Research Findings - Intramural Research

Development and Plasticity Section, Cellular Neurobiology Research Branch

Protective Effects of Delta9-Tetrahydrocannabinol against N-methyl-D-Aspartate-Induced AF5 Cell Death The neuroprotective effects of delta9-tetrahydrocannabinol (THC) were examined using an in vitro model in which the AF5 CNS cell line was exposed to toxic levels of N-methyl-D-aspartate (NMDA), an agonist of the NMDA glutamate receptor. NMDA toxicity was reduced by THC, but not by the more specific cannabinoid receptor agonist, WIN55,212-2. Addition of dibutyryl cAMP (dbcAMP) to the culture medium did not alter the neuroprotective effect of THC and did not unmask a neuroprotective effect of WIN55,212-2. The cannabinoid antagonist SR141716A did not inhibit the neuroprotection induced by THC or alter the response to WIN55,212-2, even in the presence of dbcAMP, indicating that the neuroprotective effect of THC was cannabinoid receptor-independent. On the other hand, both THC and WIN55,212-2 produced cellular toxicology at higher dosages, an effect which was blocked in part by SR141716A. Capsaicin, an antioxidant and vanilloid receptor agonist, also produced a protective effect against NMDA toxicology. The protective effect of capsaicin was blocked by co-application of ruthenium red, but was not blocked by the specific vanilloid receptor antagonist capsazepine, and the transient receptor potential vanilloid type 1 (TRPV1) and ANKTM1 transcripts were not detected in AF5 cells. Thus, the neuroprotective effects of THC and capsaicin did not appear to be mediated by TRP ion channel family receptors. The antioxidant alpha-tocopherol prevented neurotoxicity in a dose-dependent manner. Therefore, THC may function as an antioxidant to increase cell survival in NMDA-induced neurotoxicity in the AF5 cell model, while higher dosages produce toxicity mediated by CB1 receptor stimulation. Chen, J., Lee, C.T., Errico, S., Deng, X. Cadet, J.L., and Freed, W.J. Brain Research Molecular Brain Research, 134, pp. 215-225, 2005.

Development of a Focused Microarray to Assess Human Embryonic Stem Cell Differentiation Human embryonic stem cells (hESC) must be differentiated before clinical use. In addition, the extent of contamination of undifferentiated cells and the efficiency of differentiation must also be assessed prior to clinical application. In this manuscript, IRP investigators describe the development of a focused microarray that may be used to discriminate between hESC and their differentiated progeny. This array contains 755 genes including embryonic stem cell markers as well as markers of differentiation into neural, mesodermal, and endodermal phenotypes. In addition, authors have included candidate genes belonging to families of cytokines, chemokines, receptors, signaling pathways, and homeodomain proteins that are likely to be important in the process of differentiation. Testing and validation of the focused array was performed using RNA from hESC, human embryoid body (hEB) outgrowths, and a human embryonal carcinoma (hEC) cell line. Authors have compared gene expression with negative background, GAPDH, _-actin positive controls, and human universal RNA (hURNA), showing that such an array can rapidly distinguish between undifferentiated and differentiated hESC-derived cell populations. Authors expect that the described array will be extremely useful in evaluating the extent of differentiation and the state of the hESC-derived population utilized for therapeutic purposes. Yang, A.X., Mejido, J., Luo, Y., Zeng, X., Schwartz, C., Wu, T., Thies, R.S., Bhattacharya, B., Han, J., Freed, B., Rao, M., and Puri, R.K. Stem Cells and Development, 14, pp. 270-284, 2005.

Cellular Pathobiology Unit, Development and Plasticity Section, Cellular Neurobiology Research Branch

The Potential Role of Sigma-1 Receptors in Lipid Transport and Lipid Raft Reconstitution in the Brain: Implication for Drug Abuse The brain is highly enriched in lipids. However, the molecular biological roles of lipids in the brain have been largely unexplored. Although, in the 1990s, several studies demonstrated the roles of lipids in a variety of neuronal functions and certain neurological diseases, the involvement of lipids in drug dependence, if any, is almost totally unknown. Sigma-1 receptors are brain-enriched proteins that interact with psychostimulants such as cocaine and methamphetamine. Sigma-1 receptors possess a putative sterol-binding pocket and are predominantly expressed on the endoplasmic reticulum (ER) where most lipids and their precursors are synthesized. Sigma-1 receptors are involved in drug-seeking behaviors and in psychostimulant-induced behavioral sensitization. Recent studies demonstrated that sigma-1 receptors target the lipid-storing subcompartments of the ER and are colocalized with cholesterol and neutral lipids. Sigma-1 receptors form detergent-insoluble lipid microdomains (lipid rafts) on the ER subcompartments and can translocate on the ER when stimulated. Upregulation of sigma-1 receptors affect the levels of plasma membrane lipid rafts by changing the lipid components therein. The membrane reconstitution thus induced by sigma-1 receptors in turn affects functions of proteins residing in plasma membrane lipid rafts including tropic factor receptors and tyrosine kinases. Specifically, IRP scientists recently found that sigma-1 receptors modulate MAP kinase activation induced by tropic factors, neuritegenesis and oligodendrocyte differentiation-all related to lipid raft reconstitution. Sigma-1 receptors may thus play a role in psychostimulant-induced long-lasting morphological changes in the brain via the capacity of sigma-1 receptors in regulating ER lipid transport and the resultant plasma membrane lipid raft reconstitution. Hayashi, T, and Su, T.P. Life Sciences, 77, pp. 1612-1624, 2005.

Picomolar Concentrations of Hibernation Induction Delta Opioid Peptide [D-Ala2,D-Leu5]Enkephalin Increase the Nerve Growth Factor in NG-108 Cells The delta opioid peptide [D-Ala2,D-Leu5]enkephalin (DADLE) has been shown to be a neuroprotective agent via mechanisms that are not totally understood. IRP investigators previously demonstrated that the i.p. injection of DADLE in mice causes an increase of nerve growth factor (NGF) in the brain. To further clarify the NGF-increasing action of DADLE, authors examined here the NGF-increasing effect of DADLE in vitro, using cultured NG-108 cells. DADLE dose-dependently increases the immunoreactive level of NGF in NG-108 cells in a bell-shape manner, with the effective DADLE concentrations in the picomolar range (0.01-100 pM). Also, DADLE at 1 pM selectively increases c-Jun and c-Fos, but not c-Rel. These results indicate that DADLE is one of the most potent agents in increasing the NGF in the biological system and that this action of DADLE involves selective increases of c-Jun and c-Fos, transcription factors that promote the NGF expression. Tsai, S.Y., Hayashi, T., and Su, T.P. Synapse, 57, pp. 179-181, 2005.

