Research Findings - Intramural Research

Biomedical Informatics Section, Administrative Management Branch

An EHR-Based Multi-Site Recruiting System for Clinical Trials
Optimizing screening and evaluation process is essential in maximizing recruiting potential participants into clinical trials. IRP researchers developed a clinical recruiting system used in the management of human research studies whereby recruiting for various protocols is conducted at multiple sites by different groups with process interdependencies. The system is developed based on a multi-tier architecture with a series of real-time and on-demand decision support systems to increase the efficiency of screening and evaluation of participants during a recruiting process. It simulates various conditions by using derivation rules and recruiting templates for automatic generation of checklists in order to recommend the optimum selection rules based on existing participants' data and is seamlessly integrated into our large Human Research Information System, a comprehensive electronic health record (EHR) system for research participants. Vahabzadeh, M., Lin, J.-L., Mezghanni, M., Contoreggi, C., and Leff, M. Proc. 20th IEEE International Symposium on Computer-Based Medical Systems (CBMS 2007), pp. 331-336, 2007.

Computerized Contingency Management for Motivating Behavior Change: Automated Tracking and Dynamic Reward Reinforcement Management
Contingency management has been successfully used to reinforce abstinence in drug-dependent patients. The major challenge associated with this technique, however, is its likely prohibitive cost, especially when the reinforcement follows an escalating amount schedule. IRP investigators have implemented a less costly approach involving prize draws as the reinforcer. Workflow challenges of such an implementation are extensive considering varying conditions concurrently used in various protocols for meeting inclusion criteria, stratifying into various groups, tracking categories of prizes, determining eligibility for bonus draws based on laboratory results, etc. To address these challenges authors implemented the Automated Contingency Management (ACM) decision support system for dynamic reward reinforcement in the study of treatment of cocaine and opiates. Vahabzadeh, M., Lin, J.-L., Epstein, D.H., Mezghanni, M., Schmittner, J., and Preston, K.L. Proc. 20th IEEE International Symposium on Computer-Based Medical Systems (CBMS 2007), pp. 85-90, 2007.

Office of the Scientific Director

Cannabis Withdrawal among Non-Treatment-Seeking Adult Cannabis Users
Cannabis withdrawal occurs in animals and in heavy human cannabis users. However, its clinical significance has been doubted; it is not recognized in DSM-IV. This study evaluated cannabis withdrawal symptoms and subjects' response to them in a convenience sample of 104 adult cannabis smokers who reported a self-defined "serious" quit attempt without formal treatment. Ninety-eight percent experienced at least one withdrawal symptom; 49% experienced at least 4 symptoms. The three commonest withdrawal symptoms were craving for cannabis (66%), irritability (48%), and boredom (45%). Fifty-six percent of subjects experiencing withdrawal symptoms reported taking action to relieve their symptoms. The commonest action was substance use, reported by 77% of those taking action. This included 19% resuming cannabis use, 25% using alcohol, 23% using tobacco, and 23% using tranquillizers. These findings suggest that withdrawal symptoms are common among users to attempt to quit cannabis use, and that such symptoms can serve as a negative reinforcer for substance use, including relapse to cannabis use. These results therefore support the clinical significance of cannabis withdrawal. Copersino, M.L., Boyd, S.J., Tashkin, D.P., Huestis, M.A., Heishman, S.J., Dermand, J.C., Simmons, M.S., and Gorelick, D.A. American Journal on Addictions,15, pp. 8-14, 2006.

Bromocriptine Treatment for Cocaine Addiction: Association with Plasma Prolactin Levels
Bromocriptine is a dopamine (D2) receptor agonist that has been evaluated for the treatment of cocaine dependence, with inconsistent results. Prolactin is a circulating hormone whose plasma levels are reduced by activation of D2 receptors. Prolactin plasma levels have been proposed as a predictor of response to cocaine addiction treatment. This study evaluated the safety and efficacy of bromocriptine (2.5 mg orally thrice daily), along with drug abuse counseling, in 70 cocaine-dependent adult men participating in a 28-day inpatient treatment program followed by 26 weeks of outpatient treatment. Bromocriptine was given in a double-blind, placebo-controlled, randomized design. To maximize the opportunity for efficacy, medication was started during the last 2 weeks of the inpatient stay and continued as an outpatient. Both bromocriptine and placebo groups decreased their cocaine use, with no significant group differences in retention in treatment or proportion of cocaine-positive urine samples. There was no significant association between basal plasma prolactin concentrations or prolactin response to first bromocriptine dose and either outcome measure. These data do not support the efficacy of bromocriptine treatment for cocaine addiction nor a role for prolactin concentration in predicting treatment response. Gorelick, D.A., and Wilkins, J.N. Drug and Alcohol Dependence, 81, pp. 189-195, 2006.

Effect of Rate of Administration on Subjective and Physiological Effects of Intravenous Cocaine in Humans
The rate hypothesis of psychoactive drug action holds that the faster a drug is administered, gets to its site of action in the brain, and starts to act, the greater its rewarding effects and abuse liability. There is little confirmatory evidence for the rate hypothesis with cocaine in humans. This study evaluated the rate hypothesis in 17 experienced cocaine users (7 of whom completed all 10 sessions) by administering intravenous cocaine at each of 3 doses (10, 25, 50 mg) at each of 3 injection durations (10, 30, 60 seconds) in a double-blind, placebo-controlled, escalating dose design. Heart rate, blood pressure, and positive (e.g., rush, high) and negative (e.g., feel bad, anxious) subjects effects (100-mm visual analogue scales) were measured for 1 hour after dosing. Cocaine responses were evaluated with repeated measures mixed linear regression analyses, allowing use of data from all sessions for all subjects. Both dose and infusion rate (mg/sec) significantly influenced most subjective and cardiovascular variables. Analysis of the interaction suggested that dose had a stronger impact than rate. Rate had a stronger influence on positive subjective effects than on negative subjective effects or cardiovascular variables. These findings provide support for the rate hypothesis as it applies to effects of intravenous cocaine administration in humans. Nelson, R.A., Boyd, S.J., Ziegelstein, R.C., Herning, R., Cadet, J.L., Henningfield, J.E., Schuster, C.R., Contoreggi, C., and Gorelick, D.A. Drug and Alcohol Dependence, 82, pp. 19-24, 2006.

