Research Findings - International Research

NIDA-Supported Researchers Identify New Injection Practice Among Tanzanian Women

Dr. Mark Williams, University of Texas at Houston and colleagues (Dr. Sheryl McCurdy, University of Texas at Houston; Dr. Gad P. Kilonzo and Dr. M. T. Lesheaberi, University of Muhumbili, Dar Es Salaam, Tanzania) presented their recent epidemiological findings on Tanzanian IDUs and HIV at a conference December 1, 2006 - World AIDS Day - at the University of Muhumbili. The research was supported by a NIDA International Program collaborative research supplement (NOT-02-003) and a subsequent R21 (DA19394). The binational research team reports that heroin injection and risky injection practices are continuing to increase in Dar Es Salaam and spreading outward to neighboring communities. Using modified snowball sampling and outreach, the team recruited 537 heroin IDUs in Dar Es Salaam (318 male, 219 female); 42% of whom tested HIV positive (27% among males, 64% among females). The team observed a new and unusual practice among women IDUs - termed "flashblood" - where IDUs share blood-filled syringes after one has injected heroin. The research has been published in AIDS Care, June 2005; 17(Supplement 1): S65-S76; BMJ 2005; 331: 778-781; Drug and Alcohol Dependence 82 Supplement 1(2006): S23-S27; and AIDS Behavior DOI 10.1007/s10461-006-9102-x. Participants at the conference included representatives from the University of Muhumbili medical faculty; the Tanzanian Ministry of Labor, Employment, and Youth; the Tanzanian Ministry of Health; other Tanzanian officials; and invited media. The binational research team also met with Tanzanian Deputy Minister of Labor, Employment, and Youth Dr. Emmanuel Nchimbi on October 18, 2006.

DISCA-Supported Research Suggests Fluoxetine May Be an Effective Methamphetamine Pharmacotherapy

In research published by the Annals of the New York Academy of Science (2006 Aug;1074:295-302), a binational research team supported in part by a 2004 NIDA Distinguished International Scientist Collaboration Award (DISCA) concludes that fluoxetine may be a useful tool for treating methamphetamine (METH) dependence. The DISCA Scientist, Dr. Kazutaka Ikeda, Tokyo Institute of Psychiatry, Japan, his research partner, Dr. Athina Markou, The Scripps Research Institute, and their colleagues investigated the effects of intraperitoneal (i.p.) injections of 20 mg/kg fluoxetine, a selective serotonin reuptake inhibitor (SSRI), on 2 mg/kg METH (i.p.) conditioned place preference (CPP) and locomotor sensitization to 1 mg/kg METH (i.p.) in C57BL/6J mice. Fluoxetine treatment before both the conditioning and preference tests abolished METH CPP. A two-way analysis of variance (ANOVA) revealed that METH CPP tended to be lower in mice pretreated with fluoxetine before the preference test than in control mice pretreated with saline before the preference test. Furthermore, pretreatment with fluoxetine had inhibitory effects on METH-induced locomotor sensitization. Following Dr. Ikeda's DISCA research exchange visit with Dr. Markou, the team received a 3-year grant from the U.S. - Japan Brain Research Cooperation Program as well as additional support from the Tokyo Institute of Psychiatry and the Japanese Society of Pharmacopoeia.

Research Publications by International Program Alumni

Alumni of the NIDA International Program research training and exchange programs authored or coauthored the following articles indexed by PubMed:

Former NIDA INVEST Drug Abuse Research Fellows

Variations of Alcohol Impairment in Different Types, Causes and Contexts of Injuries: Results of Emergency Room Studies from 16 Countries
Macdonald, S., Cherpitel, C.J., Desouza, A., Stockwell, T., Borges, G., and Giesbrecht, N. Accid Anal Prev. July 5, 2006 [Epub ahead of print]
INVEST Fellow: Guilherme Borges, Mexico, 1997-1998
The purpose of this paper is to document alcohol impairment (based on a blood alcohol content (BAC) of at least 80mg%) for different types, causes and location contexts of injuries. Data from 45 studies with 11,536 injury patients were merged to determine variations in the percent of alcohol impairment among injury patients. In each study, emergency room (ER) injury patients were given a short interview on the circumstances of their injury and BAC was measured. Injury severity, measured by number of body regions injured was significantly associated with BACs over 80mg%. The highest percentage of injury type to involve alcohol was head injury/concussion. In terms of causes of injuries, patients with alcohol impairment were significantly more likely to be involved in violence than any other cause (i.e., vehicle, falling, poisoning or burns). Finally, injuries occurring at a bar or restaurant were significantly more likely to involve alcohol impairment than any other setting. The results demonstrate considerable variation in the circumstances where alcohol is involved in injuries. These results may be useful for the development of prevention initiatives.

Treatment and Adequacy of Treatment of Mental Disorders among Respondents to the Mexico National Comorbidity Survey
Borges, G., Medina-Mora, M.E., Wang, P.S., Lara, C., Berglund, P., and Walters, E. Am J Psychiatry. 163(8), pp. 1371-1378, August 2006.
INVEST Fellow: Guilherme Borges, Mexico, 1997-1998
This study described the rate and adequacy of mental health service use among participants in the Mexico National Comorbidity Survey and the correlates of any 12-month treatment and of adequate treatment. The authors conducted face-to-face household surveys of a probability sample of individuals ages 18 to 65 years in the noninstitutionalized population living in urban areas of Mexico from 2001 to 2002. The use of mental health services and 12-month DSM-IV disorders was assessed with the World Mental Health version of the World Health Organization Composite International Diagnostic Interview. The rates and correlates of any service use and the adequacy of treatment were identified in logistic regression analyses, taking into account the complex sample design and weighting process. The data reported here were based on 2,362 interviews. Fewer than one in five respondents with any psychiatric disorder during the last 12 months used any service during the prior year. The rates of service use by those with mood disorders were somewhat higher. About one in every two respondents who used services received minimally adequate care. The authors found large unmet needs for mental health services among those with psychiatric disorders. Those with mental illness and those who deliver or seek to improve mental health care in Mexico face enormous challenges.