Electrophysiology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Additive Effects of Endogenous Cannabinoid Anandamide and Ethanol on Alpha7-Nicotinic Acetylcholine Receptor-Mediated Responses in Xenopus Oocytes The interaction between the effects of the endogenous cannabinoid receptor agonist anandamide and ethanol on the function of homomeric alpha7-nicotinic acetylcholine (nACh) receptors expressed in Xenopus oocytes were investigated using the two-electrode voltage-clamp technique. Anandamide and ethanol reversibly inhibited currents evoked with ACh (100 iM) in a concentration-dependent manner. Coapplication of anandamide and ethanol caused a significantly greater inhibition of alpha7- nACh receptor function than anandamide or ethanol alone. The IC50 value of 238 +/- 34 nM for anandamide inhibition decreased significantly to 104 +/- 23 nM in the presence of 30 mM ethanol. The inhibition of alpha7-mediated currents by coapplication of anandamide and ethanol was not altered by phenylmethylsulfonyl fluoride, an inhibitor of anandamide hydrolyzing enzyme, or AM404, an anandamide-transport inhibitor. Analysis of oocytes by matrix-assisted laser desorption/ionization technique indicated that ethanol treatment did not alter the lipid profile of oocytes and there is negligible, if any, anandamide present in these cells. Results of studies with chimeric alpha7-nACh-5-HT3 receptors comprised of the amino-terminal domain of the alpha7-nACh receptor and the transmembrane and carboxyl-terminal domains of 5-HT3 receptors suggest that while ethanol inhibition of the alpha7-nAChR is likely to involve the N-terminal region of the receptor, the site of action for anandamide is located in the transmembrane and carboxyl-terminal domains of the receptors. These data indicate that endocannabinoids and ethanol potentiate each other's inhibitory effects on alpha7-nACh receptor function through distinct regions of the receptor. Oz, M., Jackson, S., Woods, A., Morales, M., and Zhang, L. Journal of Pharmacology Experimental Therapeutics, 313, pp. 1272-1280, 2005.

Presynaptic Angiotensin II AT1 Receptors Enhance Inhibitory and Excitatory Synaptic Neurotransmission to Motoneurons and other Ventral Horn Neurons in Neonatal Rat Spinal Cord In neonatal spinal cord, IRP scientists previously reported that exogenous angiotensin II (ANG II) acts at postsynaptic AT1 receptors to depolarize neonatal rat spinal ventral horn neurons in vitro. This study evaluated an associated increase in synaptic activity. Patch clamp recordings revealed that 38/81 thoracolumbar (T7-L5) motoneurons responded to bath applied ANG II (0.3-1 microM; 30 s) with a prolonged (5-10 min) and reversible increase in spontaneous postsynaptic activity, selectively blockable with Losartan (n = 5) but not PD123319 (n = 5). ANG-II-induced events included both spontaneous inhibitory (IPSCs; n = 6) and excitatory postsynaptic currents (EPSCs; n = 5). While most ANG induced events were tetrodotoxin-sensitive, ANG induced a significant tetrodotoxin-resistant increase in frequency but not amplitude of miniature IPSCs (n = 7/13 cells) and EPSCs (n = 2/7 cells). In 35/77 unidentified neurons, ANG II also induced a tetrodotoxin-sensitive and prolonged increase in their spontaneous synaptic activity that featured both IPSCs (n = 5) and EPSCs (n = 4) when tested in the presence of selective amino acid receptor antagonists. When tested in the presence of tetrodotoxin, ANG II was noted to induce a significant increase in the frequency but not the amplitude of mIPSCs (n = 9) and mEPSCs (n = 8). ANG also increased spontaneous motor activity from isolated mouse lumbar ventral rootlets. Collectively, these observations support the existence of a wide pre- and postsynaptic distribution of ANG II AT1 receptors in neonatal ventral spinal cord that are capable of influencing both inhibitory and excitatory neurotransmission. Oz, M., Yang, K.H., O'Donovan, M.J., and Renaud, L.P. Journal of Neurophysiology, 94, pp. 1405-1412, 2005.

Endocannabinoid Release from Midbrain Dopamine Neurons: A Potential Substrate for Cannabinoid Receptor Antagonist Treatment of Addiction Substantial evidence suggests that all commonly abused drugs act upon the brain reward circuitry to ultimately increase extracellular concentrations of the neurotransmitter dopamine in the nucleus accumbens and other forebrain areas. Many drugs of abuse appear to increase dopamine levels by dramatically increasing the firing and bursting rates of dopamine neurons located in the ventral mesencephalon. Recent clinical evidence in humans and behavioral evidence in animals indicate that cannabinoid receptor antagonists such as SR141716A (Rimonabant) can reduce the self-administration of, and craving for, several commonly addictive drugs. However, the mechanism of this potentially beneficial effect has not yet been identified. IRP researchers propose, on the basis of recent studies in their laboratory and others, that these antagonists may act by blocking the effects of endogenously released cannabinoid molecules (endocannabinoids) that are released in an activity- and calcium-dependent manner from mesencephalic dopamine neurons. It is hypothesized that, through the antagonism of cannabinoid CB1 receptors located on inhibitory and excitatory axon terminals targeting the midbrain dopamine neurons, the effects of the endocannabinoids are occluded. The data from these studies therefore suggest that the endocannabinoid system and the CB1 receptors located in the ventral mesencephalon may play an important role in regulating drug reward processes, and that this substrate is recruited whenever dopamine neuron activity is increased. Lupica, C.R. and Riegel, A.C. Neuropharmacology, 48, pp. 1105-1116, 2005.

Identification of CRF Binding Protein Expression In VTA: A Link Between Reward and Stress Systems? Corticotropin releasing factor (CRF) interacts with specific receptors and a binding protein (CRF-BP). CRF-BP is produced in peripheral tissue and brain. While a role of peripheral CRF-BP in lowering free circulating CRF levels is well established, the effect of CRF-BP in brain is less clear. Ungless et al., (Neuron, 2003) showed that application of CRF-BP to in vitro preparations of midbrain slices from mice was required for CRF to potentiate N-methyl-D-aspartate receptor mediated synaptic transmission in dopamine (DA) neurons in the ventral tegmental area (VTA). Whereas these studies highlight the participation of CRF-BP on CRF and glutamatergic transmission on DA neurons in VTA, the neuronal distribution of CRF-BP in VTA is unknown. IRP investigators detected CRF-BP immunoreactivity in Western blots prepared from VTA homogenates. Consistent with this result, CRF-BP immunoreactive cells and varicose fibers were observed in VTA of rat brain sections. To determine possible origin of CRF-BP detected in VTA, authors examined whether CRF-BP mRNA was found in neurons located in VTA or in cells projecting to this region. In situ hybridization analysis demonstrated CRF-BP mRNA in VTA cells but not in those of the substantia nigra compacta or reticulata. By combining in situ hybridization (to identify cellular expression of CRF-BP mRNA) and tract tracing immunohistochemistry (to detect projecting neurons to VTA), authors found no neurons innervating VTA that expressed CRF-BP. This suggests that CRF-BP detected within the VTA is synthesized by VTA neurons. By double in situ hybridization histochemistry authors demonstrated that in VTA, CRF-BP expression was mainly present in a subset of DAergic neurons and in some GABAergic neurons. Based on these anatomical observations and previous in vitro electrophysiological studies (Ungless et al., 2003), authors suggest that CRF-BP synthesized in VTA, mainly by DAergic neurons, is well positioned to affect the reward system in response to local release of CRF. Morales M., Roach E., and Diaz Ruiz O. Poster. Anatomy of the Soul, Ameland, The Nethetlands, May 19-25, 2005.