The Cannabinoid CB1 Receptor Antagonist Rimonabant Attenuates the Hypotensive Effect of Smoked Marijuana in Male Smokers
Animal studies suggest that cannabinoid CB1 receptors play a role in regulating blood pressure. In human studies, activation of CB1 receptors by cannabis or THC has modest and inconsistent effects on blood pressure. This study evaluated the role of CB1 receptors in blood pressure in 63 male cannabis smokers (10.3 + 5.9 years of lifetime cannabis use) by administering escalating oral doses (1, 3, 10, 30, 90 mg) of the selective CB1 receptor antagonist rimonabant (or placebo) in a randomized, parallel-group, double-blind, placebo-controlled design. Subjects smoked an active (2.64% THC) or placebo marijuana cigarette 2 and 6 hours after rimonabant dosing. Blood pressure and symptoms were monitored for 90 minutes after smoking while subjects remained seated. Marijuana smoking after placebo rimonabant had no consistent effect on blood pressure, but 22% of subjects experienced symptomatic (dizziness, lightheadedness) hypotension, as did 20-33% of subject who received pretreatment with 1, 3, or 10 mg rimonabant. No subject receiving 30 or 90 mg rimonabant before marijuana smoking experienced symptomatic hypotension. The 7 subjects who experienced symptomatic hypotension had significantly higher mean (SD) peak plasma THC concentrations than did the 33 subjects who did not. Rimonabant by itself had no effects on blood pressure and did not alter THC pharmacokinetics. These findings indicate that CB1 receptors play a role in medicating effects of cannabis smoking on blood pressure in humans. Gorelick, D.A., Heishman, S.J., Preston, K.L., Nelson, R.A., Moolchan, E.T., and Huestis, M.A. American Heart Journal, 151, pp. 754.e1-754.e5, 2006.

Quitting among Non-Treatment-Seeking Marijuana Users: Reasons and Changes in Other Substance Use
Marijuana is the most widely used illegal drug in the world, yet relatively little is known about why adult users try to quit without formal treatment or whether they change use of other substances in response to quitting. This study examined the self-reported reasons for quitting marijuana use, changes in other substance use during the quit attempt, and reasons for resumption of use in 104 non-treatment-seeking adult marijuana smokers. Quitting was primarily motivated by concerns about the negative impact of marijuana on health and on self- and social-image. Spontaneous quitting of marijuana use was often associated with increased use of legal substances such as alcohol, tobacco, and sleeping aids, but not with initiation of new substance use. These findings suggest clinically relevant areas for further research on spontaneous recovery from marijuana use. Copersino, M.L., Boyd, S.J., Tashkin, D.P., Huestis, M.A., Heishman, S.J., Dermand, J.C., Simmons, M.S., and Gorelick, D.A. American Journal on Addictions,15, pp. 297-302, 2006.

Spending of Remuneration by Subjects in Non-Treatment Drug Abuse Research Studies
Research subjects are commonly paid for participation in non-treatment research studies, but relatively little is known about subject preferences or how they spend their payments. This study examined remuneration spending by 94 adult males who participated in residential, non-treatment drug abuse research studies. Subjects earned remuneration based on length of stay and specific research procedures. Remuneration could be used for in-kind purchases and bill payments, or taken as cash after discharge. Data on subject characteristics and spending of remuneration were extracted retrospectively from subjects' charts. The influence of subject characteristics on remuneration spending was evaluated with multivariate analyses. Participants received average remuneration of $1,454, taking 59% in cash. They received remuneration in a 3:1:1 ratio among cash, in-kind housing payments, and other in-kind categories. The four most popular spending categories were cigarettes (60.6% of subjects), toiletries (60.6%), clothing (54.3%), and housing (52.1%). Primary drug of abuse, total remuneration, monthly income, length of stay on the residential research unit, age, and education were significantly associated with in-kind remuneration choices. Less total remuneration, intoxication in the month prior to study, higher IQ, and non-white race were associated with taking a greater percentage of remuneration in cash. These findings suggest that residential drug abuse research participants prefer cash to in-kind research remuneration, use in-kind spending predominantly for housing costs and personal items, and have remuneration choices influenced by drug use and economic status. These findings may help inform future research subject remuneration practices. Kurlander, J.E., Simon-Dack, S.L., and Gorelick, D.A. American Journal of Drug and Alcohol Abuse, 32, pp. 527-540, 2006.

Risk of Psychoactive Substance Dependence among Substance Users in a Trauma Inpatient Population
One measure of a substance's addictive risk is the proportion of users who become dependent. This study evaluated the lifetime and current risk of substance dependence among lifetime substance users' among trauma inpatients and provided a relative ranking of addictive risk among the substances. Data on use of 8 substance groups (alcohol, opiates, marijuana, cocaine, other stimulants, sedative-hypnotics, hallucinogens, other drugs) were obtained by interview (Structured Clinical Interview for the DSM-III-R) from 1,118 adult trauma inpatients. Prevalence of lifetime dependence among lifetime users ranged from 80.7% for opiates and 70.9% for cocaine to 33.3% for hallucinogens and 26.6% for sedative-hypnotics. The rank order of addictive risk was similar to that found in the general population. Trauma inpatients had a higher absolute addictive risk than the general population, comparable to the risk found in patients in treatment for substance use disorders. These findings suggest the importance of screening trauma inpatients for substance dependence. Martins, S.S., Copersino, M.L., Soderstrom, C.A., Smith, G.S., Dischinger, P.C., McDuff, D.R., Hebel, J.R., Kerns, T.J., Ho, S.M., Read, K.M., and Gorelick, D.A. Journal of Addictive Diseases, 26, pp. 71-77, 2007.

Sociodemographic Characteristics Associated with Substance Use Status in a Trauma Inpatient Population
Substance use is significantly associated with physical injury, yet relatively little is known about the prevalence of specific substance use disorders among trauma patients, or their associated sociodemographic characteristics. This study evaluated these issues in an unselected sample of 1,118 adult inpatients at the University of Maryland Shock Trauma Center, Baltimore, MD, who were interviewed with the psychoactive substance use disorder section of the Structured Clinical Interview for DSM-III-R. Among trauma inpatients, lifetime alcohol users (71.8% of subjects) were more likely male; users of illegal drugs (45.3%) were also more likely to be younger, unmarried, and poor. Patients with current drug abuse/dependence (18.8%) were more likely to be non-white, less educated, and poor; those with current alcohol abuse/dependence (32.1%) were also more likely male, unmarried, and older. These findings highlight the need for screening for substance use disorders in trauma settings and referral of patients to substance abuse treatment programs. Martins, S.S., Copersino, M.L., Soderstrom, C.A., Smith, G.S., Dischinger, P.C., McDuff, D.R., Hebel, J.R., Kerns, T.J., Ho, S.M., Read, K.M., and Gorelick, D.A. Journal of Addictive Diseases, 26, pp. 53-62, 2007.

