribution of neurons expressing alpha7 nACh mRNA in relation to those containing CB1 mRNA. By in situ hybridization the authors found that the alpha7 nACh mRNA is diffusely expressed in principal neurons and is highly expressed in a subset of interneurons. They observed that the pattern of distribution of hippocampal interneurons co-expressing transcripts encoding alpha7 nACh and glutamate decarboxylase (GAD; synthesizing enzyme of GABA) closely resembles the one displayed by interneurons expressing CB1 mRNA. By double in situ hybridization the authors established that the majority of hippocampal interneurons expressing alpha7 nACh mRNA have high levels of CB1 mRNA. As CB1 interneurons contain cholecystokinin (CCK), they investigated the degree of cellular co-expression of alpha7 nACh mRNA and CCK, and found that the cellular co-existence of alpha7 nACh and CCK varies within the different layers of the hippocampus. In summary, the authors established that most of the hippocampal alpha7 nACh expressing interneurons are endowed with CB1 mRNA. They found that these alpha7 nACh/CB1 interneurons are the major subpopulation of hippocampal interneurons expressing CB1 mRNA. The alpha7 nACh expressing interneurons represent half of the detected population of CCK containing neurons in the hippocampus. Since it is well established that the vast majority of hippocampal interneurons expressing CB1 mRNA have 5-HT type 3 (5-HT(3)) receptors, we conclude that these hippocampal alpha7 nACh/5HT(3)/CB1/CCK interneurons correspond to those previously postulated to relay inputs from diverse cortical and subcortical regions about emotional, motivational, and physiological states. Morales, M., Hein, K., and Vogel, Z. Neuroscience, 152(1), pp. 70-81, 2008.

Synapses Between Corticotropin-Releasing Factor-Containing Axon Terminals and Dopaminergic Neurons in the Ventral Tegmental Area are Predominantly Glutamatergic
Interactions between stress and the mesocorticolimbic dopamine (DA) system have been suggested from behavioral and electrophysiological studies. Because corticotropin-releasing factor (CRF) plays a role in stress responses, IRP scientists investigated possible interactions between neurons containing CRF and those producing DA in the ventral tegmental area (VTA). They first investigated the cellular distribution of CRF in the VTA by immunolabeling VTA sections with anti-CRF antibodies and analyzing these sections by electron microscopy. They found CRF immunoreactivity present mostly in axon terminals establishing either symmetric or asymmetric synapses with VTA dendrites. They established that nearly all CRF asymmetric synapses are glutamatergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed the vesicular glutamate transporter 2, and that the majority of CRF symmetric synapses are GABAergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed glutamic acid decarboxylase, findings that are of functional importance. The authors then looked for synaptic interactions between CRF- and DA-containing neurons, by using antibodies against CRF and tyrosine hydroxylase (TH; a marker for DA neurons). They found that most synapses between CRF-immunoreactive axon terminals and TH neurons are asymmetric (in the majority likely to be glutamatergic) and suggest that glutamatergic neurons containing CRF may be part of the neuronal circuitry that mediates stress responses involving the mesocorticolimbic DA system. The presence of CRF synapses in the VTA offers a mechanism for interactions between the stress-associated neuropeptide CRF and the mesocorticolimbic DA system. Tagliaferro, P., and Morales, M. The Journal of Comparative Neurology, 506 (4), pp. 616-626, 2008.

