www.drugabuse.gov Volume 21, Number 4 U.S. Department of Health and Human Services | National Institutes of Health

Neuropeptide Promotes Drug- ALSO IN THIS ISSUE Research Findings

Seeking and in Rats 7 Endorphin Derivative Inhibits Reward From and Orexin emerges as a link in the chain of mechanisms in Rats regulating appetite for rewards. 8 Gene Experiment Confirms a Suspected discovered that people with narcolepsy lack orex- Action BY LORI WHITTEN, NIDA Notes Staff Writer in-producing . The finding suggested an 16 Study Links Anabolic explanation for a striking observation made in the Steroids to Brain Changes rexin, a neuropeptide that stimulates mid-1970s: People with narcolepsy rarely became in Adolescent Female eating and regulates wakefulness, also addicted to the potent used to treat the Mice fosters animals’ drug seeking and disorder at the time. Perhaps, some scientists spec- Director’s Perspective craving responses to drugs, according ulated, orexin contributes to the development of O 2 NIDA Will Contribute to to two NIDA-funded studies. The research teams, drug abuse. led by Drs. Glenda Harris and Gary Aston-Jones at Obesity Research OREXIN AND DRUG SEEKING the University of Pennsylvania and Drs. Stephanie Research in Brief Borgland and Antonello Bonci at the University of An observation in animals by Drs. Harris and 3 • Tracing NET California, San Francisco (UCSF), used different Aston-Jones also seemed to suggest a possible con- • Controlling ADHD experimental procedures and studied different nection. They noted that lateral hypothalamus (LH) Symptoms • Morphine drugs. Their findings, however, point to the same cells in the same area as orexin neurons were acti- Exposure in Rats conclusion: Augmenting orexin increases drug vated during drug seeking using a behavioral assay NIDA at Work seeking, while blocking it has the opposite effect. called conditioned place preference (CPP; for more Orexin, also called hypocretin, is produced by on CPP, see “Animal Experiments in Sci- 4 Division of Clinical neurons in the hypothalamus—a brain structure ence,” NIDA Notes Vol. 20, No. 5). After repeated Neuroscience and Behavioral Research that regulates hunger, thirst, sleep, and other morphine injections in one chamber of a test cage processes essential to survival. Scientists recently and saline in the other, rats gravitate to the drug- Reference Article paired area in an effort to re-expe- 11 Impacts of Drugs on rience the effects. The time Neurotransmission OREXIN STIMULATION DRIVES DRUG-SEEKING Exposure to cues associated with drugs activate the orexin neurons (red), which stim- they spend in the area—their Bulletin Board ulates neruons in the brain’s mesolimbic reward pathway (blue). morphine place preference— 18 • Meeting Reviews indicates how intensely the drug 1 Drug Abuse and HIV Rewards (drugs or food) motivates drug seeking. When • Adolescent Cues for Rewards 3 Drs. Harris and Aston-Jones Use Chemical Stimulation Mesolimbic Reward Pathway determined that the LH neurons Prefrontal Nucleus Ventral Tearoff Cortex Accumbens Tegmental Area activated during drug seeking pro- (PFC) (NAc) Shell (VTA) 2 duce orexin, they conducted fur- 19 Journal Highlights Drug Orexin neurons in lateral ther experiments. Abuse and HIV/AIDS hypothalamus (LH) The Pennsylvania researchers Adapted from Trends in Neurosciences 29(10):571-577, 2006. 20 What the Numbers Say first demonstrated that activa- [ Continued on page 6 ]

NIDA Notes | Volume 21, Number 4 1 ■ DIRECTOR’S PERSPECTIVE By NORA D. VOLKOW, M.D., NIDA Director NIDA Notes

EDITOR David Anderson Public Health Advisor, Office of Science Policy and Communications, National Institute on Drug Abuse

MANAGING EDITOR Simon Weavers NIDA Will Contribute to MasiMax Resources, Inc. STAFF WRITERS Debra P. Davis Obesity Research Lori Whitten, Ph.D. MasiMax Resources, Inc.