Influence of Blood Loss on the Pharmacokinetics of Citalopram
Kugelberg, F.C., Alkass, K., Kingback, M., Carlsson, B., and Druid, H. Forensic Sci Int. July 11, 2006 [Epub ahead of print]
INVEST Fellow: Henrik Druid, Sweden, 2001-2002
Extended blood loss results in several compensatory physiological mechanisms, including transfer of extravascular fluid into the blood circulation. If drugs are present in the body, this fluid exchange may imply that blood drug concentrations found in a trauma victim may differ from the concentrations present at the time of the trauma. To address this issue, an animal model was used to investigate the influence of blood loss on pre-existing levels of the antidepressant drug citalopram and its demethylated metabolites. Rats were administered citalopram either acutely (40mg/kg, orally) or chronically (20mg/kg daily, subcutaneously) for 6 days using osmotic pumps. In the experimental rats, blood loss was accomplished by withdrawing 0.8mL blood at 10min intervals during 70min. In the control rats, blood was withdrawn at 0 and 70min only. Blood, brain and lung drug concentrations were analyzed with an enantioselective HPLC method. In the chronically treated rats, the ratios between final and initial citalopram concentrations were 1.08+/-0.15 and 1.01+/-0.09 in the experimental rats and controls, respectively, indicating no major effect of blood loss. In contrast, acute oral administration resulted in increased ratios in the exsanguinated rats as compared to controls (1.84+/-0.50 versus 0.73+/-0.07; p=0.0495). In conclusion, the observation of increased blood drug levels in the acute oral rats indicates that absorption of fluid from the gastrointestinal tract may be important in the intravascular refill. Further, in the interpretation of post-mortem blood levels of drugs, these physiological mechanisms should be taken into account.

The u-opioid Receptor Gene and Smoking Initiation and Nicotine Dependence
Zhang, L., Kendler, K.S., and Chen, X. Behav Brain Funct. 2(1):28, August 4, 2006 [Epub ahead of print]
INVEST Fellow: Lan Zhang, China, 2004-2005
The gene encoding the mu-opioid receptor (OPRM1) is reported to be associated with a range of substance dependence. Experiments in knockout mice indicate that the mu-opioid receptor may mediate reinforcing effects of nicotine. In humans, opioid antagonist naltrexone may reduce the reinforcing effects of tobacco smoking. Additionally, the OPRM1 gene is located in a region showing linkage to nicotine dependence. The OPRM1 is thus a plausible candidate gene for smoking behavior. To investigate whether OPRM1 contributes to the susceptibility of smoking initiation and nicotine dependence, the authors genotyped 11 SNPs in the gene for 688 Caucasian subjects of lifetime smokers and nonsmokers. Three SNPs showed nominal significance for smoking initiation and one reached significance for nicotine dependence. The global test for three-marker (rs9479757-rs2075572-rs10485057) haplotypes was significant for smoking initiation (p = 0.0022). The same three-marker haplotype test was marginal (p = 0.0514) for nicotine dependence. These results suggest that OPRM1 may be involved in smoking initiation and nicotine dependence.

Effects of NAAG Peptidase Inhibitor 2-PMPA in Model Chronic Pain - Relation to Brain Concentration
Nagel, J., Belozertseva, I., Greco, S., Kashkin, V., Malyshkin, A., Jirgensons, A., Shekunova, E., Eilbacher, B., Bespalov, A., and Danysz, W. Neuropharmacology. August 18, 2006 [Epub ahead of print].
INVEST Fellow: Anton Bespalov, Russia, 1994-1995.
N-acetylated-alpha-linked-acidic peptidase (NAAG peptidase) converts N-acetyl-aspartyl-glutamate (NAAG, mGluR3 agonist) into N-acetyl-aspartate and glutamate. The NAAG peptidase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) had neuroprotective activity in an animal model of stroke and anti-allodynic activity in CCI model despite its uncertain ability to penetrate the blood-brain barrier. The NAAG concentration in brain ECF under basal conditions and its alteration in relation to the brain ECF concentration of 2-PMPA is unclear. Authors therefore assessed those brain concentrations after i.p. administration of 2-PMPA, using in vivo microdialysis combined with LC/MS/MS analysis. Administration of 2-PMPA (50mg/kg) produced a mean peak concentration of 2-PMPA of 29.66+/-8.1muM. This concentration is about 100,000 fold more than is needed for inhibition of NAAG peptidase, and indicates very good penetration to the brain. Application of 2-PMPA was followed by a linear increase of NAAG-concentration reaching a maximum of 2.89+/-0.42muM at the end of microdialysis. However, during the time the anti-allodynic effects of 2-PMPA were observed, the NAAG concentration in the ECF did not reach levels which are likely to have an impact on any known target. It appears therefore that the observed behavioral effects of 2-PMPA may not be mediated by NAAG nor, in turn, by mGluR3 receptors.