Clinical Psychopharmacology Section, Chemical Biology Research Branch

Chronic Fenfluramine Administration Increases Plasma Serotonin (5-HT) to Non-Toxic Levels
Large elevations in blood serotonin (5-HT) can produce valvular heart disease in humans and laboratory animals. Accordingly, one prevailing hypothesis (i.e., the "5-HT hypothesis") suggests 5-HT transporter substrates like fenfluramine increase the risk for valvular heart disease by elevating plasma 5-HT, secondary to the release of 5-HT from platelets. The main purpose of this study was to determine if chronic administration of fenfluramine increases plasma 5-HT to concentrations that are associated with the development of valvular heart disease. To the best of the authors' knowledge, this is the first study to address this issue using an in vivo microdialysis method that measures plasma 5-HT in non-hypoxic rats. The IRP scientists examined the effects of chronic (+/-)-fenfluramine and fluoxetine on plasma levels of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in blood samples from conscious catheterized rats. Plasma indoles were measured by HPLC-ECD in dialysates of whole blood. Baseline plasma 5-HT was < 1.0 nM. Chronic fenfluramine (14-day minipump infusion) produced small increases in baseline plasma 5-HT (~2-to-4-fold), while chronic fluoxetine had no effect. Chronic fenfluramine and fluoxetine markedly decreased whole blood 5-HT, and reduced the ability of acute fenfluramine to evoke 5-HT release. Elevations in baseline plasma 5-HT produced by chronic fenfluramine are far below M levels necessary to produce valvular heart disease. Furthermore, chronic fenfluramine reduces the ability of acute fenfluramine to increase plasma 5-HT, suggesting the "5-HT hypothesis" can not explain the increased risk of valvular heart disease in patients treated with fenfluramine. Zolkowska, D., Baumann M.H., and Rothman R.B. J. Pharmacol. Exp. Ther., 324, pp. 791-797, 2008.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

Blockade of THC-seeking Behavior and Relapse in Monkeys by the Cannabinoid CB(1)-receptor Antagonist Rimonabant
Accumulating evidence suggests the endocannabinoid system modulates environmental cues' ability to induce seeking of drugs, including nicotine and alcohol. However, little attention has been directed toward extending these advances to the growing problem of cannabis use disorders. Therefore, IRP researchers studied intravenous self-administration of delta(9)-tetrahydrocannabinol (THC), the main psychoactive constituent of marijuana, using a second-order schedule of drug seeking. Squirrel monkeys' lever responses produced only a brief cue light until the end of the session, when the final response delivered THC along with the cue. When a reinstatement procedure was used to model relapse following a period of abstinence, THC-seeking behavior was robustly reinstated by the cue or by pre-session administration of THC, other cannabinoid agonists, or morphine, but not cocaine. The cannabinoid antagonist rimonabant blocked cue-induced drug seeking, THC-induced drug seeking, and the direct reinforcing effects of THC. Thus, rimonabant and related medications might be effective as treatments for cannabis use disorders. Justinova, Z., Munzar, P., Panlilio, L. V., Yasar, S., Redhi, G. H., Tanda, G. and Goldberg, S. R. Neuropharmacology, February 27, 2008, Epubmed ahead of print.

Blocking Striatal Adenosine A2A Receptors: A New Strategy for Basal Ganglia Disorders
Adenosine A(2A) receptors are highly concentrated in the striatum, where they play an important modulatory role of glutamatergic transmission to the GABAergic enkephalinergic neuron, which function is particularly compromised in Parkinson's disease and in the early stages of Huntington's disease. An important amount of preclinical data suggested the possible application of A(2A) receptor antagonists in Parkinson's disease, particularly as adjuvant therapy to the currently used dopaminergic agonists. Several A(2A) receptor antagonists are currently in clinical trials in patients with Parkinson's disease and initial results have been promising. In recent years, many pharmaceutical companies have started programs to develop A(2A) antagonists for Parkinson's disease and for other indications, such as neurodegenerative diseases in general, depression and restless legs syndrome. Antagonists with high A(2A) receptor affinity and selectivity have been developed from various chemical classes of compounds, including xanthenes, adenines and other amino-substituted heterocyclic compounds. Novel structures include benzothiazole and thiazolopyridine derivatives. The present review describes properties of standard A(2A) receptor antagonists including those in clinical development. Furthermore, the different chemical classes of A(2A) receptor antagonists that have been described in the literature, including recent patent literature, will be presented. Muller, C.E. and Ferre, S. Recent Patents CNS Drug Discoveries, 2, pp. 1-21, 2007.