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Direct Profiling of Lipid Distribution in Brain Tissue Using MALDI-TOFMS Recent developments in mass spectrometry have permitted direct analysis of biomolecules in tissue. However, most studies have focused on proteins with emphasis on biomarker discovery. In the present work, matrix-assisted laser desorption/ionization mass spectrometry was used for the direct analysis of lipids in rat cerebellum. The lipid bilayer role as a storage depot for small organic molecules such as therapeutic drugs and pollutants such as DDT, as well as the ability to compare lipid profiles in healthy and diseased animal models, are a few of the many reasons why the direct probing of tissue to determine the qualitative and possibly quantitative lipid content could be a very useful tool. Molecular ions corresponding to cholesterol, phosphatidylcholines, sphingomyelins, and phosphatidylethanolamines were recorded in positive ion mode, while ones corresponding to phosphatidylinositols, sulfatides, and gangliosides were recorded in negative ion mode. Overall, representatives from all major categories of brain lipids including cholesterol, 15 phospholipid species (9 phosphatidylcholines, 1 sphingomyelin, 3 phosphatidylethanol-amines, 2 phosphatidylinositols), 10 sulfatides (5 hydroxylated species and 5 nonhydroxylated species), and 7 species of gangliosides were detected. Jackson, S.N., Wang, H.Y., and Woods, A.S. Analytical Chemistry, 77, pp. 4523-4527, 2005.

Amazing Stability of the Arginine-Phosphate Electrostatic Interaction Electrostatic interactions between a basic epitope containing adjacent arginine residues and an acidic epitope containing a phosphorylated serine are involved in receptor heteromerization. In the present study, IRP researchers demonstrate that this arginine-phosphate electrostatic interaction possesses a "covalent-like" stability. Hence, these bonds can withstand fragmentation by mass spectrometric collision-induced dissociation at energies similar to those that fragment covalent bonds and they demonstrate an extremely low dissociation constant by plasmon resonance. The present work also highlights the importance of phosphorylation-dephosphorylation events in the modulation of this electrostatic attraction. Phosphorylation of the acidic epitope, a casein kinase one consensus site, makes it available to interact with the basic epitope. On the other hand, phosphorylation of serine and/or threonine residues adjacent to the basic epitope, a protein kinase A consensus site, slows down the attraction between the epitopes. Although analyzed here in the frame of receptor heteromerization, the arginine-phosphate electrostatic interaction most likely represents a general mechanism in protein-protein interactions. Woods, A.S., and Ferre, S. Journal of Proteome Research, 4, pp. 1397-1402, 2005.

Role of Electrostatic Interaction in Receptor-Receptor Heteromerization Using pull-down and mass spectrometry experiments, IRP scientists have previously demonstrated that adenosine A2A-dopamine D2 receptor-receptor heteromerization depends on an electrostatic interaction between an Arg-rich epitope from the third intracellular loop of the D2 receptor (217RRRRKR222) and two adjacent Asp residues (DD401-402) or a phosphorylated Ser (pS374) residue in the carboxyl terminus of the A2A receptor. It has been demonstrated recently that a specific region in the carboxyl terminus of the dopamine D1 receptor (L387-L416) and a specific region in the carboxyl terminus of the NR1-1 subunit of the NMDA receptor (E834-S938) are involved in D1-NMDA receptor-receptor heteromerization. Careful perusal of these interacting regions shows the presence of a phosphorylated serine (pS397) and adjacent glutamates (EE404-405) in the D1 receptor, whereas NR1-1 contains three adjacent Arg residues (RRR893-896). These epitopes are highly conserved in all species, a sign that the epitopes are likely to be involved in a physiologically significant activity. If similar epitopes are found to be involved in the formation of receptor heteromers other than A2A-D2 and D1-NMDA, the epitope-epitope electrostatic interaction might represent an important general mechanism underlying receptor- receptor interactions. Woods, A.S., Ciruela, F., Fuxe, K., Agnati, L.F., Lluis, C., Franco, R., and Ferre, S. Journal of Molecular Neuroscience, 26, pp. 125-132, 2005.

How Proteins Come Together in the Plasma Membrane and Function in Macromolecular Assemblies: Focus on Receptor Mosaics Some theoretical aspects on structure and function of proteins have been discussed previously. Proteins form multimeric complexes, as they have the capability of binding other proteins (Lego property) resulting in multimeric complexes capable of emergent functions. Multimeric proteins might have either a genomic or a postgenomic origin. Proteins spanning the plasma membrane have been analyzed by considering the effects of the microenvironment in which the protein is embedded. In particular, the different effects of the hydrophilic (extracellular and intracellular) versus the lipophilic (intramembrane) environment have been considered. These aspects have been discussed in the framework of membrane microdomains, in particular, the so-called rafts. In alpha-helix proteins the individual peptide dipoles align to produce a macrodipole crossing the entire membrane. This macrodipole has its positive (extracellular) pole at the N-terminal end of the helix and its negative (intracellular) pole at the C-terminal end. This arrangement has been analyzed in the framework of the counter-ion atmosphere, that is, the formation of a cloud of small ions bearing an opposite charge. Excitable cells reverse their resting potential during the all-or-none action potentials. Hence, the extracellular side of the plasma membrane becomes negative with respect to the intracellular side. This change of polarization affects also the direction and magnitude of the alpha-helix dipole in view of the fact that there is a displacement of the counter ions. The oscillation in the intensity of the dipole caused by the action potentials opens the possibility of an interaction among dipoles by electromagnetic waves. Agnati, L.F., Guidolin, D., Genedani, S., Ferre, S., Bigiani, A., Woods, A.S., and Fuxe, K. Journal of Molecular Neuroscience, 26, pp. 133-154, 2005.

Computer-Assisted Image Analysis of Caveolin-1 Involvement in the Internalization Process of Adenosine A2A-Dopamine D2 Receptor Heterodimers A functional aspect of horizontal molecular networks has been investigated experimentally, namely the heteromerization between adenosine A2A and dopamine D2 receptors and the possible role of caveolin-1 in the cotrafficking of these molecular complexes. This study has been carried out by means of computer-assisted image analysis procedure of laser images of membrane immunoreactivity of caveolin-1, A2A, D1, and D2 receptors obtained in two clones of Chinese hamster ovary cells one transfected with A2A and dopamine D1 receptors and the other one with A2A and D2 receptors. Cells were treated for 3 h with 10 microM D1 receptor agonist SKF 38393, 50 microM D2-D3 receptor agonist quinpirole, and 200 nM A2A receptor agonist CGS 21680. In A2A-D1-cotransfected cells, caveolin-1 was found to colocalize with both A2A and D1 receptors and treatment with SKF 38393 induced internalization of caveolin-1 and D1 receptors, with a preferential internalization of D1 receptors colocalized with caveolin-1. In A2A-D2-cotransfected cells, caveolin-1 was found to colocalize with both A2A and D2 receptors and either CGS 21680 or quinpirole treatment induced internalization of caveolin-1 and A2A and D2 receptors, with a preferential internalization of A2A and D2 receptors colocalized with caveolin-1. The results suggest that A2A and D2 receptors and caveolin-1 likely interact forming a macrocomplex that internalizes upon agonist treatment. These observations are discussed in the frame of receptor oligomerization and of the possible functional role of caveolin-1 in the process of co-internalization and, hence, in controlling the permanence of receptors at the plasma membrane level (prerequisite for receptor mosaic organization and plastic adjustments) and in the control of receptor desensitization. Genedani, S., Guidolin, D., Leo, G., Filaferro, M. Torvinen, M., Woods, A.S., Fuxe, K. Ferre, S., and Agnati, L.F. Journal of Molecular Neuroscience, 26, pp. 177-184, 2005.