Use of a "Microecology Technique" to Study Crime around Substance Abuse Treatment Centers
The issue of whether substance abuse treatment centers affect neighborhood crime is hotly debated. Empirical evidence on this issue would be useful to treatment providers, police agencies, policymakers, and community members, but is currently lacking. One reason for this lack is the difficulty of distinguishing the potential effect of a treatment center from the baseline crime rate in the high crime areas in which they are often located. An appropriate control group is needed to distinguish whether crime appears to cluster around clinics only because they have higher foot traffic than nearby locations. This study presents a method to overcome these challenges using crime databases (arrests, 911 calls, and incidents) that are geocoded by street address (i.e., latitude and longitude of each event are assigned by computerized mapping). Events per unit area are calculated in concentric circular "buffers" drawn at 25-meter intervals around each site. A "crime slope" (slope estimate = _) comparing the crimes/area among the buffers is calculated using regression analysis for treatment centers and for three types of control sites: convenience stores and hospitals (socially acceptable high foot traffic sites) and residential points (low foot traffic). This technique controls for the high crime milieu of most treatment centers by comparing crime within rather than between neighborhoods and overcomes the lack of "before and after" crime data. Use of this innovative technique should provide valid empirical evidence on crime around substance abuse treatment centers, perhaps helping resolve a long debated public policy question. Boyd, S.J., Armstrong, K.M., Fang, L.J., Medoff, D.R., Dixon, L.B., and Gorelick, D.A. Social Science Computer Review, 25, pp. 163-173, 2007.

Neural Protection and Regeneration Section, Molecular Neuropsychiatry Branch

Bone Morphogenetic Protein 7 Has Neuroprotective Effects against Methamphetamine Toxicity
Methamphetamine (MA) is a drug of abuse and can have neurotoxic effects on dopaminergic neurons. NIDA IRP has previously found that bone morphogenetic protein 7 (BMP7) has neuroprotective and neuroregenerative properties (Chou, J. Harvey, B.K., Chang, C.F., Shen, H, Morales, M., and Wang, Y. J Neurol Sci. 240, pp. 21-29, 2006) and pretreatment with BMP7 reduced 6-hydroxydopamine-mediated neurodegeneration of dopaminergic neurons in a rodent model of Parkinson's disease (Harvey, B.K., Mark, A., Chou, J., Chen, G.J., Hoffer, B.J., and Wang, Y. Brain Res. 1022, pp. 88-95, 2004). Based on these findings, the neuroprotective effect of BMP7 against MA-mediated toxicity in dopaminergic neurons was evaluated. Primary rat dopaminergic neurons treated with MA (1 mM) decreased tyrosine hydroxylase immunoreactivity (THir) while increasing TUNEL staining. Pretreatment with BMP7 antagonized the effects of MA. The effects of BMP7 on MA toxicity were next examined in CD1 mice. High doses of MA (10 mg/kg x 4 s.c.) significantly reduced locomotor activity and THir in striatum. Intra-cerebroventricular administration of BMP7 attenuated these neurodegenerative changes. In BMP7 heterozygous knockout (BMP7+/-) mice, MA greatly suppressed locomotor activity and reduced TH immunoreactivity in nigra reticulata compared to wild type BMP7 +/+ mice, suggesting that deficiency in BMP7 expression increases vulnerability to MA insults. Since BMP7+/- mice carry a LacZ gene encoding the _-galactosidase (_-gal) protein at the BMP7 locus, the expression of BMP7 was indirectly measured through the enzymatic activity of _-gal in BMP7+/- mice. High doses of MA significantly suppressed _-gal activity in striatum, suggesting that MA may inhibit BMP7 expression at the terminals of nigrostriatal pathway. Overall, BMP7 reduced MA-related degeneration caused by high doses of methamphetamine. Chou, J., Luo, Y., Kuo, CC, Powers, K., Shen, H., Harvey, B.K., Hoffer, B. and Wang, Y. Poster. Society for Neuroscience Annual Meeting, Atlanta, GA, October 14-18, 2006.

Protective Effects of Astaxanthin against Ischemic Brain Injury
Astaxanthin (ATX) is a carotenoid pigment found in crustaceans and structurally similar to other dietary carotenoids such as beta-carotene, lutein and lycopene which are potent antioxidants. The ability of astaxanthin to act as neuroprotective agent against stroke was examined using the transient middle cerebral artery (MCA) ligation model in adult rats. Anesthetized animals were given ATX or vehicle intracerebroventricularly 10-20 minutes prior to a 60 min MCA ligation. Astaxanthin did not alter blood gases (CO2 and O2), blood pH, blood pressure, body temperature, brain temperature and cerebral blood flow. Pretreatment with ATX significantly reduced ischemia -mediated glutamate release in cerebral cortex, measured by microdialysis and HPLC. ATX also antagonized ischemia/reperfusion - induced loss of aconitase activity, an indirect marker for oxidative damage, at 8 hours after stroke. At 2 days after stroke, rats that received ATX had a significant increase in locomotor activity and decrease in cerebral infarction volume compared to the vehicle controls. TUNEL labeling and cytochrome C release in the ischemic brain was suppressed by ATX. Collectively, astaxanthin has protective effects against ischemia-related injury in vivo through the inhibition of oxidative stress, inhibition of glutamate release, and anti-apoptosis. Wang, Y., Harvey, B.K., Shen, H. and Hoffer, B. Oral Presentation, 2007. American Society for Neural Transplantation and Repair, Clearwater, FL, May 3-6, 2007.

Tropism and Toxicity of Adeno-associated Viral Vectors on Neurons and Glia in vitro
To establish a platform for evaluating gene function and therapeutic potential against neurodegeneration, IRP scientists examined the infectivity (tropism) and toxicity associated with recombinant adeno-associated viral (rAAV) vectors. rAAV are frequently used for gene delivery to the central nervous system and are capable of transducing neurons and glia in vitro. In this study, seven serotypes of a rAAV vector expressing green fluorescent protein (GFP) were characterized for tropism and toxicity in primary cortical cells derived from embryonic rat brain. At 2 days after transduction, serotypes 1, 5, 6, 7 and 8 expressed GFP predominately in glia, but by 6 days post-transduction expression was neuronal except for AAV5. AAV2 and 9 produced minimal GFP expression. Using LDH and MTS assays, toxicity was observed at higher multiplicities of infection (MOI) for all serotypes except AAV2 and 9. The toxicity of AAV1 and 5-8 affected mostly glia as indicated by a loss of glial-marker immunoreactivity. A frameshift mutation in the GFP gene reduced overall toxicity for serotypes 1, 5 and 6, but not 7 and 8 suggesting that the toxicity was not solely due to the overexpression of GFP. Collectively, a differential tropism and toxicity was observed among the AAV serotypes on primary cortical cultures with an overall preferential glial transduction and toxicity. Use of cell-restricted promoters may be necessary to limit expression to neurons and reduce gliotoxicity in vitro. Howard, D.B, Powers, K., Wang, Y. and Harvey, B.K. Poster, 2007. American Society for Gene Therapy, Seattle, WA, May 30 - June 3, 2007.