Lowered Brain Stimulation Reward Thresholds in Rats Treated with a Combination of Caffeine and N-methyl-D-aspartate but not Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate or Metabotropic Glutamate Receptor-5 Receptor Antagonists
Bespalov, A., Dravolina, O., Belozertseva, I., Adamcio, B., and Zvartau, E. Behav Pharmacol. 17(4), pp. 295-302, 2006.
INVEST Fellow: Anton Bespalov, Russia, 1994-1995.
Previous studies suggested that adenosine A1 and A2A receptor agonists counteract behavioral effects of N-methyl-D-aspartate (NMDA) receptor antagonists while adenosine receptor antagonists may produce opposite effects enhancing the actions of NMDA receptor antagonists. To further evaluate the effects of combined administration of adenosine receptor antagonist caffeine and various NMDA and non-NMDA glutamate receptor antagonists on brain stimulation reward (discrete-trial threshold current intensity titration procedure), rats with electrodes implanted into the ventral tegmental area were tested after pretreatment with NMDA receptor channel blocker MK-801 (0.01-0.3 mg/kg), competitive antagonist D-CPPene (0.3-5.6 mg/kg), glycine site antagonist L-701,324 (1.25-5 mg/kg), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor antagonist GYKI-53655 (1-10 mg/kg), metabotropic glutamate receptor 5 (mGluR5) antagonist MPEP (1-10 mg/kg) alone and in combination with caffeine (1-30 mg/kg). MK-801 (0.056 and 0.1 mg/kg) was the only tested glutamate antagonist that lowered self-stimulation thresholds, while D-CPPene (5.6 mg/kg) and MPEP (5.6 and 10 mg/kg) had the opposite effects. Threshold-increasing effects of D-CPPene, but not of MPEP, however, were associated with marked impairment of operant performance, reflected by longer latencies to respond and higher rates of responding during the inter-trial intervals. Operant performance was also disrupted by the highest dose of MK-801 (0.3 mg/kg). For subsequent experiments, caffeine (1-30 mg/kg) was combined with the highest doses of NMDA receptor antagonists that did not lower the brain stimulation reward thresholds and did not impair operant performance. Caffeine had no appreciable effects on self-stimulation behavior when given alone. A low dose of caffeine (3 mg/kg) significantly lowered self-stimulation thresholds only when given together with MK-801 (0.03 mg/kg) or D-CPPene (3 mg/kg). Combined with the same antagonist drugs, higher doses of caffeine (10 and 30 mg/kg) facilitated time-out responding. These results indicate that, within a limited dose range, caffeine in combination with an NMDA receptor channel blocker and a competitive antagonist significantly lowers brain stimulation reward thresholds in rats.

Effects of Group I Metabotropic Glutamate Receptor Antagonists on the Behavioral Sensitization to Motor Effects of Cocaine in Rats
Dravolina, O.A., Danysz. W., and Bespalov, A.Y. Psychopharmacology (Berl).187(4), pp. 397-404, 2006. Epub June 20, 2006.
INVEST Fellow: Anton Bespalov, Russia, 1994-1995.
Metabotropic glutamate receptors (mGluRs) were reported to regulate various behavioral effects of addictive drugs. The present study evaluated the role of group I mGluRs in the progressive augmentation ("sensitization") of the behavioral effects observed after repeated, intermittent cocaine exposure. After habituation to handling and baseline activity measurement (days 1-2), rats received eight injections of cocaine (10 mg/kg) or saline on days 3-6, 8-11, and then, were tested twice with acute saline and cocaine given in a counterbalanced manner on days 13 and 15. Before the test sessions, subjects were pretreated with mGluR1 antagonist EMQMCM (JNJ16567083, (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate) and mGluR5 antagonist MTEP ([(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine). Pretreatment with EMQMCM (2.5-10 mg/kg) but not MTEP (2.5-10 mg/kg) significantly reduced expression of the sensitized ambulatory motor activity of the cocaine-experienced animals acutely challenged with cocaine. Both EMQMCM and MTEP significantly reduced vertical motor activity across all cocaine/saline treatment conditions. These findings indicate that the expression of behavioral sensitization to cocaine-induced stimulation of locomotor activity may be modulated by group I mGluR antagonists (mGluR1 rather than mGluR5), but these effects occur at the dose levels that attenuate vertical activity.

A Risk Index for 12-month Suicide Attempts in the National Comorbidity Survey Replication (NCS-R)
Borges, G., Angst, J., Nock, M.K., Ruscio, A.M., Walters, E.E., and Kessler, R.C. Psychol Med. August 29, 2006, pp. 1-11 [Epub ahead of print].
INVEST Fellow: Guilherme Borges, Mexico, 1997-1998
Clinical judgments about the likelihood of suicide attempt would be aided by an index of risk factors that could be quickly assessed in diverse settings. The authors sought to develop such a risk index for 12-month suicide attempts among suicide ideators. The National Comorbidity Survey Replication (NCS-R), a household survey of adults aged 18+, assessed the 12-month occurrence of suicide ideation, plans and attempts in a subsample of 5692 respondents. Retrospectively assessed correlates include history of prior suicidality, sociodemographics, parental psychopathology and 12-month DSM-IV disorders. Twelve-month prevalence estimates of suicide ideation, plans and attempts are 2.6, 0.7 and 0.4% respectively. Although ideators with a plan are more likely to make an attempt (31.9%) than those without a plan (9.6%), 43% of attempts were described as unplanned. History of prior attempts is the strongest correlate of 12-month attempts. Other significant correlates include shorter duration of ideation, presence of a suicide plan, and several sociodemographic and parental psychopathology variables. Twelve-month disorders are not powerful correlates. A four-category summary index of correlates is strongly related to attempts among ideators [area under the receiver operator characteristic curve (AUC)=0.88]. The distribution (conditional probability of attempt) of the risk index is: 19.0% very low (0.0%), 51.1% low (3.5%), 16.2% intermediate (21.3%), and 13.7% high (78.1%). Two-thirds (67.1%) of attempts were made by ideators in the high-risk category. A short, preliminary risk index based on retrospectively reported responses to fully structured questions is strongly correlated with 12-month suicide attempts among ideators, with a high concentration of attempts among high-risk ideators.

Endogenous Opioids are Involved in Morphine and Dipyrone Analgesic Potentiation in the Tail Flick Test in Rats
Hernandez-Delgadillo, G.P., and Cruz, S.L.. Eur J Pharmacol. Sepember 28, 2006, 546(1-3) pp. 54-59. Epub 2006 Jul 22.
INVEST Fellow: Silvia Cruz, Mexico, 1996-1997.
The combined administration of low doses of opiates with non-steroidal anti-inflammatory drugs can produce additive or supra-additive analgesic effects while reducing unwanted side effects. We have recently reported that co-administration of morphine with dipyrone (metamizol) produces analgesic potentiation both in naive and in morphine-tolerant rats. The purpose of this work was to determine the role of opioids on the acute potentiation observed between morphine and dipyrone i.v. in the rat tail flick test. To do this, two experiments were done. In the first one, naloxone was administered 10 min before morphine (3.1 mg/kg), dipyrone (600 mg/kg) or their combination at the same doses. Control animals received saline instead of naloxone. In the second experiment, naloxone (or saline) was given 2 min after reaching the maximal peak effect with each individual analgesic treatment. When naloxone was i.v. administered prior to analgesics, it completely blocked morphine effects, partially prevented morphine/dipyrone antinociception and delayed dipyrone-induced nociception. At 3.1 mg/kg, naloxone produced an increased nociception. When naloxone was given after analgesics, it dose-dependently blocked the effects of morphine alone and in combination with dipyrone but with different potency in each case. As to dipyrone, naloxone delayed the time to antinociceptive peak effect. Taken together, these results support the notion that endogenous opioids are involved in the analgesic potentiation observed with the combination of morphine plus dipyrone.