Adenosine A2A and Dopamine D2 Heteromeric Receptor Complexes and Their Function The existence of A2A-D2 heteromeric complexes is based on coimmunoprecipitation studies and on fluorescence resonance energy transfer and bioluminescence resonance energy transfer analyses. It has now become possible to show that A2A and D2 receptors also coimmunoprecipitate in striatal tissue, giving evidence for the existence of A2A-D2 heteromeric receptor complexes also in rat striatal tissue. The analysis gives evidence that these heteromers are constitutive, as they are observed in the absence of A2A and D2 agonists. The A2A-D2 heteromers could either be A2A-D2 heterodimers and/or higher-order A2A -D2 hetero-oligomers. In striatal neurons there are probably A2A-D2 heteromeric complexes, together with A2A-D2 homomeric complexes in the neuronal surface membrane. Their stoichiometry in various microdomains will have a major role in determining A2A and D2 signaling in the striatopallidal GABA neurons. Through the use of D2/D1 chimeras, evidence has been obtained that the fifth transmembrane (TM) domain and/or the I3 of the D2 receptor are part of the A2A-D2 receptor interface, where electrostatic epitope-epitope interactions involving the N-terminal part of I3 of the D2 receptor (arginine-rich epitope) play a major role, interacting with the carboxyl terminus of the A2A receptor. Computerized modeling of A2A-D2 heteromers are in line with these findings. It seems likely that A2A receptor-induced reduction of D2 receptor recognition, G protein coupling, and signaling, as well as the existence of A2A-D2 co-trafficking, are the consequence of the existence of an A2A-D2 receptor heteromer. The relevance of A2A-D2 heteromeric receptor complexes for Parkinson's disease and schizophrenia is emphasized as well as for the treatment of these diseases. Finally, recent evidence for the existence of antagonistic A2A-D3 heteromeric receptor complexes in cotransfected cell lines has been summarized. Fuxe, K., Ferre, S., Canals, M., Torvinen, M. Terasmaa, A., Marcellino, D., Goldberg, S.R., Staines, W., Jacobsen, K.X., Woods, A.S., Agnati, L.F., and Franco, R. Journal of Molecular Neuroscience, 26, pp. 209-220, 2005.

Heptaspanning Membrane Receptors and Cytoskeletal/Scaffolding Proteins: Focus on Adenosine, Dopamine, and Metabotropic Glutamate Receptor Function Most cellular functions are mediated by multiprotein complexes. In neurons, these complexes are directly involved in the proper neuronal transmission, which is responsible for phenomena like learning, memory, and development. In recent years studies based on two-hybrid screens and proteomic, biochemical, and cell biology approaches have shown that intracellular domains of G protein-coupled receptors (GPCRs) or heptaspanning membrane receptors (HSMRs) interact with intracellular proteins. These interactions are the basis of a protein network associated with these receptors, which includes scaffolding proteins containing one or several PDZ (postsynaptic-density-95/discs-large/zona occludens-1) domains, signaling proteins, and proteins of the cytoskeleton. The present article is focused on the emerging evidence for interactions of adenosine, dopamine, and metabotropic glutamate receptors, with scaffolding and cytoskeletal proteins that play a role in the targeting and anchoring of these receptors to the plasma membrane, thus contributing to neuronal development and plasticity. Finally, given the complexity of neurological disorders such as ischemic stroke, Alzheimer's disease, and epilepsy, exploitation of these HSMR-associated interactions might prove to be efficient in the treatment of such disorders. Ciruela, F., Canela, L., Burgueno, J., Soriguera, A., Cabello, N., Canela, E.I., Casado, V., Cortes, A., Mallol, J., Woods, A.S., Ferre, S., Lluis, C., and Franco, R. Journal of Molecular Neuroscience, 26, pp. 277-292, 2005.

Clinical Psychopharmacology Section, Medications Discovery Research Branch

Chronic Morphine Upregulates Ga12 and Cytoskeletal Proteins in CHO Cells Expressing the Cloned Mu Opioid Receptor A growing body of literature indicates that chronic morphine exposure alters the expression and function of cytoskeletal proteins in addition to the well-established interactions between mu opioid receptors and G proteins. In the present study, IRP scientists hypothesized that chronic morphine alters the expression and functional effects of G_12, a G protein that regulates downstream cytoskeletal proteins via its control of RhoA. Results showed that chronic morphine treatment decreased the expression of G_i2 (64%) and G_i3 (60%), had no effect of G_o and increased G_12 (66%) expression in CHO cells expressing the cloned human mu opioid receptors (hMOR-CHO cells), but not in cells expressing a mutant mu opioid receptor that do not develop morphine tolerance and dependence (T394A-CHO cells). Morphine treatment had no significant effect on PAR-1 thrombin receptor-activated G protein activity, as measured by thrombin-stimulated [35S]GTP-__S binding. Chronic morphine treatment significantly enhanced thrombin-stimulated RhoA activity and thrombin-stimulated expression of _-actinin, a cytoskeletal anchoring protein, in hMOR-CHO cells. Proteomic analysis of 2D spots prepared from hMOR-CHO cells showed that morphine treatment affected the expression of a number of proteins associated with morphological changes. Up-regulation of G_12 and _-actinin by chronic morphine was also observed in mouse brain. Viewed collectively, these findings indicate, for the first time, that chronic morphine enhances the G_12-associated signaling system, which is involved in regulating cellular morphology and growth, supporting other findings that chronic morphine may alter cellular morphology, in addition to cellular function. Xu H., Wang X, Zimmerman D., Boja E.S., Wang J.B., Bilsky E.J. and Rothman R.B. Journal of Pharmacology and Experimental Therapeutics June 29, 2005 [Epub ahead of print].

Medicinal Chemistry Section, Medications Discovery Research Branch

Radioiodinated Azide and Isothiocyanate Derivatives of Cocaine for Irreversible Labeling of Dopamine Transporters: Synthesis and Covalent Binding Studies Two novel N-substituted-3b-phenyl tropane alkaloids have been labeled with iodine-125 for use as irreversible probes of dopamine transporter (DAT) binding sites. One contains an iodoarylazide moiety for photolabeling, while the other bears an iodoaryl isothiocyanate for direct conjugation. Both radioligands were prepared in a one-flask procedure by electrophilic radioiodination of the corresponding aniline under no-carrier-added conditions, followed either by diazotization and treatment with sodium azide, or by addition of thiophosgene under basic conditions. Specifically, (-)-N-[4-(3'-[125I]-iodo-4'-azidophenyl) butyl]-2b-carbomethoxy-3b-(4-chlorophenyl)tropane ([125I]-MFZ-2-24) and (-)-N-[4-(3'-[125I]-iodo-4'-isothiocyanophenyl) butyl]-2b-carbomethoxy-3b-(4-chlorophenyl) tropane ([125I]-MFZ 3-37) were synthesized. Isolation by reverse phase HPLC and solid-phase extraction gave good average yields of [125I]-MFZ-2-24 (67%, n = 5) and [125I]-MFZ-3-37 (45%, n = 3) with high radiochemical purities (96 - 99%) and specific radioactivities (> 2000 mCi / _mol). The utility of the radioligands was demonstrated by their covalent linkage to rat striatal membranes, and immunoprecipitation of a single radiolabeled band at 80 kDa corresponding to the full-length DAT. Lever, J. R., Zou, M.-F., Parnas, M. L., Duval, R. A., Wirtz, S. E., Justice, J. B., Vaughan, R. A., Newman, A. H.. Bioconjugate Chemistry, 16, pp. 644-649, 2005.