An Update on the Mechanisms of the Psychostimulant Effects of Caffeine
There has been a long debate about the predominant involvement of the different adenosine receptor subtypes and the preferential role of pre- versus post-synaptic mechanisms in the psychostimulant effects of the adenosine receptor antagonist caffeine. Both striatal A(1) and A(2A) receptors are involved in the motor-activating and probably reinforcing effects of caffeine, although they play a different role under conditions of acute or chronic caffeine administration. The present review emphasizes the key integrative role of adenosine and adenosine receptor heteromers in the computation of information at the level of the striatal spine module (SSM). This local module is mostly represented by the dendritic spine of the medium spiny neuron with its glutamatergic and dopaminergic synapses and astroglial processes that wrap the glutamatergic synapse. In the SSM, adenosine acts both pre- and post-synaptically through multiple mechanisms, which depend on heteromerization of A(1) and A(2A) receptors among themselves and with D(1) and D(2) receptors, respectively. A critical aspect of the mechanisms of the psychostimulant effects of caffeine is its ability to release the pre- and post-synaptic brakes that adenosine imposes on dopaminergic neurotransmission by acting on different adenosine receptor heteromers localized in different elements of the SSM. Ferre, S. Journal of Neurochemistry, January 17, 2008, Epubmed ahead of print.

Topiramate Does Not Alter Nicotine or Cocaine Discrimination in Rats
Two groups of rats trained to discriminate the administration of either 0.4 mg/kg nicotine or 10 mg/kg cocaine from that of saline, under a fixed-ratio 10 schedule of food delivery. Topiramate (1-60 mg/kg, intraperitoneal) did not produce any nicotine-like or cocaine-like discriminative effects by itself and did not produce any shift in the dose-response curves for nicotine or cocaine discrimination. Furthermore, topiramate, given either alone or in combination with nicotine or cocaine, did not depress rates of responding. These experiments indicate that topiramate does not enhance or reduce the ability of rats to discriminate the effects of nicotine or cocaine. Le Foll, B., Justinova, Z., Wertheim, C.E., Barnes, C. and Goldberg, S.R. Behavioral Pharmacology, 19, pp. 13-20, 2008.

Light Resonance Energy Transfer-based Methods in the Study of G protein-coupled Receptor Oligomerization
Oligomerization Since most of the functions in cells are mediated by multimeric protein complexes, the determination of protein-protein interactions is an important step in the study of cellular mechanisms. Traditionally, after screening for possible target interactors by means of a yeast two-hybrid screen, several methods are used to validate the initial result before carrying out functional experiments. Nowadays, non-invasive fluorescence-based methods like Bioluminescence Resonance Energy Transfer (BRET) and Fluorescence Resonance Energy Transfer (FRET) are widely used in the study of protein-protein interactions in living cells. In the present review, the authors address the individual strengths and weaknesses of both RET approaches, providing information on their possible future use in the study of G protein-coupled receptor oligomerization. Gandia, J., Lluis, C., Ferre, S., Franco, R. and Ciruela, F. Bioessays, 30, pp. 82-89, 2008.

Nicotine Psychopharmacology Unit, Treatment Section, Clinical Pharmacology and Therapeutics Research Branch

Nicotine Enhances Mood and Cognition in Smokers
The discovery of the role of nicotinic receptors in attention and memory has led to the testing of nicotinic analogs as cognitive enhancing agents in patient populations. Empirical information about nicotine's ability to enhance elements of attention and memory in normal individuals might guide development of therapeutic uses of nicotine in cognitively-impaired populations. The purpose of this study was to determine the effect of nicotine on continuous attention, working memory, and computational processing in tobacco-deprived and nondeprived smokers. A total of 28 smokers (14 men, 14 women) participated in a double-blind, placebo-controlled, within-subject study, in which they were overnight (12 h) tobacco deprived at one session and smoked ad libitum before the other session. At each session, participants received 0, 1, and 2 mg nicotine via nasal spray in random order at 90-min intervals. Before and after each dose, a battery of cognitive, subjective, and physiological measures was administered, and blood samples were taken for plasma nicotine concentration. Overnight tobacco deprivation resulted in impaired functioning on all cognitive tests and increased self-reports of tobacco craving and negative mood; nicotine normalized these deficits. In the nondeprived condition, nicotine enhanced performance on the Continuous Performance Test and Arithmetic Test in a dose-related manner, but had no effect on working memory. In general, women were more sensitive than men to the subjective effects of nicotine. These results provide an unequivocal determination that nicotine enhanced attentional and computational abilities in nondeprived smokers and suggest these cognitive domains as substrates for novel therapeutic indications. Myers, C.S., Taylor, R.C., Moolchan, E.T., and Heishman, S.J. Neuropsychopharmacology, 33, pp. 588-598, 2008.