CONTRIBUTING WRITERS Carl Sherman The has a serious weight problem. Two-thirds of our adults are overweight Sarah Teagle, Dr. Ph. or obese. The prevalence of overweight among our children has nearly tripled since 1970. COPY EDITOR The consequences for the Nation’s health and economy are grave. Obesity has been Debra P. Davis MasiMax Resources, Inc. shown to decrease overall life expectancy and to increase the risk for cardiovascular dis- DESIGN/LAYOUT ease, type 2 diabetes, and other chronic conditions. Annually, it costs an estimated $117 bil- Maggie Bray lion or more in lost productivity and future earnings. To identify new strategies for preven- MasiMax Resources, Inc. tion and treatment, the National Institutes of Health (NIH) has established an Agency-wide EDITORIAL BOARD obesity task force and research plan (see obesityresearch.nih.gov). David Anderson, Chair; Public NIDA’s assignment on the 25-Institute task force represents a natural extension of the Health Advisor, Public Information and Liaison Branch, Office of Science Policy Institute’s research agenda. Addiction and compulsive eating both involve impaired impulse and Communications control and distorted valuation of the rewards to be derived from a certain behavior— Jessica Campbell Chambers, Ph.D., i.e., drug-taking or eating. The two conditions have roots in some of the same brain areas and Health Science Administrator, Division of Epidemiology, Services and Prevention circuits, including the hypothalamus, prefrontal cortex, and limbic system. The knowledge Research NIDA-funded scientists have developed of how those areas function normally and in the J.C. Comolli, Public Health Advisor, Division of Pharmacotherapies and Medical context of drug abuse undoubtedly will have great application to understanding the other Consequences of Drug Abuse compulsive behavior. Gaya Dowling, Ph.D., Deputy Branch In this issue, we report an exemplary illustration of the neurobiological overlap between Chief, Science Policy Branch, Office of Science Policy and Communications addiction and eating disorders: The hormone orexin appears to foster cravings for both food Lynda Erinoff, Ph.D., Associate Director, and drugs (see p. 1). Several other NIDA-supported studies are investigating compounds AIDS Research Program, Division of Epidemiology, Services and Prevention found to be effective in suppressing appetite and food intake. Some of the most promising Research produce their effects by blocking the brain’s cannabinoid receptors, which are targeted by Marsha F. Lopez, Ph.D. , Health Scientist THC, the active ingredient in marijuana (), a drug with marked effects on appetite. Administrator, Division of Epidemiology, Services and Prevention Research Researchers hope to translate this knowledge into medications for weight control, drug Gerald McLaughlin, Ph.D. , Chief, Grants abuse treatment, or both. Review Branch, Office of Extramural Affairs Mary Ellen Michel, Ph.D., Deputy EFFORTS BY OTHER INSTITUTES IN THE COLLABORATION INCLUDE: Director, Center for the Clinical Trials Network Ivan Montoya, M.D., M.P.H., Medical ■ A program, led by the National Institute of Diabetes and Digestive and Kidney Diseases Officer, Division of Pharmacotherapies and (NIDDK), to foster a multidisciplinary approach to clinical obesity research in areas such Medical Consequences of Drug Abuse Joni Rutter, Ph.D., Program Director, as metabolism, endocrinology, nutrition, cardiovascular biology, digestive diseases, Genetics and Molecular Neurobiology Research genetics, and behavioral sciences; and Branch, Division of Basic Neuroscience and Projects to combat pediatric obesity in primary care, day care, schools, and other commu- Behavioral Research ■ Vincent Smeriglio, Ph.D., nity settings by exploring effective methods to curb weight gain, including changes in Research Psychologist, Division of Clinical food and lifestyle choices. Neuroscience and Behavioral Research Frank Vocci, Ph.D., Director, We hope that further research into the many factors that contribute to obesity— Division of Pharmacotherapies and Medical including neural systems that modulate appetite and eating behavior—will lead to the devel- Consequences of Drug Abuse opment of new therapies for obesity as well as addiction. Cora Lee Wetherington, Ph.D., ■ Psychologist, Behavioral and Cognitive Science Research Branch, Division of Basic Neuroscience and Behavioral Research

This publication was produced and printed by MasiMax Resources, Inc., under Contract No. N01DA-4-1117 from the National Institute on Drug Abuse. 2 NIDA Notes | Volume 21, Number 4 ■ RESEARCH IN BRIEF Highlights of recently published NIDA-supported studies

disrupted in depression, atten- (besides and Holtzman and David White tion disorders, and marijuana) during the past first established that both peri- abuse. Researchers previously year. Of the 76 respondents adolescent (7 to 10 days before developed radiotracers for the who said they had been puberty onset) and adult male dopamine and serotonin trans- prescribed ADHD medications rats receiving 10 mg/kg/day of porters; the ability to image at some point during their morphine for 1 day or 3 consec- all three transporters in the lives, one-quarter said they had utive days exhibited similar brain will allow researchers to abused their medication to get increases in locomotor activity determine the role of each high, and 29 percent said they relative to age-matched rats neurochemical in the had given or sold it to someone receiving only saline. When the Tracing NET disorders. else. Dr. Himanshu Upadhyaya researchers reexposed the ani- Researchers have developed > Journal of Neurochemistry and colleagues at the Medical mals to morphine 5 weeks later, and successfully tested a new 94(2):337-351, 2005. University of South Carolina significant age- and exposure- tool for studying the neurobiol- conducted the study. Appropri- related differences emerged. ogy of depression, attention ate treatment of ADHD may All of the rats were more active, deficit hyperactivity disorder, reduce college students’ risk of but a relatively small dose of and stimulant abuse. drug abuse. morphine (0.3 mg/kg) Researchers will be able to use > Journal of Child and Adolescent triggered significantly more the tool, a new radiotracer, to Psychopharmacology 15(5):799- activity in the rats that map the location and 809, 2005 previously were exposed to circulation of a protein that morphine for 3 days as plays a central role in those periadolescents, relative to conditions: the norepinephrine their 1-day and unexposed transporter (NET). Drs. Yu- counterparts. By contrast, rats Shin Ding, Jean Logan, and Controlling College previously exposed for 3 days colleagues at Brookhaven Students’ ADHD as adults required 10 times that National Laboratory screened Symptoms May dose to exhibit more activity six molecules to see whether Protect Them Against than those in the 1-day and any of them did the things a unexposed groups. The radiotracer for NET must do: In a survey of 334 students at a findings suggest that, during get into the brain, spread to college in the Southeast, those adolescence, even a relatively regions of the brain with high who reported having Exposure to low level of exposure to concentrations of NET, bind experienced symptoms of Morphine During morphine can have profound, selectively to the protein, and attention deficit hyperactivity Early Adolescence long-lasting effects. leave the brain in a reasonable disorder (ADHD) during the Sensitizes Rats as > European Journal of amount of time. Of the six, past 6 months were three times Adults Pharmacology 528(1-3):119-123, (S,S)-[11C]methylreboxetine as likely as those whose A study conducted at Emory 2005. (MRB) was by far the best symptoms were controlled to University School of Medicine candidate, binding tightly and say they had intensified their indicates that exposure to mor- specifically to NET. The smoking, and six times as likely phine during adolescence may norepinephrine, dopamine, to report increased frequency increase sensitivity to the drug and serotonin transporters are of abuse of other drugs during adulthood. Drs. Stephen