Neocortical Neurogenesis in Humans is Restricted to Development
Bhardwaj, R.D., Curtis, M.A., Spalding, K.L., Buchholz, B.A., Fink, D., Bjork-Eriksson, T., Nordborg, C., Gage, F.H., Druid, H., Eriksson, P.S., and Frisen, J. Proc Natl Acad Sci U S A. August 15, 2006, 103(33):12564-125688. Epub 2006 Aug 10.
INVEST Fellow: Henrik Druid, Sweden, 2000-2001.
Stem cells generate neurons in discrete regions in the postnatal mammalian brain. However, the extent of neurogenesis in the adult human brain has been difficult to establish. We have taken advantage of the integration of (14)C, generated by nuclear bomb tests during the Cold War, in DNA to establish the age of neurons in the major areas of the human cerebral neocortex. Together with the analysis of the neocortex from patients who received BrdU, which integrates in the DNA of dividing cells, our results demonstrate that, whereas nonneuronal cells turn over, neurons in the human cerebral neocortex are not generated in adulthood at detectable levels but are generated perinatally.

The Selective Dopamine D3 Receptor Antagonist SB-277011A Reduces
Nicotine-enhanced Brain Reward and Nicotine-paired Environmental Cue Functions Pak, A.C., Ashby, C.R., Heidbreder, C.A., Pilla, M., Gilbert, J., Xi, Z.X., and Gardner, E.L. Int J Neuropsychopharmacol. August 31, 2006, pp. 1-18 [Epub ahead of print].
INVEST Fellow: Zhengxiong Xi, China, 1995-1996.
Increasing evidence suggests that enhanced dopamine (DA) neurotransmission in the nucleus accumbens (NAc) may play a role in mediating the reward and reinforcement produced by addictive drugs and in the attentional processing of drug-associated environmental cues. The meso-accumbens DA system is selectively enriched with DA D3 receptors, a DA receptor subtype increasingly implicated in reward-related brain and behavioral processes. From a variety of evidence, it has been suggested that selective DA D3 receptor antagonism may be a useful pharmacotherapeutic approach for treating addiction. The present experiments tested the efficacy of SB-277011A, a selective DA D3 receptor antagonist, in rat models of nicotine-enhanced electrical brain-stimulation reward (BSR), nicotine-induced conditioned locomotor activity (LMA), and nicotine-induced conditioned place preference (CPP). Nicotine was given subcutaneously within the dose range of 0.25-0.6 mg/kg (nicotine-free base). SB-277011A, given intraperitoneally within the dose range of 1-12 mg/kg, dose-dependently reduced nicotine-enhanced BSR, nicotine-induced conditioned LMA, and nicotine-induced CPP. The results suggest that selective D3 receptor antagonism constitutes a new and promising pharmacotherapeutic approach to the treatment of nicotine dependence.

Cannabinoid CB1 Receptor Antagonist AM251 Inhibits Cocaine-primed Relapse in Rats: Role of Glutamate in the Nucleus Accumbens
Xi, Z.X., Gilbert, J.G., Peng, X.Q., Pak, A.C., Li, X., and Gardner, E.L. J Neurosci. August 16, 2006, 26(33) pp. 8531-8536. INVEST Fellow: Zhengxiong Xi, China, 1995-1996.
Blockade of cannabinoid CB1 receptors has been reported to inhibit cocaine- or cocaine cue-induced reinstatement of drug seeking. However, the mechanisms underlying this action are poorly understood. Given the importance of dopamine, glutamate, and GABA in cocaine reward and relapse, the authors studied the effects of AM251 [N-(piperidin-1-yl)-5-(4-iodophonyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], a novel highly selective CB1 receptor antagonist, on cocaine-primed reinstatement of drug-seeking behavior and on cocaine-induced changes in extracellular DA, glutamate, and GABA in the nucleus accumbens (NAc) under reinstatement conditions. The authors found that systemic administration of AM251 selectively inhibited cocaine-induced, but not sucrose plus sucrose cue-induced, reinstatement of reward-seeking behavior. AM251 alone did not trigger reinstatement. Local perfusion of AM251 into the NAc or the dorsal striatum also inhibited cocaine-triggered reinstatement. AM251 alone dose dependently elevated NAc glutamate in a voltage-dependent Na+ channel-dependent manner. AM251 did not affect NAc DA or GABA. Pretreatment with AM251 dose dependently inhibited cocaine-induced increases in NAc glutamate but not in DA. Blockade of NAc metabotropic glutamate mGluR2/3 receptors by LY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid] slightly facilitated cocaine-enhanced glutamate release but blocked the antagonism of cocaine-induced reinstatement by AM251. These data suggest the following: (1) CB1 receptors exert tonic inhibition over NAc glutamate release under cocaine-extinction conditions; (2) blockade of CB1 receptors by AM251 inhibits cocaine-enhanced NAc glutamate release and cocaine-triggered reinstatement; and (3) these effects appear to be mediated by activation of presynaptic mGluR2/3 autoreceptors secondary to AM251-induced increase (disinhibition) of NAc glutamate release.