Development and Plasticity Section, Cellular Neurobiology Research Branch

Increases in Expression of 14-3-3 Eta and 14-3-3 Zeta Transcripts during Neuroprotection Induced by Delta9-Tetrahydrocannabinol in AF5 Cells
The molecular mechanisms involved in N-methyl-D-aspartate (NMDA)-induced cell death and Delta9-tetrahydrocannabinol (THC)-induced neuroprotection were investigated in vitro with an AF5 neural progenitor cell line model. By microarray analysis, Ywhah, CK1, Hsp60, Pdcd 4, and Pdcd 7 were identified as being strongly regulated by both NMDA toxicity and THC neuroprotection. The 14-3-3 eta (14-3-3eta; gene symbol Ywhah) and 14-3-3 zeta (14-3-3zeta; gene symbol Ywhaz) transcripts were deceased by NMDA treatment and increased by THC treatment prior to NMDA, as measured by cDNA microarray analysis and quantitative real-time RT-PCR. Other 14-3-3 isoforms were unchanged. Whereas up-regulation of 14-3-3zeta expression was observed 30 min after treatment with THC plus NMDA, down-regulation by NMDA alone was not seen until 16 hr after treatment. By Western blotting, THC increased 14-3-3 protein only in cells that were also treated with NMDA. Overexpression of 14-3-3eta or 14-3-3zeta by transient plasmid transfection increased 14-3-3 protein levels and decreased NMDA-induced cell death. These data suggest that increases in 14-3-3 proteins mediate THC-induced neuroprotection under conditions of NMDA-induced cellular stress. Chen, J., Lee, C.T., Errico, S.L., Becker, K.G., and Freed, W.J. Journal of Neuroscience Research, 85(8), pp. 1724-1733, 2007.

Microarray Analysis of Oxidative Stress Regulated Genes in Mesencephalic Dopaminergic Neuronal Cells: Relevance to Oxidative Damage in Parkinson's Disease
Oxidative stress and apoptotic cell death have been implicated in the dopaminergic cell loss that characterizes Parkinson's disease. While factors contributing to apoptotic cell death are not well characterized, oxidative stress is known to activate an array of cell signaling molecules that participate in apoptotic cell death mechanisms. IRP researchers investigated oxidative stress-induced cytotoxicity of hydrogen peroxide (H2O2) in three cell lines, the dopaminergic mesencephalon-derived N27 cell line, the GABAergic striatum-derived M213-20 cell line, and the hippocampal HN2-5 cell line. N27 cells were more sensitive to H2O2-induced cell death than M213-20 and HN2-5 cells. H2O2 induced significantly greater increases in caspase-3 activity in N27 cells than in M213-20 cells. H2O2-induced apoptotic cell death in N27 cells was mediated by caspase-3-dependent proteolytic activation of PKCdelta. Gene expression microarrays were employed to examine the specific transcriptional changes in N27 cells exposed to 100 microM H2O2 for 4 h. Changes in genes encoding pro- or anti-apoptotic proteins included up-regulation of BIK, PAWR, STAT5B, NPAS2, Jun B, MEK4, CCT7, PPP3CC, and PSDM3, while key down-regulated genes included BNIP3, NPTXR, RAGA, STK6, YWHAH, and MAP2K1. Overall, the changes indicate a modulation of transcriptional activity, chaperone activity, kinase activity, and apoptotic activity that appears highly specific, coordinated and relevant to cell survival. Utilizing this in vitro model to identify novel oxidative stress-regulated genes may be useful in unraveling the molecular mechanisms underlying dopaminergic degeneration in Parkinson's disease. Anantharam, V., Lehrmann, E., Kanthasamy, A., Yang, Y., Banerjee, P., Becker, K.G., Freed, W.J., and Kanthasamy, A.G. Neurochemistry International, 50(6), pp. 834-847, 2007.

Role of Heparin Binding Growth Factors in Nigrostriatal Dopamine System Development and Parkinson's Disease
The developmental biology of the dopamine (DA) system may hold important clues to its reconstruction. IRP scientists hypothesized that factors highly expressed during nigrostriatal development and re-expressed after injury and disease may play a role in protection and reconstruction of the nigrostriatal system. Examination of gene expression in the developing striatum suggested an important role for the heparin binding growth factor family at time points relevant to establishment of dopaminergic innervation. Midkine, pleiotrophin (PTN), and their receptors syndecan-3 and receptor protein tyrosine phosphatase beta/zeta, were highly expressed in the striatum during development. Furthermore, PTN was up-regulated in the degenerating substantia nigra of Parkinson's patients. The addition of PTN to ventral mesencephalic cultures augmented DA neuron survival and neurite outgrowth. Thus, PTN was identified as a factor that plays a role in the nigrostriatal system during development and in response to disease, and may therefore be useful for neuroprotection or reconstruction of the DA system. Marchionini, D.M., Lehrmann, E., Chu, Y., He, B., Sortwell, C.E., Becker, K.G., Freed, W.J., Kordower, J.H., and Collier, T.J. Brain Research, 25:1147, pp. 77-88, 2007.

Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Apoptotic and Behavioural Consequences of Mild Brain Trauma in Mice: Markers for Prospective Intervention
Mild traumatic brain injury (mTBI) is a not uncommon event in adolescents and young adults, and although not resulting in clear morphological brain defects, it is associated with long-term cognitive, emotional and behavioural problems. The focus of the present study was to characterize the biochemical and behavioural changes associated with experimental mTBI in mice that may act as either targets or surrogate markers for interventional therapy. Specifically, mTBI was induced by 30 g and 50 g weight drop and, at 8 and 72 hr thereafter, markers of cellular apoptosis - caspase-3, Bax, apoptosis inducing factor (AIF) and cytochrome-c (Cyt-c) - were quantified by Western blot analysis in hippocampus ipsilateral to impact. Levels of amyloid- precursor protein (APP) were also measured, and specific behavioural tests, the passive avoidance, open field and forced swimming (Porsolt) paradigms, were undertaken to assess learning, emotionality and emotional memory. In the absence of hemorrhage or infarcts, as assessed by triphenyltetrazolium chloride staining, procaspase-3 and Bax levels were dramatically altered following mTBI at both times. No cleaved caspase-3 was detected, and levels of AIF and Cyt-c, but not APP, were significantly changed at 72 hr. Mice subjected to mTBI were indistinguishable from controls by neurological examination at 1 and 24 hr, and by passive avoidance/open field at 72 hr; but could be differentiated in the forced swimming paradigm. In general, this model mimics the diffuse effects of mTBI on brain function associated with the human condition, and highlights specific apoptotic proteins and a behavioural paradigm as potential markers for prospective interventional strategies. Tweedie, D., Milman, A., Holloway, H.W., Li, Y., Harvey, B.K., Shen, H., Pistell, P.J., Lahiri, D.K., Hoffer, B.J., Wang, Y., Pick, C.G., and Greig, N.H. Journal of Neuroscience Research, 85(4), pp. 805-815, 2007.