NIDA Notes | Volume 21, Number 4 3 ■ NIDA AT WORK: NIDA’s Division of Clinical Neuroscience and of awake and functioning people and Behavioral Research obtain specific measures on how drugs affect the brain,” Dr. Frascella says. Imag- ing studies indicate that the human brain undergoes major changes as a conse- NIDA’s Newest quence of drug exposure and that the adolescent brain may be particularly Division Mines pliant, “which may help explain why ado- lescence is the period when most new Clinical Applications cases of drug addiction occur,” Dr. Frascella adds. “Thus, we’ve become From Basic Research increasingly committed to using brain imaging to get a sharper picture of how the brain changes with development and in response to active and passive BY DEBRA P. DAVIS, Illinois University demonstrated that exposure to drugs of abuse.” NIDA Notes Staff Writer nicotine exposure and both influence the ability to pay atten- THREE BRANCHES OF CLINICAL IDA’s Division of Clinical tion. Now, NIDA’s Division of Pharma- RESEARCH Neuroscience and Behav- cotherapies and Medical Consequences DCNBR, formed in May 2004, has ioral Research (DCNBR) of Drug Abuse is exploring the clinical three Branches: Nidentifies, validates, and impact of these observations. They are Clinical Neuroscience: Under Dr. explores the clinical implications of basic testing whether bupropion and nicotine Steven Grant’s leadership, the Clinical science discoveries. Much of the patches affect attention during smoking Neuroscience Branch focuses on how Division’s work consists of replicating cessation, and whether such effects drug abuse affects the human central results obtained in laboratory and animal correlate with success in quitting. nervous system, including brain changes studies in human subjects. A DCNBR In another DCNBR-supported study, during different stages and states of project typically culminates in one of two Dr. Robert Risinger and colleagues at abuse, such as addiction, withdrawal, outcomes: carrying a new discovery the Medical College of Wisconsin docu- abstinence, craving, and . It also forward for development into actual mented a pattern of fluctuating activa- studies:

interventions or referring it back to basic tion of the mesolimbic as ■ The factors that make some individu- scientists for further investigation. six cocaine-abusing men transitioned als more vulnerable and others more “We are uniquely positioned to through cycles of cocaine craving, self- resilient to drug abuse and addiction;

uncover the factors in humans—neuro- administration, and highs. The results, ■ The impact of drugs on learning, biologic, genetic, social-behavioral—that obtained with functional magnetic reso- memory, judgment, and decision- help explain the development and effects nance imaging (fMRI), confirmed previ- making;

of drug abuse,” says Director Dr. Joseph ous research linking those brain areas to ■ Co-occurrence of drug addiction with Frascella. “Being positioned between drug self-administration in animals, and other mental disorders;

NIDA’s basic research division and other extended them by correlating them with ■ Neurobiological changes that result more applied programs, our research drug abusers’ subjective feelings and from behavioral and pharmacological programs inform basic science and pro- responses. (see “Cocaine Craving Acti- treatments for drug abuse; and

mote the development and implementa- vates Brain Reward Structures; Cocaine ■ Interventions to ameliorate the tion of new medications and behavioral ‘High’ Dampens Them,” NIDA Notes, Vol. negative health consequences of treatments across NIDA.” 21, No. 2). drugs, whether self-administered or For example, building on basic Neuroimaging studies comprise one- incurred prenatally. research that linked nicotine acetyl- third of the Division’s research portfolio. As an example of a recent Branch choline receptors to the regulation of “Brain imaging has pushed drug abuse achievement, Dr. Grant points to a study attention, DCNBR-sponsored researcher research in the last 15 to 20 years—allow- by Dr. Martin Paulus and colleagues at David Gilbert and colleagues at Southern ing us to directly observe neural activity the University of California, San Diego.

4 NIDA Notes | Volume 21, Number 4 The researchers performed fMRI while Onken’s direction, aims to develop new times, they are too complicated and 46 men who had been abstinent from and improved treatments for drug abuse expensive, and require extensive training for about a month and addiction, including behavioral, com- for clinicians,” Dr. Frascella notes. One took a decisionmaking test. The bined behavioral-pharmacological, and way of developing a community-friendly researchers found that patterns of acti- complementary and alternative treat- treatment is to identify the active vation in certain brain regions predicted ments. The Branch designs new interven- ingredients of effective treatments, with high accuracy which men would and tions, tests them for efficacy, and evalu- explains Dr. Onken. “In this way, we may would not relapse 1 year later (see “Brain ates strategies to improve treatment be able to create more streamlined treat- Activity Patterns Signal Risk of Relapse engagement, adherence, and retention— ments that retain their potency. Another to Methamphetamine,” NIDA Notes, Vol. key factors in success. The Branch also novel approach to making a treatment 20, No. 5). “This use of fMRI, if replicated, supports research that prepares treat- more community-friendly is to use could be adapted for treatment and pre- ments for use in community settings, computers to help counselors deliver ventive interventions,” Dr. Frascella says. where cost, training, and fit with existing treatment. For example, Dr. Kathleen Behavioral and Brain Development: services can be constraints. Carroll at Yale University is testing the Led by Dr. Vincent Smeriglio, this Branch “Behavioral therapies that work well efficacy of computer-assisted cognitive- examines drug exposure and abuse, and in a research setting cannot necessarily behavioral therapy in preventing cocaine their health and social consequences, be taken into the community. Often- relapse.” ■ through the course of human develop- ment—from the prenatal period through childhood, adolescence, and young adult- BRAIN IMAGING IS A KEY TOOL IN STUDIES SPONSORED BY THE hood. Research focuses on: DIVISION OF CLINICAL NEUROSCIENCE AND BEHAVIORAL RESEARCH The images below—used in a recent Division presentation—show that repeated exposure to ■ The effects of drugs on behavioral and drugs depletes the brain's dopamine receptors, which are critical for one’s ability to experience brain development; pleasure and reward.