Methadone Doses upon Multiple Readmissions to Inpatient Detoxification: Clinical Evidence for Very Moderate Opioid Tolerance
Madlung, E., Haring, C., Crespo, J.A., Saria, A., Grubinger, P., and Zernig, G. Pharmacology. 78(1) pp. 38-43, 2006. Epub 2006 August 15.
INVEST Fellow: Gerald Zernig, Austria, 1993-1994.
Escalation of drug use by addicts has traditionally been interpreted as tolerance to the drug's effects. On the basis of animal behavioral data, several groups have recently proposed alternative explanations, i.e., that such an escalation of drug dose might not be based on tolerance but rather be indicative of (i) sensitization to the reinforcing effect or the incentive salience of the drug of abuse or (ii) shifts in baseline mood, i.e., allostasis. In the present study, the emergence of opioid tolerance or sensitization during the progression of opioid dependence was assessed by comparing the methadone doses that were initially required to alleviate the opioid withdrawal symptoms of intravenous opioid users who presented for detoxification upon 3-7 consecutive admissions over the course of up to 84 months. Upon the second admission, the 48 surveyed male patients needed 115 +/- 6% (p = 0.012) and the 32 female patients needed 121 +/- 12% (p = 0.01) of the methadone dose required upon the first admission. Upon the third admission, the respective values were 121 +/- 8% (males; p = 0.013) and 111 +/- 12% (females; n.s.), and upon the fourth admission, 125 +/- 14% (males; p = 0.026) and 131 +/- 14 (females; p = 0.01). Inter-admission intervals averaged 14 +/- 1 months (n = 135) for males and 13 +/- 1 months (n = 91) for females and were not significantly different across consecutive admissions, suggesting that tolerance did not develop faster upon repetition of abuse-withdrawal cycles. In conclusion, the intravenous opioid users surveyed in the present study developed only very moderate tolerance during the repeated abuse-detoxification cycles that were typical for their disease progression. The present data do not support the notion that sensitization to the opioids' reinforcing effects occurred in this naturalistic clinical sample.

mGlu1 Receptor Blockade Attenuates Cue- and Nicotine-Induced Reinstatement of Extinguished Nicotine Self-administration Behavior in Rats
Dravolina, O.A., Zakharova, E.S., Shekunova, E.V., Zvartau, E.E., Danysz, W., and Bespalov, A.Y. Neuropharmacology. September 7, 2006; [Epub ahead of print]
INVEST Fellow: Anton Bespalov, Russia, 1994-1995.
Glutamatergic neurotransmission is believed to be critically involved in the acquisition and maintenance of drug addiction. The present study evaluated the role of metabotropic glutamate (mGlu) 1 receptors in the reinstatement of nicotine-seeking behavior. Rats were trained to nose-poke to receive response-contingent intravenous infusions of nicotine (0.01mg/kg/infusion, free base). Following the subsequent extinction phase, reinstatement tests were conducted in animals that were exposed either to response-contingent presentations of the nicotine-associated discrete light cues or to non-contingent nicotine priming injection (0.3mg/kg, s.c., salt) just prior to the test session. In a separate experiment, rats were subjected to the nearly identical response-reinstatement procedure but operant responding was established using food pellets instead of nicotine infusions. Pretreatment with the mGlu1 receptor antagonist EMQMCM (JNJ16567083, (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate) significantly inhibited cue-induced reinstatement of nicotine-seeking behavior (5 and 10, but not 2.5mg/kg). EMQMCM (5mg/kg) also prevented nicotine priming-induced reinstatement of nicotine-seeking behavior. At the highest tested dose only (10mg/kg), EMQMCM attenuated cue-induced reinstatement of food-seeking behavior. Taken together with the previous reports, the present findings further suggest that blockade of mGlu1 receptors may be beneficial for preventing relapse to tobacco smoking in nicotine-dependent individuals.

Cause of Death and Drug Use Pattern in Deceased Drug Addicts in Sweden, 2002-2003
Jonsson, A.K., Holmgren, P., Druid, H., and Ahlner, J. Forensic Sci Int. Sepember 9, 2006; [Epub ahead of print].
INVEST Fellow: Henrik Druid, Sweden, 2001-2002.
Compared with their contemporaries, individuals abusing illicit drugs suffer a higher risk of premature death. In Sweden, a simple protocol for registration of fatalities among abusers of alcohol, pharmaceuticals, illicit drugs, or other substances, has been used by the forensic pathologists since 2001. This routine was introduced to allow for an evaluation of the cause and manner of death, and patterns of abuse among different groups of abusers. The authors explored the data on drug abusers (i.e. abusers of illicit drugs) subjected to a forensic autopsy 2002-2003. The Swedish forensic pathologists examined 10,273 dead victims during the study period and 7% (743/10,273) of the cases were classified as drug abusers. Toxicological analyses were carried out in 99% (736/743) and illicit drugs were detected in 70% (514/736) of these. On average, 3.8 substances (legal or illegal) were found per case. The most common substances were ethanol and morphine, detected in 43 and 35% of the cases, respectively. When exploring the importance of the different substances for the cause of death, the authors found that the detection of some substances, such as fentanyl and morphine, strongly indicated a poisoning, whereas certain other substances, such as benzodiazepines more often were incidental findings. In total, 50% (372/743) died of poisoning, whereas only 22% (161/743) died of natural causes. Death was considered to be directly or indirectly due to drug abuse in 47% (346/743), whereas evidence of drug abuse was an incidental finding in 21% (153/743) or based on case history alone in 33% (244/743). The authors believe that this strategy to prospectively categorize deaths among drug addicts constitutes a simple means of standardizing the surveillance of the death toll among drug addicts that could allow for comparisons over time and between countries.