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

MALDI-Ion Mobility-TOFMS Imaging of Lipids in Rat Brain Tissue
While maintaining anatomical integrity, matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) has allowed researchers to directly probe tissue, map the distribution of analytes and elucidate molecular structure with minimal preparation. MALDI-ion mobility (IM)-orthogonal time-of-flight mass spectrometry (oTOFMS) provides an advantage by initially separating different classes of biomolecules such as lipids, peptides, and nucleotides by their IM drift times prior to mass analysis. In the present work, the distribution of phosphatidlycholine and cerebroside species was mapped from 16 microm thick coronal rat brain sections using MALDI-IM-oTOFMS. Furthermore, the use of gold nanoparticles as a matrix enables detection of cerebrosides, which although highly concentrated in brain tissue, are not easily observed as positive ions because of intense signals from lipids such as phosphatidlycholines and sphingomyelins. Jackson, S.N., Ugarov, M., Egan, T., Post, J.D., Langlais, D., Albert Schultz, J., and Woods, A.S. Journal of the American Society for Mass Spectrometry, 42(8), pp. 1093-1098, 2007.

Tag-Mass: Specific Molecular Imaging of Transcriptome and Proteome by Mass Spectrometry Based on Photocleavable Tag
MALDI tissue imaging of tissues has become a promising technique for tracking biomarkers while determining their location and structural characterization. IRP scientists have now developed specific targeting probes (oligonucleotides, antibodies), named Tag-Mass. This approach is based on probes modified with a photocleavable linker coupled with a tag cleaved and detected using mass spectrometry. Tag-Mass development is the key for a rapid, sensitive, and accurate approach to correlate levels of expression of different mRNA or proteins in diseases. Lemaire, R., Stauber, J., Wisztorski, M., Van Camp, C., Desmons, A., Deschamps, M., Proess, G., Rudlof, I., Woods, A.S., Day, R., Salzet, M., and Fournier, I. Journal of Proteome Research, 6(6), pp. 2057-2067, 2007.

Molecular Neurobiology Research Branch

Molecular Genetics of Nicotine Dependence and Abstinence Genome Wide Association
Classical genetic studies indicate that nicotine dependence is a substantially heritable complex disorder. Genetic vulnerabilities to nicotine dependence largely overlap with genetic vulnerabilities to dependence on other addictive substances. Successful abstinence from nicotine displays substantial heritable components as well. Some of the heritability for the ability to quit smoking appears to overlap with the genetics of nicotine dependence and some does not. IRP researchers now report genome wide association studies of nicotine dependent individuals who were successful in abstaining from cigarette smoking, nicotine dependent individuals who were not successful in abstaining and ethnically-matched control subjects free from substantial lifetime use of any addictive substance. These data, and their comparison with data that the authors have previously obtained from comparisons of four other substance dependent vs. control samples support two main ideas: 1) Single nucleotide polymorphisms (SNPs) whose allele frequencies distinguish nicotine-dependent from control individuals identify a set of genes that overlaps significantly with the set of genes that contain markers whose allelic frequencies distinguish the four other substance dependent vs. control groups (p < 0.018). 2) SNPs whose allelic frequencies distinguish successful vs. unsuccessful abstainers cluster in small genomic regions in ways that are highly unlikely to be due to chance (Monte Carlo p < 0.00001). These clustered SNPs nominate candidate genes for successful abstinence from smoking that are implicated in interesting functions: cell adhesion, enzymes, transcriptional regulators, neurotransmitters and receptors and regulation of DNA, RNA and proteins. As these observations are replicated, they will provide an increasingly-strong basis for understanding mechanisms of successful abstinence, for identifying individuals more or less likely to succeed in smoking cessation efforts and for tailoring therapies so that genotypes can help match smokers with the treatments that are most likely to benefit them. Uhl, G.R., Liu, Q.R., Drgon, T., Johnson, C., Walther, D., and Rose, J.E. BMC Genetics, 8, pp. 10 - 15, 2007.

Molecular Genetics of Nicotine Dependence and Abstinence Genome Wide Association
Addictions are substantially heritable complex disorders. IRP investigatorsd report whole genome association studies that identify 89 genes likely to contain variants that contribute to addiction vulnerability, using previously- and newly-validated microarray based pooling assays. Each gene contains clustered single nucleotide polymorphisms (SNPs) that display significant allele frequency differences between abusers and controls in each of the two samples studied with 639,401 SNP arrays and confirmatory SNPs from each of two other abuser/control samples. These genes are implicated in interesting functions, including "cell adhesion" processes that help to establish and maintain neuronal connections of special relevance to addiction's memory-like features. Liu Q.R., Drgon T., Johnson C., Walther D., Hess J., and Uhl G.R. Am J Med Genet B Neuropsychiatr Genet. 141, pp. 918-925, 2006.

Medicinal Chemistry Section, Medications Discovery Research Branch

Novel Metabotropic Glutamate Receptor Subtype 5 Antagonists
The metabotropic glutamate receptor subtype 5 (mGluR5) has been implicated in drug abuse and other neuropsychiatric disorders. The lead compounds for this receptor subtype, MPEP and MTEP, have provided important tools with which to study the role these receptors play in the central nervous system, but have limited therapeutic application. In this report, IRP scientists followed a lead from our previously described heterobicyclic amides by incorporating a heterobicyclic ring structure into the molecule. One novel quinoline analogue demonstrated high affinity for mGluR5 receptors (Ki=110 nM) and moderately potent antagonist activity (IC50 = 260 nM) in the functional assay measuring the hydrolysis of phosphoinositide at mGluR5 in CHO cells. Hence a new template for further structure-activity relationship investigation was discovered and the resulting compounds will provide new molecular tools with which to further study the mGluR5 and its role in CNS disorders. Kulkarni, S.S., and Newman A.H. Bioorganic Medicinal Chemistry Letters, 17, pp. 2987-2991, 2007.