■ The role of genetic, neurobiological, and environmental factors in youths’ vulnerability to drug abuse and addic- tion; and

■ The developmental effects of co- occurring exposure to drugs and infectious diseases as well as the impact of drug abuse on youths with mental illness. One theme of the Branch’s work is research to tailor drug abuse treatments to individual needs. As a recent example, a Branch-supported study by Dr. Leslie Jacobsen and colleagues at Yale Universi- ty School of Medicine found that adoles- cent smokers experienced greater mem- ory impairment during if their mothers had smoked while pregnant. The study also produced fMRI evidence implicating particular brain regions in the deficits, which may be useful diagnostically as well as thera- peutically. Behavioral and Integrative Treat- ment: This Branch, under Dr. Lisa

NIDA Notes | Volume 21, Number 4 5 ■ RESEARCH FINDINGS

■ OREXIN [ Continued from page 1 ] for drugs and their risk for relapse. “It makes To explore the cellular bases for their tion of orexin neurons in the LH was tightly sense, anatomically and physiologically, that behavioral observations, the UCSF group coupled with rats’ place preferences for mor- orexin might play a role in reward-seeking measured orexin’s effects on the electro- phine, cocaine, and sweet food. Next, they and craving,” says Dr. Harris, now at the Cen- physiological properties of dopamine-pro- gave a different group of rats morphine for 3 tre de Regulacio Genomica in Spain. “Neu- ducing cells in brain slices removed from the days to establish place preference, then rons in this part of the brain stimulate eat- VTA of rats. The results showed that orexin stopped the drug and injected some rats with ing; intense cravings for food and water increased the number of receptors for neural a compound (SB334867) that prevents orexin originate here. Our findings suggest that excitation on the surfaces of these cells. Such from interacting with brain cells. Following orexin from the lateral hypothalamus affects strengthening of intercellular connections treatment with SB334867, rats spent 58 per- the reward pathway. Perhaps drugs take over occurs during learning. Scientists believe it cent less time in the morphine-associated the brain system for feeding and craving just may foster the development of drug craving. cage area—indicating a halving of their drug as they usurp neural systems for reward.” When the researchers pretreated the rats seeking. Rats given inert vehicle showed no “These behavioral findings extend the with an orexin blocker, cocaine lost its ability significant change in drug seeking. team’s important anatomical work differen- to alter dopamine-producing cells in the The investigators also tested orexin’s tiating two populations of orexin-producing VTA, suggesting that orexin may be neces- impact on the tendency of a new group of neurons in the hypothalamus. One popula- sary for cocaine-induced neuroplasticity and rats to revert to drug seeking after CPP tion, located in the lateral hypothalamus, is its behavioral consequences. waned following extended testing without involved in feeding, reward, and drug seek- “Our findings point to a key role for orex- drug administration (extinction). In con- ing, while the other regulates sleep and in in the neural changes in the reward path- trast to the first experiment, this time the arousal,” says Dr. Susan Volman of NIDA’s way that underlie craving and relapse,” says investigators injected a compound (rat Division of Basic Neuroscience and Behav- Dr. Borgland. “The physiological alterations pancreatic polypeptide, rPP) that stimu- ioral Research. “The findings identify new we observed likely influence those cells’ lates orexin neurons into the LH of some neural pathways involved in drug abuse, dopamine release, perhaps affecting the animals. These rats quickly resumed CPP— craving, and relapse, and may ultimately help activity of the reward pathway in a way that indicated by the difference in time spent in scientists find more effective therapies. increases the likelihood of relapse. One the morphine versus saline chambers—as implication of our findings is that addiction marked as that of another group that A ROLE IN COCAINE CRAVING? medication development efforts might do received a morphine priming injection Drs. Borgland and Bonci and colleagues well to target orexin receptors,” she says. (353 seconds and 424 seconds for rPP and at the UCSF Ernest Gallo Clinic and The work of the Pennsylvania and UCSF morphine, respectively). Rats that received Research Center demonstrated orexin teams points to orexin involvement in a vehicle injection did not renew morphine effects on cocaine-related behaviors remark- reward-seeking in general. Researchers CPP. ably consistent with those the Pennsylvania studying the effects of orexin-blocking com- To establish that stimulation of orexin team showed with respect to drug-related pounds in animal models of and neurons by rPP, and not some other behaviors. They also provided evidence that obesity have reported preliminary but prom- unidentified factor, was responsible for the orexin produces these effects at least in part ising findings. Both teams are currently effects in their second experiment, the by altering neurons in the VTA. The UCSF determining whether giving such com- investigators repeated the procedure. This team used behavioral to evalu- pounds to animals reduces self-administra- time they blocked the extinction of orexin ate orexin’s impact on rats’ responses to tion of cocaine, say Dr. Bonci and Dr. Aston- by giving the rats SB334867 prior to rPP. cocaine. Scientists generally think animals’ Jones, the latter now at the Medical These rats did not resume CPP. Finally, the behavioral sensitization—increased loco- University of South Carolina. ■ researchers infused orexin directly into motor activity following repeated exposure rats’ ventral tegmental area (VTA), the ori- to a drug—reflects drug-induced neural SOURCES gin of the dopamine-rich reward pathway, changes and corresponds to human craving Harris, G.C., Wimmer, M., and Aston-Jones, G. A role for and observed a resumption of drug-seeking for the drug. In the UCSF experiment, rats lateral hypothalamic orexin neurons in reward seeking. behavior. pretreated with an orexin blocker displayed Nature 437(7058):556-559, 2005. only half as much increase in locomotor Borgland, S.L., et al. Orexin A in the VTA is critical for the DRUGS MAY USURP FEEDING SYSTEM induction of synaptic plasticity and behavioral sensitiza- activity (138 percent) following five daily tion to cocaine. 49(4):589-601, 2006. The results, although in animals, suggest cocaine infusions (15 mg/kg) as rats pretreat- that orexin promotes drug abusers’ desire ed with an inert vehicle (257 percent).