Pharmacological MRI in Awake Rats Reveals Neural Activity in Area Postrema and Nucleus Tractus Solitarius: Relevance as a Potential Biomarker for Detecting Drug-induced Emesis
Chin, C.L., Fox, G.B., Hradil, V.P., Osinski, M.A., McGaraughty, S.P., Skoubis, P.D., Cox, B.F., and Luo, Y. Neuroimage. October 3, 2006; [Epub ahead of print]
INVEST Fellow: Steven P. McGaraughty, Canada, 1995-1996.
Drug-induced vomiting (emesis) is a major concern in patient care and a significant hurdle in the development of novel therapeutics. With respect to the latter, rodents, such as the rat and mouse, are typically used in efficacy and safety studies; however, drug-induced emesis cannot be readily observed in these species due to the lack of an emetic reflex. It is known that emesis can be triggered by neural activity in brain regions including area postrema (AP) and nucleus tractus solitarius (NTS). In this study, using pharmacological magnetic resonance imaging (phMRI) and a blood-pool contrast agent, authors imaged the hemodynamic consequences of brain activity in awake rats initiated by the administration of compounds (apomorphine 0.1, 0.3 micromol/kg i.v. and ABT-594 0.03, 0.1, 0.3 micromol/kg i.v.) that elicit emesis in other species. Regional drug-induced relative cerebral blood volume (rCBV) changes and percent activated area within the AP and NTS were calculated, in which a dose-dependent relationship was evident for both apomorphine and ABT-594. Additionally, to correlate with behavioral readouts, it was found that the activation of AP and NTS was observed at plasma concentrations consistent with those that induced emesis in ferrets for both drugs. These data thus suggest that phMRI in awake rats may be a useful tool for predicting emetic liability of CNS-acting drugs and may provide insights into depicting the underlying emetic neural pathways in vivo.

Blockade of mGluR1 Receptor Results in Analgesia and Disruption of Motor and Cognitive Performances: Effects of A-841720, A Novel Non-competitive mGluR1 Receptor Antagonist
El-Kouhen, O., Lehto, S.G., Pan, J.B., Chang, R., Baker, S.J., Zhong, C., Hollingsworth, P.R., Mikusa, J.P., Cronin, E.A., Chu, K.L., McGaraughty, S.P., Uchic, M.E., Miller, L.N., Rodell, N.M., Patel, M., Bhatia, P., Mezler, M., Kolasa, T., Zheng, G.Z., Fox, G.B., Stewart, A.O., Decker, M.W., Moreland, R.B., Brioni, J.D., and Honore, P. Br J Pharmacol. October 3, 2006; [Epub ahead of print]. INVEST Fellow: Steven P. McGaraughty, Canada, 1995-1996.
The purpose of this study was to further assess the clinical potential of the blockade of metabotropic glutamate receptors (mGluR1) for the treatment of pain. Authors characterized the effects of A-841720, a novel, potent and non-competitive mGluR1 antagonist in models of pain and of motor and cognitive function. At recombinant human and native rat mGluR1 receptors, A-841720 inhibited agonist-induced calcium mobilization, with IC(50) values of 10.7+/-3.9 and 1.0+/-0.2 nM, respectively, while showing selectivity over other mGluR receptors, in addition to other neurotransmitter receptors, ion channels, and transporters. Intraperitoneal injection of A-841720 potently reduced complete Freund's adjuvant-induced inflammatory pain (ED(50)=23 mumol kg(-1)) and monoiodoacetate-induced joint pain (ED(50)=43 mumol kg(-1)). A-841720 also decreased mechanical allodynia observed in both the sciatic nerve chronic constriction injury and L5-L6 spinal nerve ligation (SNL) models of neuropathic pain (ED(50)=28 and 27 mumol kg(-1), respectively). Electrophysiological studies demonstrated that systemic administration of A-841720 in SNL animals significantly reduced evoked firing in spinal wide dynamic range neurons. Significant motor side effects were observed at analgesic doses and A-841720 also impaired cognitive function in the Y-maze and the Water Maze tests. The analgesic effects of a selective mGluR1 receptor antagonist are associated with motor and cognitive side effects. The lack of separation between efficacy and side effects in pre-clinical models indicates that mGluR1 antagonism may not provide an adequate therapeutic window for the development of such antagonists as novel analgesic agents in humans. British Journal of Pharmacology advance online publication, 3 October 2006; doi:10.1038/sj.bjp.0706877.

PMA and Doxorubicin Decrease Viability, MTT Activity and Expression of CD10 Marker on NALM-1 Leukemic Cells
Martin-Kleiner, I,., Svoboda-Beusan, I. and Gabrilovac, J. Immunopharmacol Immunotoxicol. 28(3), pp. 411-420, 2006.
INVEST Fellow: Irena Martin-Kleiner, Croatia, 1995-1996.
PMA (10, 20 ng/ml) and doxorubicin (5-20 ng/ml) decreased the viability and MTT-activity of NALM-1 pre-B leukemic cells (3 days' treatment). Further, CD10 was downregulated, suggesting that PMA and doxorubicin induced differentiation of NALM-1 cells. However, PMA did not alter expression of B cell markers CD20 and of mIgM. In contrast to PMA, another differentiation agent ATRA did not alter CD10 expression on NALM-1 cells but affected viability after 6 days (5, 10 ng/ml). The data in this study are the first evidence that PMA and doxorubicin inhibited viability and MTT activity and induced partial differentiation, by decreasing CD10 on NALM-1 cells.

Peripheral Formalin Injection Induces Long-Lasting Increases in Cyclooxygenase 1 Expression by Microglia in the Spinal Cord
Zhang, F., Wan, Y., Zhang, Z.K., Light, A.R., and Fu, K.Y. J Pain. Sepember 1, 2006; [Epub ahead of print].
INVEST Fellow: You Wan, China, 1998-1999.
Activated glia are a source of substances known to enhance pain, including centrally synthesized prostaglandins. The authors have previously shown that microglia are activated in the spinal cord following peripheral formalin injection. In the present study, they investigated cyclooxygenase (COX-1 and COX-2) expression in the spinal cord using immunohistochemistry and Western blots in the formalin pain model, to further understand how spinal glia modulate pain processing. The authors show that both COX-1 and COX-2 are constitutively expressed in the spinal cord. Hind paw formalin injection increased COX-1 expression, beginning at 1 day after injection and lasting at least 2 weeks, the duration of experiments. The COX-2 expression changed considerably less, with a significant increase of COX-2 protein level only observed at 2 h after injection. Double labeling studies showed that COX-1 was expressed in microglia and COX-2 was expressed in neurons. These data indicate that both COX-1 and COX-2 are increased in the spinal cord following formalin injection, but the time course and cellular sources are different, suggesting that both COX-1 (longer time points) and COX-2 (very short time points) may be involved in spinal modulation in the formalin pain model. Our study also suggests that spinal microglial activation may play a role in long-term hyperalgesia through the increased expression of COX-1. This article reports that COX-1 expression by microglia is increased in the spinal cord after peripheral formalin injection into the rat hind paw. This result could potentially help clinicians understand how COX-1 may be involved in pain processing and the role microglial activation plays in pain mechanisms.