Yawning in Rats: A Dopamine D3 Receptor Mediated Behavior A specific role for the dopamine D3 receptor in behavior has not been definitively defined. IRP researchers now report that dopamine D2/D3 agonists elicit dose dependent yawning behavior in rats, resulting in an inverted U-shaped dose-response curve. A series of experiments was directed toward testing the hypothesis that this induction of yawning is a D3 receptor mediated effect, while the inhibition of the yawning seen at higher doses is due to competing D2 receptor activity. Authors compared several dopaminergic agonists with a wide range of in vitro D3 selectivity, including; PD-128,907, PD-128,908, quinelorane, pramipexole, 7-OH-DPAT, quinpirole, bromocriptine, and apomorphine with respect to their ability to induce yawning in rats. A series of D2/D3 antagonists differing in selectivity for D3 over D2 receptors were evaluated for their ability to alter the effects of the dopamine agonists. The antagonists studied were L-741,626, haloperidol, nafadotride, U99194, SB-277011A, and PG01037; they have been used to determine effects on dose-response curves for D2/D3 agonist induced yawning. In addition, the potential contribution of cholinergic and/or serotonergic mechanisms to the yawning response was investigated using selective ligands including scopolamine, mianserin, and the D3-preferring antagonists; nafadotride, U99194, SB-277011A, and PG01037 to differentially modulate yawning induced by the PD-128,907, physostigmine, and TFMPP. The results of these experiments provide convergent evidence that dopamine D2/D3 agonist-induced yawning is a D3 agonist induced behavior, with subsequent inhibition of yawning at higher doses being driven by competing D2 agonist activity. Thus, dopamine agonist-induced yawning may represent an in vivo method for selectively identifying D3 and D2 receptor-mediated activities. Collins, G. T., Witkin, J. M., Newman, A. H., Svensson, K. A., Grundt, P., Cao, J., Woods, J. H. Journal of Pharmacology and Experimental Therapeutics, 314, pp. 310-319, 2005.

Recognition of Benztropine by the Dopamine Transporter (DAT) Differs from that of the Classical Dopamine Uptake Inhibitors Cocaine, Methylphenidate and Mazindol as a Function of a DAT Transmembrane 1 Aspartic Acid Residue Binding of cocaine to the dopamine transporter (DAT) protein blocks synaptic dopamine clearance, triggering the psychoactive effects associated with the drug; the discrete drug-protein interactions, however, remain poorly understood. A longstanding postulate holds that cocaine inhibits DAT-mediated dopamine transport via competition with dopamine for formation of an ionic bond with the DAT transmembrane aspartic acid residue D79. In the present study, DAT mutations of this residue were generated and assayed for translocation of radiolabeled dopamine and binding of radiolabeled DAT inhibitors under identical conditions. When feasible, dopamine uptake inhibition potency and apparent binding affinity Ki values were determined for structurally diverse DAT inhibitors. The glutamic acid substitution mutant (D79E) displayed values indistinguishable from wildtype DAT in both assays for the charge-neutral cocaine analog 8-oxa-norcocaine, a finding not supportive of the D79 "salt bridge" ligand docking model. In addressing whether the D79 side chain contributes to the DAT binding sites of other portions of the cocaine pharmacophore, only inhibitors with modifications of the tropane ring C-3 substituent, i.e., benztropine and its analogs, displayed a substantially altered dopamine uptake inhibition potency as a function of the D79E mutation. A single conservative amino acid substitution thus differentiated structural requirements for benztropine function relative to those for all other classical DAT inhibitors. Distinguishing the precise mechanism of action of this DAT inhibitor with relatively low abuse liability from that of cocaine may be attainable using DAT mutagenesis and other structure-function studies, opening the door to rational design of therapeutic agents for cocaine abuse. Ukairo, O. T., Bondi, C. D., Newman, A. H., Kulkarni, S.S., Kozikowski, A. P., Pan,S., Surratt, C. K. Journal of Pharmacology and Experimental Therapeutics, 314, pp. 575-583, 2005.

fMRI Section, Neuroimaging Research Branch

Neural Correlates of Cocaine Self-administration Modern theories of drug dependence hold the hedonic effects of drugtaking central to understanding the motivation for compulsive drug use. Previous neuroimaging studies have begun to identify brain regions associated with acute drug effects after passive delivery. In this study, a more naturalistic model of cocaine self-administration (SA) was employed in order to identify those sites associated with drug-induced high and craving as measures of reward and motivation. Nontreatment seeking cocaine-dependent subjects chose both when and how often i.v. cocaine administration occurred within a medically supervised SA procedure. Both functional magnetic resonance imaging (fMRI) data and real-time behavioral ratings were acquired during the 1-h SA period. Drug-induced HIGH was found to correlate negatively with activity in limbic, paralimbic, and mesocortical regions including the nucleus accumbens (NAc), inferior frontal/orbitofrontal gyrus (OFC), and anterior cingulate (AC), while CRAVING correlated positively with activity in these regions. This study provides the first evidence in humans that changes in subjective state surrounding cocaine self-administration reflect neural activity of the endogenous reward system. Risinger, R.C., Salmeron, B.J., Ross, T.J., Amen, S.L., Sanfilipo, M., Hoffmann, R., Bloom, A.S., Garavan, H., and Stein, E.A., NeuroImage, 26, pp. 1097-1108, 2005.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

Self-administration of Cannabinoids by Experimental Animals and Human Marijuana Smokers Drug self-administration behavior has been one of the most direct and productive approaches for studying the reinforcing effects of psychoactive drugs, which are critical in determining their abuse potential. Cannabinoids, which are usually abused by humans in the form of marijuana, have become the most frequently abused illicit class of drugs in the United States. The early elucidation of the structure and stereochemistry of delta-9-tetrahydrocannabinol (THC) in 1964, which is now recognized as the principal psychoactive ingredient in marijuana, activated cannabinoid research worldwide. This review examines advances in research on cannabinoid self-administration behavior by humans and laboratory animals. There have been numerous laboratory demonstrations of the reinforcing effects of cannabinoids in human subjects, but reliable self-administration of cannabinoids by laboratory animals has only recently been demonstrated. It has now been shown that strong and persistent self-administration behavior can be maintained in experimentally and drug-naive squirrel monkeys by doses of THC comparable to those in marijuana smoke inhaled by humans. Furthermore, reinforcing effects of some synthetic CB1 cannabinoid agonists have been recently reported using intravenous and intracerebroventricular self-administration procedures in rats and mice. These findings support previous conclusions that THC has a pronounced abuse liability comparable to other drugs of abuse under certain experimental conditions. Self-administration of THC by squirrel monkeys provides the most reliable animal model for human marijuana abuse available to date. This animal model now makes it possible to study the relative abuse liability of other natural and synthetic cannabinoids and to preclinically assess new therapeutic strategies for the treatment or prevention of marijuana abuse in humans. Justinova, Z., Goldberg, S.R., Heishman, S.J. and Tanda, G. Pharmacol Biochem Behav. May 30, 2005 (Epub ahead of print) PMID 15932767.