6 NIDA Notes | Volume 21, Number 4 ■ RESEARCH FINDINGS

SAFE ANALGESIA, TOO? Endorphin Derivative Inhibits In separate studies, Dr. Millington and col- leagues found evidence that Gly-Gln has Reward From Morphine and potential for improving pain treatment by slowing the development of morphine toler- Nicotine in Rats ance. The investigators treated rats with mor- phine twice daily for 7 days and, each day, Recent studies suggest therapeutic potential for measured the rats’ reaction to pain with tail- glycyl-glutamine in opiate and nicotine addiction. flick latency tests. They observed that the pain-relieving effects of morphine declined 20

BY SARAH TEAGLE, percent by the second day, an indication that NIDA Notes Contributing Writer GLY-GLN BLOCKS THE REWARDING tolerance had developed rapidly. However, EFFECTS OF NICOTINE AND MORPHINE Rats trained to associate nicotine or morphine rats pretreated with Gly-Gln did not begin naturally occurring brain chem- infusions with a particular chamber showed a showing evidence of morphine tolerance until clear preference for the drug-linked chamber ical has shown early promise as when later allowed to roam freely between it the fourth day of treatment. Their level of pain a treatment for addiction. and a second chamber. Rats that were pre- relief had dropped to 75 percent of the maxi- NIDA-funded researcher Dr. treated with Gly-Gln prior to receiving nicotine mum by day 4, compared with 39 percent for A or morphine divided their time roughly equally William Millington and colleagues at Albany between drug-linked and saline-linked rats that were not pretreated. College of Pharmacy demonstrated that chambers. These findings offer new hope for making glycyl-glutamine (Gly-Gln), a product of the pain treatment more effective, but without conversion of one form of beta endorphin to Nicotine Experiment adding to the problems of painkiller abuse and another, reduces the rewarding effects of 300 prescription drug diversion. Dr. David Saline + Saline morphine and nicotine and the severity of 250 Gly-Gln + Saline Thomas of NIDA’s Division of Basic Neuro- 200 Saline + Nicotine withdrawal from these drugs in rats. r science and Behavioral Research explains, “It e b 150 Gly-Gln + Nicotine The researchers utilized an animal model m is like a tug-of-war; we want better pain treat- a r h e c

100 b

called conditioned place preference (CPP; d ment, but we do not want more addiction. If m e a k 50 h n i l for more on CPP, see “Animal Experiments c we can find a medication that improves the

- d g 0 e u r in Addiction Science,” NIDA Notes, Vol. 20, k management of without causing d n

i l n

- -50 i

e No. 5). When pretreated with Gly-Gln, rats t addiction or the negative physical side effects, n n i l e a p s Morphine Experiment stopped preferring a cage in which they s then it really is a win-win situation. Gly-Gln

n i s

t t a n r received morphine infusions over another in 300 may give us that.” e ) Saline + Saline s p s d

n which they received a physiologically inert e 250 Gly-Gln + Saline “We need to learn much more about Gly- o c m i e t

Saline + Morphine s 200

substance. The investigators concluded that s Gln’s pharmacology before we can develop it n u a n Gly-Gln + Morphine i Gly-Gln completely blocked the brain- e 150 into a drug that is useful clinically,” says Dr. m m ( rewarding effects of morphine. They used the e 100 Thomas. “The profile of its effects brought out m i same experimental technique to demon- T 50 by Dr. Millington’s work is striking. It makes a strate that Gly-Gln pretreatment also blocks 0 strong argument for taking the research to the the rewarding effects of nicotine in rats. -50 next step: understanding its mechanism of Some chemicals that block the rewarding action in the brain. In our world of preclinical effects of drugs of abuse also take away sub- morphine dependence in the rats, injected animal research, we really could not ask for jects’ pleasure in normal healthy activities. some with Gly-Gln 72 hours later, and then better findings. So far, everything we know Again using CPP, Dr. Millington’s team administered the antagonist about Gly-Gln is promising.” ■ demonstrated that Gly-Gln does not have to induce withdrawal. Across all measures, SOURCES this drawback, at least in regard to the Gly-Gln pretreated rats exhibited signifi- Cavun, S.; Goktalay, G.; and Millington, W.R. Glycyl-glut- one food—a sweetened cereal—that rats cantly fewer behavioral and physiological amine, an endogenous beta-endorphin-derived peptide, inhibits morphine-induced conditioned place prefer- enjoy. signs of withdrawal than the others. The ence, tolerance, dependence, and withdrawal. Journal of Subsequently, Dr. Millington and his coin- researchers observed a similar effect Pharmacology and Experimental Therapeutics 315(2):949- 958, 2005. vestigators examined Gly-Gln’s effect on when they induced withdrawal from nicotine Goktalay, G., et al. Glycyl-glutamine inhibits nicotine withdrawal from morphine. They induced with the agent mecamylamine. conditioned place preference and withdrawal. European Journal of Pharmacology 530(1-2):95-102, 2005.