Highly Variable mRNA Expression and Splicing of L-type Voltage-dependent Calcium Channel Alpha Subunit 1C in Human Heart Tissues
Wang, D., Papp, A.C., Binkley, P.F., Johnson, J.A., and Sadee, W. Pharmacogenet Genomics. 16(10), pp. 735-745, October 2007.
INVEST Fellow: Danxin Wang, China, 1996-1997.
The voltage-dependent L-type calcium channel alpha-subunit 1c (Cav1.2, CACNA1C) undergoes extensive mRNA splicing, leading to numerous isoforms with different functions. L-type calcium channel blockers are used in the treatment of hypertension and arrhythmias, but response varies between individuals. Authors have studied the interindividual variability in mRNA expression and splicing of CACNA1C, in 65 heart tissue samples, taken from heart transplant recipients. Splice variants were measured quantitatively by polymerase chain reaction in 12 splicing loci of CACNA1C mRNA. To search for functional cis-acting polymorphisms, we determined allelic expression ratios for total CACNA1C mRNA and several splice variants using marker single nucleotide polymorphisms in exon 4 and exon 30. Total CACNA1C mRNA levels varied approximately 50-fold. Substantial splicing occurred in six loci generating two or more splice variants, some with known functional differences. Splice patterns varied broadly between individuals. Two heart tissues expressed predominantly the dihydropyridine-sensitive smooth muscle isoform of CACNA1C (containing exon 8), rather than the cardiac isoform (containing exon 8a). Lack of significant allelic expression imbalance, observed with total mRNA and several splice variants, argued against CACNA1C polymorphisms as a cause of variability. Taken together, highly variable splicing can cause profound phenotypic variations of CACNA1C function, potentially associated with disease susceptibility and response to L-type calcium channel blockers.

The Reovirus {micro}1 Structural Rearrangements that Mediate Membrane Penetration
Zhang, L., Chandran, K., Nibert, M.L., and Harrison, S.C. J Virol. Sepember 27, 2006; [Epub ahead of print].
INVEST Fellow: Lan Zhang, China, 2004-2005.
Membrane penetration by nonenveloped reoviruses is mediated by the outer-capsid protein, micro 1 (76 kDa). Previous evidence has suggested that an autolytic cleavage in micro 1 allows release of its N-terminally myristoylated peptide, micro 1N (4 kDa), which probably then interacts with the target-cell membrane. A substantial rearrangement of the remaining portion of micro 1, micro 1C (72 kDa), must also have occurred for micro 1N to be released, and some regions in micro 1C may make additional contacts with the membrane. Authors describe here a particle-free system to study conformational rearrangements of micro 1. The authors show that removal of the protector protein sigma3 is not sufficient to trigger rearrangement of free micro 1 trimer and that free micro 1 trimer undergoes conformational changes similar to those of particle-associated micro 1 when induced by similar conditions. The micro 1 rearrangements require separation of the micro 1 trimer head domains, but not the micro 1N/C autocleavage. The authors have also obtained a relatively homogeneous form of the structurally rearranged micro 1 (micro 1*) in solution. It is an elongated monomer and retains substantial alpha-helix content. The authors have identified a protease-resistant, approximately 23-kDa fragment of micro 1*, which contains the largely alpha-helical regions designated as domains I and II in the conformation of micro 1 prior to rearrangement. The authors propose that the micro 1 conformational changes preceding membrane penetration or disruption during cell entry involve: (i) separation of the beta-barrel head domains in the micro 1 trimer; (ii) autolytic cleavage at the micro 1N/C junction, associated with partial unfolding of micro 1C and release of micro 1N; and (iii) refolding of the N-terminal helical domains of micro 1C, with which micro 1N was previously complexed, accompanied by dissociation of the micro 1 trimer.

The Last Decade of Solvent Research in Animal Models of Abuse: Mechanistic and Behavioral Studies
Bowen, S.E., Batis, J.C., Paez-Martinez, N., and Cruz, S.L. Neurotoxicol Teratol. Sepember 20, 2006; [Epub ahead of print]
INVEST Fellow: Silvia Cruz, Mexico, 1996-1997.
The abuse of volatile organic solvents (inhalants) leads to diverse sequelae at levels ranging from the cell to the whole organism. This paper reviews findings from the last 10 years of animal models investigating the behavioral and mechanistic effects of solvent abuse. In research with animal models of inhalant abuse, NMDA, GABA(A), glycine, nicotine, and 5HT(3) receptors appear to be important targets of action for several abused solvents with emerging evidence suggesting that other receptor subtypes and nerve membrane ion channels may be involved as well. The behavioral effects vary in magnitude and duration among the solvents investigated. The behavioral effects of acute and chronic inhalant abuse include motor impairment, alterations in spontaneous motor activity, anticonvulsant effects, anxiolytic effects, sensory effects, and effects on learning, memory and operant behavior (e.g., response rates and discriminative stimulus effects). In addition, repeated exposure to these solvents may produce tolerance, dependence and/or sensitization to these effects.

Measuring Air Quality to Protect Children from Secondhand Smoke in Cars
Rees, V.W., and Connolly, G.N. Am J Prev Med. 31(5), pp. 363-368, November 2006.
INVEST Fellow: Vaughan W. Rees, Australia, 1999-2000
Secondhand tobacco smoke (SHS) is a major, preventable contributor to acute and chronic adverse health outcomes that affect children disproportionately. The predominant source of SHS among children is domestic exposure, and while up to two thirds of U.S. households have car smoking bans, an unacceptable number of children remain vulnerable. To help promote more effective protection through legislation, health communication strategies, or behavioral interventions, data demonstrating the adverse effect of SHS on air quality in cars are needed. Secondhand tobacco smoke in a motor vehicle under actual driving conditions was monitored by measuring respirable suspended particles (RSPs) of less than 2.5 microns in diameter, and carbon monoxide. Forty-five driving trials were conducted, using teams of volunteer drivers and smokers recruited from the general community. Three smoking conditions (nonsmoking baseline, active smoking, and immediate post-smoking period, each 5 minutes) were crossed with two ventilation conditions (windows open, closed) in a 3 x 2 within-sessions factorial design. The highest mean observed RSP level was 271 mug/m(3), which is unsafe, particularly for children. Peak RSP levels were considerably higher. RSPs and carbon monoxide increased significantly from baseline after smoking, and these increases were greatest during the closed ventilation condition, compared with open ventilation. Authors concluded that private passenger cars are a domestic environment with the potential to yield unsafe levels of SHS contaminants. These data may assist policymakers and health advocates to promote protective strategies to ensure smoke-free domestic environments for children.