Neuropeptide Y Protects Against Methamphetamine-induced Neuronal Apoptosis in the Mouse Striatum Methamphetamine (METH) is an illicit drug that causes neuronal apoptosis in the mouse striatum, in a manner similar to the neuronal loss observed in neurodegenerative diseases. In the present study, injections of METH to mice were found to cause the death of enkephalin-positive projection neurons but not the death of neuropeptide Y (NPY)/nitric oxide synthase-positive striatal interneurons. In addition, these METH injections were associated with increased expression of neuropeptide Y mRNA and changes in the expression of the NPY receptors Y1 and Y2. Administration of NPY in the cerebral ventricles blocked METH-induced apoptosis, an effect that was mediated mainly by stimulation of NPY Y2 receptors and, to a lesser extent, of NPY Y1 receptors. Finally, authors also found that neuropeptide Y knock-out mice were more sensitive than wild-type mice to METH-induced neuronal apoptosis of both enkephalin- and nitric oxide synthase-containing neurons, suggesting that NPY plays a general neuroprotective role within the striatum. Together, these results demonstrate that neuropeptide Y belongs to the class of factors that maintain neuronal integrity during cellular stresses. Given the similarity between the cell death patterns induced by METH and by disorders such as Huntington's disease, these results suggest that NPY analogs might be useful therapeutic agents against some neurodegenerative processes. Thiriet, N., Deng, X., Solinas, M., Ladenheim, B., Curtis, W., Goldberg, S.R., Palmiter, R.D. and Cadet, J.L. J Neurosci, 25, pp. 5273-5279, 2005.

Control of the Reinforcing Effects of Nicotine by Associated Environmental Stimuli in Animals and Humans Tobacco dependence through cigarette smoking is the leading preventable cause of death in the world and kills nearly 4 million people annually. Nicotine, a psychoactive component of tobacco, is thought to have a major role in tobacco dependence by acting directly as a reinforcer of drug-seeking and drug-taking behavior. However, recent findings obtained with two procedures that are used widely to assess reinforcing effects of drugs in experimental animals, intravenous drug self-administration and conditioned place-preference procedures, demonstrate that environmental factors have a major influence on the reinforcing effects of nicotine. Under some experimental conditions, nicotine is also self-administered reliably by humans. Environmental stimuli that have been associated previously with the self-administration of nicotine can reinstate extinguished drug-seeking behavior in animals and precipitate relapse to smoking behavior in ex-smokers. Innovative medications that target cannabinoid CB(1) and dopamine D(3) receptors and might block specifically the influence of such conditioned environmental stimuli in smokers are in development. LeFoll, B. and Goldberg, S.R. Trends Pharmacol Sci, 26, pp. 287-293, 2005.

Cannabinoid Agonists but not Inhibitors of Endogenous Cannabinoid Transport or Metabolism Enhance the Reinforcing Efficacy of Heroin in Rats Accumulating evidence suggests that the endogenous cannabinoid system is involved in the reinforcing effects of heroin. In rats intravenously self-administering heroin, IRP scientists investigated effects of cannabinoid CB(1) receptor agonists and compounds that block transport or metabolism of the endogenous cannabinoid anandamide. The natural cannnabinoid CB(1) receptor agonist delta-9-tetrahydrocannabinol (THC, 0.3-3 mg/kg i.p.) did not alter self-administration of heroin under a fixed-ratio one (FR1) schedule, except at a high 3 mg/kg dose which decreased heroin self-administration. Under a progressive-ratio schedule, however, THC dose-dependently increased the number of 50 mug/kg heroin injections self-administered per session and the maximal ratio completed (break-point), with peak increases at 1 mg/kg THC. In addition, 1 mg/kg THC increased break-points and injections self-administered over a wide range of heroin injection doses (25-100 mug/kg), indicating an increase in heroin's reinforcing efficacy and not its potency. The synthetic cannabinoid CB(1) receptor agonist WIN55,212-2 (0.3-3 mg/kg i.p.) had effects similar to THC under the progressive-ratio schedule. In contrast, AM-404 (1-10 mg/kg i.p.), an inhibitor of transport of anandamide, and URB-597 (0.01-0.3 mg/kg i.p.), an inhibitor of the enzyme fatty acid amide hydrolase (FAAH) that degrades anandamide, or their combination, did not increase reinforcing efficacy of heroin at any dose tested. Thus, activation of cannabinoid CB(1) receptors facilitates the reinforcing efficacy of heroin and this appears to be mediated by interactions between cannabinoid CB(1) receptors and mu-opioid receptors and their signaling pathways, rather than by an opioid-induced release of endogenous cannabinoids. Solinas, M., Panlilio, L.V., Tanda, G., Makriyannis, A., Matthews, S.A. and Goldberg, S.R. Neuropsychopharmacology, May 4, 2005 (Epub ahead of print) PMID: 15870833.

Motivational Effects of Cannabinoids and Opioids on Food Reinforcement Depend on Simultaneous Activation of Cannabinoid and Opioid Systems Strong functional interactions exist between endogenous cannabinoid and opioid systems. Here, IRP researchers investigated whether cannabinoid-opioid interactions modulate motivational effects of food reinforcement. In rats responding for food under a progressive-ratio schedule, the maximal effort (break point) expended to obtain 45 mg pellets depended on the level of food deprivation, with free-feeding reducing break points and food-deprivation increasing break points. Delta-9-tetrahydro-cannabinol (THC; 0.3-5.6 mg/kg intrapeitoneally (i.p.)) and morphine (1-10 mg/kg i.p.) dose-dependently increased break points for food reinforcement, while the cannabinoid CB1 receptor antagonist rimonabant (SR-141716A; 0.3-3 mg/kg i.p.) and the preferential mu-opioid receptor antagonist naloxone (0.3-3 mg/kg i.p.) dose-dependently decreased break points. THC and morphine only increased break points when food was delivered during testing, suggesting that these treatments directly influenced reinforcing effects of food, rather than increasing behavior in a nonspecific manner. Effects of THC were blocked by rimonabant and effects of morphine were blocked by naloxone, demonstrating that THC's effects depended on cannabinoid CB1 receptor activation and morphine's effects depended on opioid-receptor activation. Furthermore, THC's effects were blocked by naloxone and morphine's effects were blocked by rimonabant, demonstrating that mu-opioid receptors were involved in the effects of THC and cannabinoid CB1 receptors were involved in the effects of morphine on food-reinforced behavior. Thus, activation of both endogenous cannabinoid and opioid systems appears to jointly facilitate motivational effects of food measured under progressive-ratio schedules of reinforcement and this facilitatory modulation appears to critically depend on interactions between these two systems. These findings support the proposed therapeutic utility of cannabinoid agonists and antagonists in eating disorders. Solinas, M. and Goldberg, S.R. Neuropsychopharmacology, April 6, 2005 (Epub ahead of print) PMID: 15812567.

Nanomolar Concentrations of Kynurenic Acid Reduce Extracellular Dopamine Levels in the Striatum Precise regulation of dopaminergic activity is of obvious importance for the physiology and pathology of basal ganglia. IRP investigators report here that nanomolar concentrations of the astrocyte-derived neuroinhibitory metabolite kynurenic acid (KYNA) potently reduce the extracellular levels of striatal dopamine in unanesthetized rats in vivo. This effect, which is initiated by the KYNA-induced blockade of alpha7 nicotinic acetylcholine receptors, highlights the functional relevance of glia-neuron interactions in the striatum and indicates that even modest increases in the brain levels of endogenous KYNA are capable of interfering with dopaminergic neurotransmission. Rassoulpour, A., Wu, H.Q., Ferre, S. and Schwarcz, R. J Neurochem, 93, pp. 762-765, 2005.