NIDA Notes | Volume 21, Number 4 7 ■ RESEARCH FINDINGS Gene Experiment Confirms a Suspected Cocaine Action

Building on knowledge from developmental and cancer biology, addiction researchers are learning how acute and chronic cocaine exposure regulates certain genes.

BY LORI WHITTEN, CHROMATIN AND DNA HELIX FORM CHROMOSOMES Nucleosomes slide apart during NIDA Notes Staff Writer chromatin remodeling, increasing transcription factors’ access to a gene and thereby activating it. The DNA will then make mRNA, the blueprint for protein production. ocaine produces the long-term brain changes that underlie addiction in part by activating Ccertain genes. Dr. Eric Nestler and colleagues at the University of Texas Southwestern Medical Center and Harvard Medical School have shown that the drug achieves this activation at least in part through a process called chromatin remodeling. The finding opens up a new avenue for potential therapies for addiction. “Our research suggests that testing chemical compounds that reverse chromatin remod- eling is a promising approach to seeking treatments for drug abuse. This is already a major strategy for cancer therapy devel- opment,” says Dr. Nestler.

GENES REGULATE CRUCIAL PROTEINS Cocaine activates the genes that provide related inquiry: How does it happen? Their less compact. As the nucleosomes bunch the templates for building the proteins candidate explanation was chromatin up or spread out, some genes move into cFos, FosB, BDNF, and Cdk5, among oth- remodeling, a basic mechanism cells use to positions that increase—and others into ers. Researchers have linked the resulting alter levels of the body’s vast array of pro- positions that decrease—their ability to higher brain levels of some of these pro- teins to suit new circumstances and chal- interact with RNA polymerase, the enzyme teins with long-term consequences of lenges (see “Experience Restructures that executes the first step in protein build- chronic drug abuse. For example, accumu- Chromatin”). ing. This helps determine how much of the lation of long-lasting FosB correlates with Chromatin consists of the deoxyribonu- protein blueprinted by each gene will cocaine craving and drug self-administra- cleic acid (DNA) double helix that carries be made. tion in animals, and may contribute to long- an organism's genes wrapped around com- To test their hypothesis that cocaine lasting structural changes in cocaine plexes of histone proteins. The unit of chro- activates genes by inducing chromatin abusers’ brain reward systems. As matin is called the nucleosome, and chem- remodeling, Dr. Nestler and his team com- researchers continue to trace out the con- ical processes control how tightly packed pared tissue taken from the striatum of rats sequences of cocaine-induced gene activa- nucleosomes are. Chromatin remodeling exposed to the drug and others given saline. tion, Dr. Nestler and colleagues pursued a occurs when this packing becomes more or Specifically, they assayed the tissue for the

8 NIDA Notes | Volume 21, Number 4 end products of two chemical reactions tion from drug abuse to addiction,” says Dr. ■ Elevations in the FosB protein selec- known to modify chromatin’s shape: acety- Nestler. The experiments and assays tively activated Cdk5—the only gene lation and phosphoacetylation of its pri- also showed: examined in the study that was turned mary molecular components, histone 3 ■ A single cocaine injection did not affect on in this way. This finding suggests that (H3) and histone 4 (H4). Both of these reac- BDNF or Cdk5 gene-associated chro- FosB may influence histone modifica- tions remodel chromatin in ways that matin, but chronic exposure induced H3 tions by recruiting the chemical agents increase gene expression. acetylation of both. Once initiated, the of chromatin remodeling to some The findings bore out the hypothesis. effects were long-lasting. The quantities target genes. Within 30 minutes of a single injection, the of modified H3 in BDNF gene-associat- “Understanding how cocaine turns on chromatin associated with the cFos gene in ed chromatin in exposed animals these genes could help addiction the cocaine-exposed animals contained increased from 3-fold of those of saline- researchers develop potential treatments twice as much acetylated H4 than that in the treated animals at day 1 to 14-fold at day that counteract the effects of drug abuse at control animals, and phosphoacetylated H3 7. Acetylated H3 related to the Cdk5 the molecular level. Agents that reverse also was higher. The time course of these gene were more than two-fold those of chromatin remodeling are available, and we effects jibed with previous observations saline 1 day after the last injection, and are examining whether they block cocaine’s that cocaine induces a rapid, transient started to return to control levels only cellular effects,” says Dr. Nestler. increase in levels of the cFos protein. They 7 days after cocaine cessation. Such “Taken together with other studies were no longer present in tissues taken 3 persistent and robust gene activation showing that drugs induce long-term struc- hours after the injection, and they stopped long after the last dose of cocaine is tural changes to brain cells, Dr. Nestler’s occurring when animals were given repeat- striking in contrast with the relatively findings show that chromatin remodeling ed cocaine doses over an extended period. short-lived activation observed for cFos is one way that such neural modification These data support scientists’ conception and FosB. might occur. Such alterations are not of the cFos gene as an early responder to acute neural disruptions, with little or no direct role in situations of recurrent disruption. Experience Restructures Chromatin As with cFos, a single cocaine injection elevated acetylated H4 in chromatin linked to the FosB gene, but the levels returned to Chromosomes (pictured) are very long, contin- baseline within 3 hours. Repeated cocaine uous pieces of DNA that contain genes that did not induce H4 acetylation in FosB gene- help determine an individual’s identity. Humans associated chromatin, but did cause H3 have 23 chromosome pairs with an estimated acetylation. The researchers say that the 30,000 genes. The DNA sequence wraps around switch from H4 acetylation after a single proteins that give the chromosome a structure; cocaine exposure to H3 acetylation after together, they form chromatin. Cocaine and chronic exposure may mark a turning point other external agents and experiences can alter in developing addiction. the configurations of these proteins. Depending “More research is needed to identify the on the type of chemical change, the chromatin either bunches up or stretches out, activat- specific molecular basis of this switch,” says ing or silencing genes along the DNA sequence. Dr. Nestler. “However, prior work in my Chromatin reshaping seems to underlie healthy adaptations such as learning and mem- laboratory and with collaborators is start- ory as well as disease processes—including cancer, seizures, , and depression. ing to fill in a picture of why H3 acetylation In another study, for example, Dr. Nestler’s team found that social stress turned on a partic- and FosB’s activation and subsequent trig- ular gene in the of mice through chromatin remodeling, a long-lasting change that cor- gering of FosB after chronic cocaine responded with a behavioral indicator of depression. Antidepressant medication reversed might be important. We believe that this both the behavioral sign of depression and the elevated gene activity, underscoring a key point series of molecular events, and probably about the modifications: experience and chemical agents can alter gene expression through others, mediate the long-term behavioral chromatin remodeling, but such changes are reversible. and neural changes that underlie the transi-