Clinical Psychopharmacology Section, Chemical Biology Research Branch

Restoration of 3,4-methylenedioxymethamphetamine-induced 5-HT Depletion by the Administration of l-5-hydroxytryptophan
3,4-methylenedioxymethamphetamine (MDMA) causes persistent decreases in brain serotonin (5-HT) content and 5-HT transporter (SERT) binding, with no detectable changes in SERT protein. Such data suggest that MDMA impairs 5-HT transmission but leaves 5-HT nerve terminals intact. To further test this hypothesis, IRP researchers carried out two types of experiments in rats exposed to high-dose MDMA. First, they examined the effects of MDMA on SERT binding and function using different in vitro assay conditions. Next, they treated rats with the 5-HT precursor, L-5-hydroxytryptophan (5-HTP), in an attempt to restore MDMA-induced depletions of 5-HT. Rats received saline or MDMA injections (7.5 mg/kg ip, q 2 h x 3 doses); two weeks later, rats were allocated to groups. Rats in one group were decapitated, and brain tissue was assayed for SERT binding and [3H]5-HT uptake under conditions of normal (100 or 126 mM) and low (20 mM) NaCl concentration. Rats from another group received saline or 5-HTP/benserazide (5-HTP-B), each drug at 50 mg/kg ip, and were sacrificed 2 hr later. MDMA reduced SERT binding to 10% of control when assayed in 100 mM NaCl, but this reduction was only 55% of control in 20 mM NaCl. MDMA decreased immunoreactive 5-HT in caudate and hippocampus to about 35% of control. Administration of 5-HTP-B to MDMA-pretreated rats significantly increased the 5-HT signal towards normal levels in caudate (85 % of control) and hippocampus (66 % of control). Authors concluded that: 1) Following high-dose MDMA treatment sufficient to reduce SERT binding by 90%, a significant number of functionally intact 5-HT nerve terminals survive. 2) The degree of MDMA-induced decreases in SERT binding depends on the in vitro assay conditions. 3) 5-HTP-B restores brain 5-HT depleted by MDMA, suggesting that this approach might be clinically useful in abstinent MDMA users. Wang, X., Baumann, M.H., Dersch, C.M., and Rothman, R.B. J Pharmacol Exp Ther [2007 Jul 11, epub], 2007.

Amphetamine Analogs Increase Plasma Serotonin: Implications for Cardiac and Pulmonary Disease
Elevations in plasma serotonin (5-HT) have been implicated in the pathogenesis of cardiac and pulmonary disease. Normally, plasma 5-HT concentrations are kept low by transporter-mediated uptake of 5-HT into platelets and by metabolism to 5-hydroxyindoleacetic acid (5-HIAA). Many abused drugs (e.g., substituted amphetamines) and prescribed medications (e.g., fluoxetine) target 5-HT transporters and could thereby influence circulating 5-HT. IRP investigators evaluated the effects of amphetamines analogs [(+/-)-fenfluramine, (+/-)-3,4-methylenedioxymethamphetamine, (+)-methamphetamine, (+)-amphetamine, phentermine] on extracellular levels (i.e., plasma levels) of 5-HT and 5-HIAA in blood from catheterized rats. Effects of the 5-HT uptake blocker fluoxetine were examined for comparison. Drugs were tested in vivo and in vitro; plasma indoles were measured using a novel microdialysis method in whole blood. The authors found that baseline dialysate levels of 5-HT are approximately 0.22 nM, and amphetamine analogs evoke large dose-dependent increases in plasma 5-HT ranging from 4 to 20 nM. The ability of drugs to elevate plasma 5-HT is positively correlated with their potency as 5-HT transporter substrates. Fluoxetine produced small, but significant, increases in plasma 5-HT. Although the drug-evoked 5-HT concentrations are below the micromolar levels required for contraction of pulmonary arteries, they approach concentrations reported to stimulate mitogenesis in pulmonary artery smooth muscle cells. Additional studies are needed to determine the effects of chronic administration of amphetamines on circulating 5-HT. Zolkowska, D., Rothman, R.B., and Baumann, M.H. J Pharmacol Exp Ther. 318, pp. 604-610, 2006.

Behavioral Neuroscience Section, Behavioral Neuroscience Research Branch

Stress-induced Relapse to Cocaine Seeking: Roles for the CRF(2) Receptor and CRF-binding Protein in the Ventral Tegmental Area of the Rat
Footshock reinstates cocaine seeking in cocaine-experienced rats by inducing corticotropin-releasing factor (CRF) and glutamate release in the ventral tegmental area (VTA) and thus activating VTA dopaminergic neurons. Footshock-induced VTA glutamate release, dopamine activation and reinstatements are blocked by VTA administration of an alpha-helical CRF, a nonselective CRF receptor antagonist. The effects of selective CRF antagonists have not yet been reported. The present studies were designed to explore the roles of VTA CRF receptor subtypes and CRF-BP in these effects induced by footshock. Rats were first trained to lever-press for intravenous cocaine (1 mg/infusion/0.13 ml, FR-1 schedule), and then tested under extinction conditions until response rates returned to the pretraining baseline. Reinstatements, VTA glutamate and dopamine levels [microdialysis with high performance liquid chromatography (HPLC)] were then assessed, under various pharmacological conditions, after mild inescapable footshock. Footshock-induced reinstatement of cocaine seeking and release of VTA glutamate and dopamine were blocked by selective blockade of VTA CRF(2) receptors (CRF(2)Rs) but not CRF(1)Rs. VTA perfusion of CRF or CRF(2)R agonists that have strong affinity for CRF-BP mimicked the effects induced by footshock while CRFR agonists that do not bind CRF-BP were ineffective. CRF(6-33), which competes for the CRF binding site on CRF-BP, attenuated the effects of CRF or urocortin I on VTA glutamate and dopamine release and on reinstatement of cocaine seeking. The present studies revealed a role of VTA CRF-BP and suggest an involvement of CRF(2)R in the effectiveness of stress in triggering glutamate and dopamine release and cocaine seeking in drug-experienced animals. Wang, B., You, Z.B., Rice, K.C., and Wise, R.A. Psychopharmacology, 193, pp. 283-294, 2007.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

Blocking of Conditioning to a Cocaine-Paired Stimulus: Testing the Hypothesis that Cocaine Perpetually Produces a Signal of Larger-Than-Expected Reward
According to a recent account of addiction, dopaminergic effects of drugs like cocaine mimic the neuronal signal that occurs when a natural reward has a larger value than expected. Consequently, the drug's expected reward value increases with each administration, leading to an over-selection of drug-seeking behavior. One prediction of this hypothesis is that the blocking effect, a cornerstone of contemporary learning theory, should not occur with drug reinforcers. To test this prediction, two groups of rats were trained to self-administer cocaine with a nose-poking response. For 5 sessions, a tone was paired with each self-administered injection (blocking group), or no stimulus was paired with injection (non-blocking group). Then, in both groups, the tone and a light were both paired with each injection for 5 sessions. In subsequent testing, the light functioned as a conditioned reinforcer for a new response (lever-pressing) in the non-blocking group, but not the blocking group. Thus, contrary to prediction, pre-training with the tone blocked conditioning to the light. Although these results fail to support a potentially powerful explanation of addiction, they are consistent with the fact that most conditioning and learning phenomena that occur with non-drug reinforcers can also be demonstrated with drug reinforcers. Panlilio, L.V., Thorndike, E.B. and Schindler, C.W. Pharmacology, Biochemistry and Behavior, 86, pp. 774-777, 2007.