How Receptor Mosaics Decode Transmitter Signals. Possible Relevance of Cooperativity It has been demonstrated that receptor-receptor interactions between G-protein-coupled receptors (GPCRs) occur at the plasma-membrane level. It has also been shown that clustering of GPCRs in aggregates or receptor mosaics (RMs) results in the reciprocal modulation of their binding and decoding characteristics. It is hypothesized that cooperativity plays an important part in the decoding of signals processed by RMs of GPCRs. Thus, the binding of the ligand at one receptor alters the likelihood of the same ligand binding at the next site, in the case of RMs, formed by identical receptors and/or by iso-receptors (receptors that bind the same ligand). Agnati, L.F., Fuxe, K. and Ferre, S. Trends Biochem Sci, 30, pp.188-193, 2005.

The Endogenous Cannabinoid Anandamide and Its Synthetic Analog R(+)-Methanand-amide are Intravenously Self-Administered by Squirrel Monkeys Anandamide, an endogenous ligand for brain cannabinoid CB1 receptors, produces many behavioral effects similar to those of _9-tetrahydrocannabinol (THC), the main psychoactive ingredient in marijuana. Reinforcing effects of THC have been demonstrated in experimental animals, but there is only indirect evidence that endogenous cannabinoids like anandamide participate in brain reward processes. IRP scientists now show that anandamide serves as an effective reinforcer of drug-taking behavior when self-administered intravenously by squirrel monkeys. They also show that methanandamide, a synthetic long-lasting anandamide analog, similarly serves as a reinforcer of drug-taking behavior. Finally, they show that the reinforcing effects of both anandamide and methanandamide are blocked by pretreatment with the cannabinoid CB1 receptor antagonist rimonabant (SR141716). These findings strongly suggest that release of endogenous cannabinoids is involved in brain reward processes and that activation of cannabinoid CB1 receptors by anandamide could be part of the signaling of natural rewarding events. Justinova, Z., Solinas, M., Tanda, G., Redhi, G.H. and Goldberg, S.R. J Neurosci, 25, pp. 5645-5650, 2005.

Treatment Section, Clinical Pharmacology and Therapeutics Research Branch

Cyclazocine: Comparison to Hydromorphone and Interaction with Cocaine Kappa opioid agonists produce neurobiological and behavioral effects opposite those of cocaine and may be useful for the treatment of cocaine dependence. To evaluate the kappa and mu agonist effects of cyclazocine and test whether cyclazocine pretreatment would attenuate the effects of cocaine, the acute effects of cyclazocine and the mu agonist hydromorphone were compared and the effects of repeated administration of cyclazocine on response to cocaine were evaluated in experienced opiate and cocaine users. Cocaine was given 2 hours after oral pretreatment with cyclazocine (0, 0.1, 0.2, 0.4, 0.8, and 0 mg, in that order) in sessions conducted daily Monday to Friday and the following Monday. Physiological, subjective, and behavioral measures were collected in each session. Hydromorphone produced prototypic mu agonist effects. Cyclazocine exhibited modest kappa-like effects; on most measures, its effects tended to be similar to those of hydromorphone (15 mg), but lower in magnitude. Cyclazocine also had only modest, non-dose-related effects on response to cocaine. However, cocaine effects were consistently lower on the last administration (cyclazocine 0 mg pretreatment) following 4 days of cyclazocine pretreatment compared to the first administration (0 mg pretreatment). This finding is unlikely to be fully attributable to cocaine tolerance, and is not accounted for by pharmacokinetic changes; plasma concentrations of cocaine and its major metabolites were not altered by cyclazocine. The results were consistent with preclinical studies showing that repeated administration of a kappa agonist alters responses to cocaine. Preston, K.L. Umbricht, A, Schroeder, J., Abreu, M., and Pickworth, W.B. Behavioural Pharmacology, 5, pp. 91-102, 2004.

Effects of High-Dose Intravenous Buprenorphine in Experienced Opioid Abusers Sublingual (SL) buprenorphine, a long-acting, partial mu-opioid agonist, is as effective as methadone in the treatment of heroin dependence, with a better safety profile due to its antagonist activity. However, the safety of therapeutic doses (8 to 16 mg) that might be diverted for intravenous (IV) use had not been demonstrated. Buprenorphine was administered to 6 non-dependent opioid abusers residing on a research unit; the doses tested, in separate sessions, were 12 mg buprenorphine SL, IV/SL placebo, and escalating IV buprenorphine (2 to 16 mg). Physiological and subjective measures were collected for 72 hours post drug administration. Buprenorphine minimally but significantly increased systolic blood pressure with no other statistically significant changes in blood pressure, heart rate or oxygen (02) saturation among the 7 drug conditions. The mean maximum decrease in 02 saturation from baseline was greatest for the 8 mg IV dose. Buprenorphine produced positive mood effects, although with substantial variability among participants. Onset and peak effects occurred earlier following IV than SL administration: peak IV effects occurred between 0.25 and 3 hrs; peak SL effects occurred at 3 to 7 hrs. Duration of effects varied among the outcome measures. The dose-response curves were flat for most parameters, particularly subjective measures. Side effects were mild except in one participant who experienced severe nausea and vomiting after the 12 mg IV dose. Buprenorphine appears to have a ceiling for cardio-respiratory and subjective effects and a high safety margin even when taken by the IV route. Umbricht, A., Huestis, M.A., Cone, E.J., and Preston, K.L. Journal of Clinical Psychopharmacology, 24, pp. 479-487, 2004.

Menstrual Cycle Length during Methadone Maintenance While heroin's menstrual disruption has been demonstrated, there are few published data concerning methadone maintenance (MM) and menstrual function. This study was conducted to evaluate whether cycles were more regular during MM. Start/end dates of each menses were collected from 191 drug-using women from two clinical trials, lasting 25-29 weeks, while on 70-100 mg of methadone. Participants were classified as regular, irregular, transient amenorrhea, persistent amenorrhea, or cycle restarters. Repeated-measures regression modeling was used to determine correlates of cycle length, probability of long cycles (>40 days), and short cycles (<20 days). Bleeding episodes (days from "start" to "stop") were defined as one or more bleeding days, bound by at least two non-bleeding days. Correlates of cycle length, body mass index, drug use, methadone dose, and race were calculated. Women had a high prevalence of cycle length irregularity; 133 participants: regular 37 (27.8%); irregular 62 (46.7%); transient amenorrhea 7 (5.3%); persistent amenorrhea 11 (8.3%); cycle restarters 16 (12%). Each additional week on MM was associated with decreased risk of long (OR=0.96, p=0.001 and short (OR=0.92, p=0.001) cycles. Of 27 women with secondary amenorrhea pre-study, 16 (59%) restarted menses. Positivity for opioids or cocaine was not significantly associated with short or long cycles. Cycle length begins to normalize during MM. Menses resumption may occur. MM, despite interfering with menstrual function in an absolute sense, may interfere less than illicit heroin abuse. Schmittner, J., Schroeder, J.R., Epstein, D.H., and Preston, K.L. Addiction, 100(6), pp. 829-836, 2005.