Searching for Polymorphisms that Affect Gene Expression and mRNA Processing: Example ABCB1 (MDR1)
Wang, D., and Sadee, W. AAPS J. 8(3), pp. E515-520, August 2006. Review.
INVEST Fellow: Danxin Wang, China, 1996-1997.
Cis-acting genetic variations can affect the amount and structure of mRNA/protein. Genomic surveys indicate that polymorphisms affecting transcription and mRNA processing, including splicing and turnover, may account for the main share of genetic factors in human phenotypic variability; however, most of these polymorphisms remain yet to be discovered. The authors use allelic expression imbalance (AEI) as a quantitative phenotype in the search for functional cis-acting polymorphisms in many genes, including ABCB1 (multidrug resistance 1 gene, MDR1, Pgp). Previous studies have shown that ABCB1 activity correlates with a synonymous polymorphism, C3435T; however, the functional polymorphism and molecular mechanism underlying this clinical association remained unknown. Analysis of allele-specific expression in liver autopsy samples and in vitro expression experiments showed that C3435T represents a main functional polymorphism, accounting for 1.5- to 2-fold changes in mRNA levels. The mechanism appears to involve increased mRNA turnover, probably as a result of different folding structures calculated for mRNA with the Mfold program. Other examples of the successful application of AEI analysis for studying functional polymorphism include 5-HTT (serotonin transporter, SLC6A4) and OPRM1 (mu opioid receptor). AEI is therefore a powerful approach for detecting cis-acting polymorphisms affecting gene expression and mRNA processing.

Former Hubert H. Humphrey Drug Abuse Research Fellows

Marked Ethnic Differences in HIV Prevalence and Risk Behaviors among Injection Drug Users in Dushanbe, Tajikistan, 2004
Stachowiak, J.A., Tishkova, F.K., Strathdee, S.A., Stibich, M.A., Latypov, A., Mogilnii, V., and Beyrer, C. Drug Alcohol Depend. 82 Suppl 1:S7-14, April 2006.
HHH Fellow: Alisher Latypov, Tajikistan, 2002-2003
The purpose of this study was to examine differences by ethnicity of HIV prevalence and correlates among injection drug users (IDUs) in Dushanbe, Tajikistan. The researchers enrolled 489 active adult IDUs in a cross-sectional risk factor study of HIV infection. Participants were provided HIV pre-and posttest counseling and risk reduction counseling and answered an interviewer-administered questionnaire. HIV-1 status was determined with rapid tests and confirmed with ELISA. Participants included four ethnicities: 204 Tajiks (49.1%), 145 Russians (29.7%), 58 Uzbeks (11.9%), and 46 participants of other nationalities (9.4%). Overall prevalence of HIV-1 infection was 12% and varied significantly by ethnicity: it was highest among ethnic Tajiks, at 19.2%; lowest among Russians and Uzbeks, at 3.4%; and 13% among other nationalities. Ethnic groups differed significantly in years injecting, receiving a needle from a needle exchange program (NEP), injecting in groups, having undergone drug treatment, reported condom use, and arrest history. Among Tajiks, HIV infection was significantly associated with daily injecting (OR 2.16); reporting that narcotics were very easy to obtain (OR 2.46); having undergone drug treatment (OR 2.75), and injecting "alone" (OR 3.12). The authors concluded that ethnic differences were strongly associated with HIV prevalence and risk behaviors in this multiethnic study, and prevention efforts might need to be targeted by ethnicity.

Hepatitis C Virus Infection among Injecting Drug Users in the Czech Republic -- Prevalence and Associated Factors
Zabransky, T., Mravcik, V., Korcisova, B., and Rehak, V. Czech National Focal Point for Drugs and Drug Addiction, Praha-Mala Strana, Czech Republic. Eur Addict Res. 12(3), pp. 151-160, 2006.
HHH Fellow: Tomas Zabransky, Czech Republic, 2003-2004
The aim of this study was to determine the prevalence of, and factors associated with, hepatitis C virus (HCV) infection in the population of Czech injecting drug users (IDUs) in a multicentric cross-sectional study. A convenience sample of injecting drug users was recruited using the snowball sampling method. Participants comprised a sample of 760 IDUs from 9 different Czech regions. Authors used one-drop instant blood tests to determine the anti-HCV antibodies status; a structured questionnaire was completed during the interview with the researcher. They calculated the ratio of positive findings and performed univariate analyses of correlations between predictors and independent variables. Finally, they created a logistic regression model that controlled for age, region of residence, reported sharing of injection paraphernalia, and length of injection drug use and for the interaction between length of injection use and imprisonment in order to assess the predictive value of imprisonment in an individual's history. 226 participants (29.74% of the tested sample) were found to be anti-HCV positive. After adjusting for the sensitivity of the test, the 'true proportion' was 34.97% (95% CI: 31.56-38.35). Many correlated independent variables were found in the univariate analyses. In this logistic regression model, the authors have found that imprisonment increases the odds of being anti-HCV positive by a factor of 4.3. The authors concluded that anti-HCV seroprevalence remains relatively low in the Czech IDUs population compared to similar populations in the developed countries. Regional differences exist in anti-HCV prevalence within the Czech Republic. The strong association of anti-HCV prevalence with imprisonment history when controlled for other potentially clinically important factors suggests the need for more effective preventive measures in Czech prisons.