NIDA Notes | Volume 21, Number 4 9 ■ RESEARCH FINDINGS

■ GENE EXPERIMENT [ Continued from page 9 ] COCAINE TURNS ON GENES BY ALTERING CHROMOSOMAL PROTEINS The researchers found chemical modifications to histone 3 (H3) and histone 4 (H4)—major proteins that form the structure of chromosomes—at areas linked with four genes. Acetylation of H3 and H4 and phosphoacetylation of H3 alter the proteins’ chemical structure, facilitating gene activation. necessarily permanent, and studies are needed to determine whether abstinence or other behavioral mod- Experiment Gene Results ifications further restructure chro- 30 min. 1 hr. 90 min. 3 hr. 1 day 7 days matin to a state similar to that seen Acute | AcetylatedAcetylated H4 H4 | Normal Normal | Phosphoacetylated H3 | Histones Histones ---- prior to drug exposure,” says Dr. Cocaine cFos Phosphoacetylated H3 Joni Rutter of NIDA’s Division of Rats received a Basic Neuroscience and Behavioral single injection, Acetylated H4 either saline or FosB | Acetylated H4 | Research. Whether nonstimulant cocaine (20 drugs of abuse also act through mg/kg). BDNF No change No change chromatin remodeling is another important area for future research, No change No change she says. Cdk5

Chronic No change No change A CONNECTION WITH Cocaine cFos COCAINE-RELATED BEHAVIOR Rats received an injection of Normal FosB Acetylated H3 In other experiments, Dr. Nestler either saline or Histones and colleagues linked chromatin cocaine (20 mg/kg) daily Normal remodeling to cocaine’s behavioral Acetylated H3 for 7 days. BDNF Histones effects by examining its role in a lab- oratory stand-in for human cue- Normal Cdk5 Acetylated H3 Histones induced drug seeking called condi- tioned place preference (CPP). By Cocaine exhibiting CPP—lingering in a part Withdrawal cFos Effects too transient to examine of a cage where it has received a Rats received an injection of drug—an animal indicates that it is either saline or FosB Effects too transient to examine seeking more of the drug (see “Ani- cocaine (20 mg/kg) daily for Acetylated H3: Levels rise to 3-fold those associated with saline injection mal Experiments in Addiction Sci- 7 days and did BDNF after 1 day, and to 14-fold after 7 days. ence,” NIDA Notes, Vol. 20, No. 5). not receive the drug again. Normal The researchers hypothesized that Acetylated H3: Levels rise to 3-fold those associated with saline injection after 1 day, and return to normal after 7 days. Histones augmenting or preventing histone Cdk5 modifications during drug adminis- tration sessions would increase or decrease ated cage, on average, relative to the con- including a group of animals that self- CPP, respectively. trol group. In contrast, the group given the administered the drug in the study. Their The investigators administered cocaine HSV vector lingered in the test cage for CPP experiment further strengthens the to mice daily for 4 days. Before each admin- one-third of the time spent by its control connection between histone restructuring istration, they treated one group with a group. and behavioral aspects of addiction and drug that enhances histone acetylation (tri- The findings suggest a causal link suggests that agents that reverse chromatin chostatin A, TSA) and another with a virus between histone acetylation in the striatum restructuring hold promise as potential that expresses an enzyme that blocks this and sensitivity to cocaine’s behavioral therapies,” says Dr. Rutter. ■ particular modification (herpes simplex effects. virus, HSV). When placed back in the test “The team had already demonstrated SOURCE cage on day 5, the group given TSA that chromatin remodeling plays a role in Kumar, A., et al. Chromatin remodeling is a key mech- doubled the time spent in the drug-associ- the rewarding aspects of cocaine abuse by anism underlying cocaine-induced plasticity in stria- tum. Neuron 48(2):303-314, 2005.

10 NIDA Notes | Volume 21, Number 4 ■ NIDA NOTES REFERENCE ARTICLE Impacts of Drugs on Neurotransmission The defining features of drug intoxication and addiction can be traced to disruptions in cell-to-cell signaling.