Clinical Pharmacology Section, Clinical Pharmacology and Therapeutics Research Branch

Calorie Restriction Increases Cigarette Use In Adult Smokers Cigarette smokers weigh less than nonsmokers, and smokers often gain weight when they quit. This is a major barrier to smoking cessation, especially among women. However, strict dieting is not recommended during smoking cessation out of concern that it might promote relapse. One reason is that calorie restriction increases self-administration of drugs of abuse in animals. This relationship has never been experimentally demonstrated in humans. This study evaluated whether calorie restriction increases cigarette smoking in humans. Seventeen (9M, 8F) healthy, normal-weight smokers not attempting to quit were cycled in partially counterbalanced order, double-blind, through four diets: normal calorie (2000-2800 kcal/day), low calorie (700 kcal/day deficit), low carbohydrate (CHO)-normal calorie, and low CHO-low calorie, for six days per diet on an inpatient research ward. Smoking was assessed by cigarette counts, breath carbon monoxide (CO) levels, and cigarette craving. Compared with the normal calorie diet, while on the low calorie diet, subjects smoked 8% more cigarettes (p<0.02) and had 11% higher breath CO levels (p<0.01). The low CHO- normal calorie diet showed no significant effect on either variable, but there was a 15% increase in breath CO levels (p<0.05) on the low CHO-low calorie diet. There were no changes in self-reported cigarette craving or mood. Consistent with animal studies, moderate calorie restriction was associated with a small but statistically significant increase in cigarette smoking, with no independent effect of carbohydrate deprivation. These findings suggest that dieting may increase smoking behavior, and could impede smoking cessation attempts. Cheskin, L.J., Hess, J.M., Henningfield, J., and Gorelick, D.A. Psychopharmacology 179, pp. 430-436, 2005.

Influence of Psychotherapy Attendance on Buprenorphine Treatment Outcome Buprenorphine is a partial mu-opioid receptor agonist approved for the treatment of opiate dependence. This study evaluated the influence of psychotherapy attendance on treatment outcome in 90 dually (cocaine and heroin) dependent outpatients who completed 70 days of a controlled clinical trial of sublingual buprenorphine (16 mg, 8 mg, or 2 mg daily, or 16 mg every other day) plus weekly individual standardized interpersonal cognitive psychotherapy. Treatment outcome was evaluated by quantitative urine benzoylecgonine (BZE) and morphine levels (log-transformed), performed three times per week. Repeated-measures linear regression was used to assess the effects of psychotherapy attendance (percent of visits kept), medication group, and study week on urine drug metabolite levels. Mean psychotherapy attendance was 71% of scheduled visits. Higher psychotherapy attendance was associated with lower urine BZE levels, and this association grew more pronounced as the study progressed (p=0.04). The inverse relationship between psychotherapy attendance and urine morphine levels varied by medication group, being most pronounced for subjects receiving 16 mg every other day (p = 0.02). These results suggest that psychotherapy can improve the outcome of buprenorphine maintenance treatment for patients with dual (cocaine and opioid) dependence. Montoya, I.D., Schroeder, J.R., Preston, K.L., Umbricht, A., Fudala, P., Johnson, R., Contoreggi, C., and Gorelick, D.A. Journal of Substance Abuse Treatment, 28, pp. 247-254, 2005.

Imaging Brain Mu-opioid Receptors in Abstinent Cocaine Users: Time Course and Relation to Cocaine Craving Cocaine treatment upregulates brain mu-opioid receptors (mOR) in animals. Human data regarding this phenomenon are limited. IRP scientists previously used positron emission tomography (PET) with 11C-carfentanil to show increased mOR binding in brain regions of 10 cocaine-dependent men after 1 and 28 days of abstinence. The present study, in collaboration with scientists at the Johns Hopkins PET Center, measured regional brain mOR binding potential (BP) using [11C]carfentanil PET scanning in 17 cocaine users over 12 weeks of enforced abstinence on a closed research ward and in 16 healthy control subjects. mOR BP was increased in the frontal, anterior cingulate, and lateral temporal cortex after one day of abstinence. mOR BP remained elevated in the first two regions after one week and in the anterior cingulate and anterior frontal cortex after 12 weeks of abstinence. Increased binding in some regions at one day and one week was positively correlated with self-reported cocaine craving. mOR BP was significantly correlated with percentage of days with cocaine use and amount of cocaine used per day of use during the two weeks prior to admission, and urine benzoylecgonine concentration at the first PET scan. These results suggest that chronic cocaine use influences endogenous opioid systems in the human brain, and may explain mechanisms of cocaine craving and reinforcement. Gorelick, D.A., Kim, Y.-K., Bencherif, B., Boyd, S.J., Nelson, R., Copersino, M., Endres, C.J., Dannals, R.F., and Frost, J.J. Biological Psychiatry, 57, pp. 1573-1582, 2005.

Chemistry and Drug Metabolism Section, Clinical Pharmacology and Therapeutics Research Branch

Pharmacological Treatment of Adolescent Tobacco Addiction To determine the safety and efficacy of the nicotine patch and gum for adolescents who want to quit smoking, IRP researchers conducted a clinical trial with nicotine patch (21 mg), nicotine gum (2 and 4 mg), or a placebo patch and gum. All participants received cognitive-behavioral group therapy and were 13-17-year-old adolescents who smoked at least 10 cigarettes per day (CPD), scored at least 5 on the Fagerstrom Test of Nicotine Dependence, and were motivated to quit smoking. Participants were treated for 12 weeks with nicotine patch or gum with cognitive-behavioral therapy, with a follow-up visit at 6 months (3 months after the end of treatment). Safety was assessed on the basis of adverse event reports for all 3 groups, prolonged abstinence, assessed through self-report and verified with exhaled carbon monoxide (CO) levels of less than 6 ppm, in intent-to-treat analyses, and smoking reduction (CPD and thiocyanate concentrations) among trial completers. A total of 120 participants were randomized (72% white, 70% female; age: 15.2 +/- 1.33 years; smoking: 18.8 +/- 8.56 CPD; Fagerstrom Test of Nicotine Dependence score: 7.04 +/- 1.29) from 1999 to 2003. Participants started smoking at 11.2 +/- 1.98 years of age and had been smoking daily for 2.66 +/- 1.56 years; 75% had at least 1 current psychiatric diagnosis. Mean compliance across groups was higher for the patch (mean: 78.4-82.8%) than for the gum (mean: 38.5-50.7%). Both the patch and gum were well tolerated, and adverse events were similar to those reported in adult trials. Changes in mean saliva cotinine concentrations throughout treatment were not statistically significant. Intent-to-treat analyses of all randomized participants showed CO-confirmed prolonged abstinence rates of 18% for the active-patch group, 6.5% for the active-gum group, and 2.5% for the placebo group; the difference between the active-patch and placebo arms was statistically significant. There was no significant effect of patch versus gum or gum versus placebo on cessation outcomes. Abstinence rates at the 3-month follow-up assessment were sustained but were not significantly associated with treatment group. Mean smoking rates, but not CO or thiocyanate concentrations, decreased significantly in all 3 arms but not as a function of treatment group. Nicotine patch therapy combined with cognitive-behavioral intervention was effective, compared with placebo, for treatment of tobacco dependence among adolescent smokers. Decreases in the numbers of cigarettes smoked appeared to be offset by compensatory smoking. Additional study of nicotine gum, with enhanced instructional support, is needed to assess its efficacy among adolescent smokers. Moolchan, E.T., Robinson, M.L., Ernst, M., Cadet, J.L., Pickworth, W.B., Heishman, S.J. and Schroeder, J.R. Pediatrics, 115, pp. 407-414, 2005.