Adenosine Receptor-Dopamine Receptor Interactions in the Basal Ganglia and Their Relevance for Brain Function
The dopamine D(1) and D(2) receptors are major receptors in the regulation of striatal function and striatal adenosine A(1) and A(2A) receptors are major modulators of their signaling. The evidence suggests the existence of antagonistic A(1)-D(1) heteromeric receptor complexes in the basal ganglia and prefrontal cortex and especially in the direct striatonigral-striatoentopeduncular GABA pathways. The neurochemical and behavioral findings showing antagonistic A(1)-D(1) receptor interactions can be explained by the existence of such A(1)-D(1) heteromeric receptor complexes and of antagonistic interactions at the level of the second messengers. In contrast, A(2A)-D(2) receptor heteromers may exist in the dorsal and ventral striato-pallidal GABA pathways, where activation of A(2A) receptors reduces D(2) receptor recognition, coupling and signaling. As a result of the A(2A) receptor-induced reduction of D(2) receptor signaling, the activity of these GABA neurons is increased resulting in reduced motor and reward functions mediated via the indirect pathway, causing a reduced glutamate drive to the prefrontal and motor areas of the cerebral cortex. Thus, A(2A) receptor antagonists and A(2A) receptor agonists, respectively, may offer novel treatments of Parkinson's disease (reduced D(2) receptor signaling) and of schizophrenia and drug addiction (increased D(2) receptor signaling). Fuxe, K., Ferre, S., Genedani, S., Franco, R. and Agnati, L.F. Physiol Behav, May 21, 2007, Epubmed ahead of print, PMID 17572452.

Adenosine A(2A) Receptors in Ventral Striatum, Hypothalamus and Nociceptive Circuitry: Implications for Drug Addiction, Sleep and Pain
Adenosine A(2A) receptors localized in the dorsal striatum are considered as a new target for the development of antiparkinsonian drugs. Co-administration of A(2A) receptor antagonists has shown a significant improvement of the effects of l-DOPA. The present review emphasizes the possible application of A(2A) receptor antagonists in pathological conditions other than parkinsonism, including drug addiction, sleep disorders and pain. In addition to the dorsal striatum, the ventral striatum (nucleus accumbens) contains a high density of A(2A) receptors, which presynaptically and postsynaptically regulate glutamatergic transmission in the cortical glutamatergic projections to the nucleus accumbens. It is currently believed that molecular adaptations of the cortico-accumbens glutamatergic synapses are involved in compulsive drug seeking and relapse. Here IRP scientists review recent experimental evidence suggesting that A(2A) antagonists could become new therapeutic agents for drug addiction. Morphological and functional studies have identified lower levels of A(2A) receptors in brain areas other than the striatum, such as the ventrolateral preoptic area of the hypothalamus, where adenosine plays an important role in sleep regulation. Although initially believed to be mostly dependent on A(1) receptors, here the authors review recent studies that demonstrate that the somnogenic effects of adenosine are largely mediated by hypothalamic A(2A) receptors. A(2A) receptor antagonists could therefore be considered as a possible treatment for narcolepsy and other sleep-related disorders. Finally, nociception is another adenosine-regulated neural function previously thought to mostly involve A(1) receptors. Although there is some conflicting literature on the effects of agonists and antagonists, which may partly be due to the lack of selectivity of available drugs, the studies in A(2A) receptor knockout mice suggest that A(2A) receptor antagonists might have some therapeutic potential in pain states, in particular where high intensity stimuli are prevalent. Ferre, S., Diamond, I., Goldberg, S.R., Yao, L., Hourani, S.M., Huang, Z.L., Urade, Y. and Kitchen, I. Prog Neurobiol, May 1, 2007, Epubmed ahead of print, PMID 17532111.

5-HT(1B) Receptor-Mediated Serotoninergic Modulation of Methylphenidate-Induced Locomotor Activation in Rats
Previous studies have shown that the dopamine (DA) uptake blocker methylphenidate, a psychostimulant widely used for the treatment of attention-deficit hyperactivity disorder (ADHD), prevents the neurotoxic effects of the highly abused DA releaser methamphetamine. However, there is a lack of information about the pharmacological interactions of these two drugs at the behavioral level. When systemically administered within an interval of 2 h, previous administration of methylphenidate (10 mg/kg, intraperitoneal (i.p.)) did not modify locomotor activation induced by methamphetamine. On the other hand, previous administration of methamphetamine (1 mg/kg, i.p.) markedly potentiated methylphenidate-induced motor activation. With in vivo microdialysis experiments, methamphetamine and methylphenidate were found to increase DA extracellular levels in the nucleus accumbens (NAs). Methamphetamine, but not methylphenidate, significantly increased the extracellular levels of serotonin (5-HT) in the NAs. Methamphetamine-induced 5-HT release remained significantly elevated for more than 2 h after its administration, suggesting that the increased 5-HT could be responsible for the potentiation of methylphenidate-induced locomotor activation. In fact, previous administration of the 5-HT uptake blocker fluoxetine (10 mg/kg, i.p.) also potentiated the motor activation induced by methylphenidate. A selective 5-HT(1B) receptor antagonist (GR 55562; 1 mg/kg), but not a 5-HT(2) receptor antagonist (ritanserin; 2 mg/kg, i.p.), counteracted the effects of methamphetamine and fluoxetine on the motor activation induced by methylphenidate. Furthermore, a 5-HT(1B) receptor agonist (CP 94253; 1-10 mg/kg, i.p.) strongly and dose-dependently potentiated methylphenidate-induced locomotor activation. The 5-HT(1B) receptor-mediated modulation of methylphenidate-induced locomotor activation in rat could have implications for the treatment of ADHD. Borycz, J., Zapata, A., Quiroz, C., Volkow, N.D. and Ferre, S. Neuropsychopharmacology, May 9, 2007, Epubmed ahead of print, PMID 17487226.

Neurotransmitter Receptor Heteromers and Their Integrative Role in 'Local Modules': The Striatal Spine Module
'Local module' is a fundamental functional unit of the central nervous system that can be defined as the minimal portion of one or more neurons and/or one or more glial cells that operates as an independent integrative unit. This review focuses on the importance of neurotransmitter receptor heteromers for the operation of local modules. To illustrate this, IRP scientists use the striatal spine module (SSM), comprised of the dendritic spine of the medium spiny neuron (MSN), its glutamatergic and dopaminergic terminals and astroglial processes. The SSM is found in the striatum, and although aspects such as neurotransmitters and receptors will be specific to the SSM, some general principles should apply to any local module in the brain. The analysis of some of the receptor heteromers in the SSM shows that receptor heteromerization is associated with particular elaborated functions in this local module. Adenosine A(2A) receptor-dopamine D(2) receptor-glutamate metabotropic mGlu(5) receptor heteromers are located adjacent to the glutamatergic synapse of the dendritic spine of the enkephalin MSN, and their cross-talk within the receptor heteromers helps to modulate postsynaptic plastic changes at the glutamatergic synapse. A(1) receptor-A(2A) receptor heteromers are found in the glutamatergic terminals and the molecular cross-talk between the two receptors in the heteromer helps to modulate glutamate release. Finally, dopamine D(2) receptor-non-alpha(7) nicotinic acetylcholine receptor heteromers, which are located in dopaminergic terminals, introduce the new concept of autoreceptor heteromer. Ferre, S., Agnati, L.F., Ciruela, F., Lluis, C., Woods, A. S., Fuxe, K. and Franco, R. Brain Res Rev, January 27, 2007, Epubmed ahead of print, PMID 17408563.

Striatal Adenosine A(2A) and Cannabinoid CB(1) Receptors Form Functional Heteromeric Complexes that Mediate the Motor Effects of Cannabinoids
The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB(1) receptors, is still a matter of debate. In the present study, IRP researchers report that CB(1) and adenosine A(2A) receptors form heteromeric complexes in co-transfected HEK-293T cells and rat striatum, where they colocalize in fibrilar structures. In a human neuroblastoma cell line, CB(1) receptor signaling was found to be completely dependent on A(2A) receptor activation. Accordingly, blockade of A(2A) receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB(1) receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A(2A) and CB(1) receptors. Carriba, P., Ortiz, O., Patkar, K., Justinova, Z., Stroik, J., Thermann, A., Muller, C., Woods, A.S., Hope, B.T., Ciruela, F., Casado, V., Canela, E.I., Lluis, C., Goldberg, S.R., Moratalla, R., Franco, R. and Ferre, S. Neuropsychopharmacology, Mar 14, 2007; Epubmed ahead of print, PMID 17356572.

Nicotinic Alpha 7 Receptors as a New Target for Treatment of Cannabis Abuse
Increasing use of cannabis makes the search for medications to reduce cannabis abuse extremely important. Here, IRP scientists show that homomeric alpha7 nicotinic receptors are novel molecular entities that could be targeted in the development of new drugs for the treatment of cannabis dependence. In rats, systemic administration of the selective alpha7 nicotinic acetylcholine receptor antagonist methyllycaconitine (MLA), but not the selective heteromeric non-alpha7 nicotinic acetylcholine receptor antagonist dihydrobetaerythroidine, (1) antagonized the discriminative effects of delta-9-tetrahydrocannabinol (THC), the main active ingredient in cannabis, (2) reduced intravenous self-administration of the synthetic cannabinoid CB1 receptor agonist WIN55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone, mesylate salt], and (3) decreased THC-induced dopamine elevations in the shell of the nucleus accumbens. Altogether, these results indicate that blockade of alpha7 nicotinic receptors reverses abuse-related behavioral and neurochemical effects of cannabinoids. Importantly, MLA reversed the effects of cannabinoids at doses that did not produce depressant or toxic effects, further pointing to alpha7 nicotinic antagonists as potentially useful agents in the treatment of cannabis abuse in humans. Solinas, M., Scherma, M., Fattore, L., Stroik, J., Wertheim, C., Tanda, G., Fratta, W. and Goldberg, S.R. Journal of Neuroscience, 27, pp. 5615-5620, 2007.

Neurobiology of Relapse Section, Behavioral Neuroscience Research Branch

Systemic and Central Amygdala Injections of the mGluR(2/3) Agonist LY379268 Attenuate the Expression of Incubation of Sucrose Craving in Rats
IRP researchers previously reported that systemic or central amygdala injections of the mGluR(2/3) agonist LY379268 (which decreases glutamate release) prevented enhanced cue-induced cocaine seeking in extinction tests after prolonged withdrawal (incubation of cocaine craving). Here, authors report that systemic and central amygdala injections of LY379268 also prevented the enhanced cue-induced sucrose seeking in extinction tests after prolonged sucrose-free period (incubation of sucrose craving). These findings suggest that central amygdala glutamate plays an important role in the incubation of reward craving after withdrawal from both drug and non-drug rewards. Uejima, J.L., Bossert, J.M., Poles, G.C., and Lu, L. Behavioral Brain Research, 181, pp. 292-296, 2007.

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

Consistency and Reliability of Subjective Responses to Imagery-induced Tobacco Craving
Although studies have demonstrated the validity of imagery procedures to elicit tobacco craving responses in single sessions, few studies have examined the consistency of responding in the same individuals over multiple experimental sessions. In this study, nondeprived smokers were presented with a randomized series of imagery scripts that varied in the intensity of smoking urge content. At each of five sessions spaced over several weeks, participants were exposed to six imagery trials (two each of no-, low-, and high-intensity imagery scripts). After each trial, participants completed subjective measures of tobacco craving and mood. Ratings of craving and negative mood significantly increased as a function of smoking-urge intensity, which was consistent across the five sessions. Further, significant intraclass correlations indicated that craving and mood responses were highly reliable over the five sessions, as well as across two, three, and four sessions. These results have practical implications for examining individual differences in sensitivity to smoking cues and for studies involving repeated measurement of elicited craving over time. Lee, D.C., Myers, C.S., Taylor, R.C., Moolchan, E.T. and Heishman, S.J. Addictive Behaviors, Epub ahead of print, 2007.

Tobacco Craving Predicts Lapse to Smoking in Adolescent Smokers
Previous research indicates that tobacco craving predicts relapse to smoking among adult smokers attempting to quit. IRP scientists hypothesized a similar relationship between craving and lapse among adolescent smokers during the treatment phase of a clinical trial. A visit was considered a lapse visit if the participant reported smoking or had a CO >= 7 ppm subsequent to an abstinent visit. Thirty-four participants (mean +/- SD, age 14.9 +/- 1.3 years, cigarettes per day 18.0 +/- 7.6, Fagerstroem Test for Nicotine Dependence 6.8 +/- 1.34, 65% female), were included in the current analysis of 167 treatment visits. Logistic regression analyses showed a positive relationship between degree of craving, measured by the Questionnaire on Smoking Urges, and lapse during smoking cessation treatment (p = 0.013). Additionally, linear regression analyses demonstrated a strong positive association between cigarettes smoked per day and craving scores (p < 0.001). Taken together with other data, these findings suggest that degree of craving might influence tobacco abstinence for adolescent smokers. Thus, monitoring and addressing craving appears useful to increase the success of adolescent smoking cessation. Bagot, K.S., Heishman, S.J. and Moolchan, E.T. Nicotine and Tobacco Research, 9, pp. 647-652, 2007.