BY CARL SHERMAN, NIDA Notes Contributing Writer

rugs of abuse alter the way people think, feel, and behave by disrupting neurotransmission, the process of communication between brain cells. Over the past few decades, studies have established that drug dependence and Daddiction are features of an organic brain disease caused by drugs’ cumulative impacts on neurotransmission. Scientists continue to build on this essential understanding with experiments to further elucidate the physiological bases for drug abuse vulnerability as well as the full dimensions and progression of the disease. The find- ings provide powerful leads to new medications and behavioral treatments. This second article in our NIDA Notes Reference Series discusses the central impor- tance of studying drugs’ effects on neurotransmission and describes some of the most common experimental methods used in this research. As with other articles in the series, we provide illustrative references from articles published in NIDA Notes.

WHAT IS NEUROTRANSMISSION? A person reads. The words on the page enter the brain through the eyes and are trans- formed into information that is relayed, from cell to cell, to regions that process visual input and attach meaning and memory. When inside cells, the information takes the form of an electrical signal. To cross the tiny intercellular gap that separates one cell from the next, the information takes the form of a chemical signal. The specialized chemicals that carry the signals across the intercellular gaps, or synapses, are called neurotransmitters. The ebb and flow of neurotransmitters—neurotransmission—is thus an essential fea- ture of the brain’s response to experience and the environment. To grasp the basic idea of neurotransmission, compare the brain to a computer. A computer consists of basic units (semiconductors) that are organized into circuits; it processes information by relaying electric current from unit to unit; the amount of current and its route through the circuitry determine the final output. The brain’s corresponding basic units are the neurons—100 billion of them; the brain relays information from neuron to neuron using electricity and neurotransmitters; the volume of these signals and their routes through the organ determine what we perceive, think, feel, and do. Of course, the brain, a living organ, is much more complex and capable than any machine. Brain cells respond with greater versatility to more types of input than any semi- conductor; they also can change, grow, and reconfigure their own circuits.

NIDA Notes | Volume 21, Number 4 11 ■ REFERENCE SERIES: NEUROTRANSMISSION

THE BASIC RESEARCH QUESTIONS How Does the Drug Alter Neurotransmission? Neuroscientists seeking to understand why a drug is abused and Neurotransmission is a cyclic process that transpires in several the consequences of that abuse focus on two issues: steps utilizing specialized components of the sending and receiv-

■ Which neurotransmitter or neurotransmitters does it affect? ing cells (see “Getting the Message Across”). Identifying the pre- ■ How does it alter neurotransmission? cise step that a drug disrupts, and how, provides crucial insight into

Which Neurotransmitter or Distribution in the Functions Drugs That Neurotransmitter Central Nervous Neurotransmitters Does the Drug Affect? Affected Affect It System A person’s experiences when abusing a drug Dopamine Midbrain, Ventral Pleasure and reward Cocaine, reflect the functional roles of the particular neu- tegmental area (VTA), Movement, Attention, Methamphetamine, rotransmitter whose activity it disrupts. Each Cerebral cortex, Memory . Hypothalamus In addition, virtually all individual neuron manufactures one or more drugs of abuse directly neurotransmitters: dopamine, serotonin, or indirectly augment dopamine in the reward acetylcholine, or any one of a dozen others that pathway scientists have discovered to date. Each neuro- Serotonin Midbrain, VTA, Mood, Sleep, MDMA (ecstasy), LSD, transmitter is associated with particular effects Cerebral cortex, Sexual desire, Cocaine Hypothalamus Appetite depending on its distribution among the brain’s Norepinephrine Midbrain, VTA, Sensory processing, Cocaine, various functional areas. Dopamine, for exam- Cerebral cortex, Movement, Sleep, Methamphetamine, ple, is highly concentrated in regions that regu- Hypothalamus Mood, Memory, Amphetamine Anxiety late motivation and feelings of reward, account- Endogenous Widely distributed in Analgesia, Sedation, Heroin, Morphine, ing for its importance in compulsive behaviors (endorphin and brain but regions vary Rate of bodily Prescription painkillers such as drug abuse. A neurotransmitter’s impact enkephalin) in type of receptors, functions, Mood (Oxycodone) Spinal cord also depends on whether it stimulates or damp- Acetylcholine Hippocampus, Memory, Arousal, Nicotine ens activity in its target neurons. Cerebral cortex, Attention, Mood Some drugs primarily disrupt one neuro- Thalamus, Basal ganglia, Cerebellum transmitter or class of neurotransmitters. For Endogenous cannabi- Cerebral cortex, Movement, Marijuana example, opioid drug abusers experience noids (anandamide) Hippocampus, Cognition and changes that are similar to—but more pro- Thalamus, Basal ganglia memory nounced than—those that accompany normal Glutamate Widely distributed in Neuron activity , fluctuations in the brain’s natural opioid-like brain (increased rate), , Learning, Cognition, Alcohol neurotransmitters, endorphin and enkephalin: Memory increased analgesia, decreased alertness, and Gamma-aminobutyric Widely distributed in Neuron activity , slowed respiration (see table). Other drugs acid (GABA) brain (slowed), Anxiety, Tranquilizers, Memory, Anesthesia Alcohol interact with more than one type of neurotrans- mitter. Cocaine, for example, attaches to struc- tures that regulate dopamine, thereby producing euphoria; howev- its impact on abusers and is key to identifying medical and behav- er, cocaine also produces changes in norepinephrine and ioral interventions to inhibit, counter, or reverse the disruption. glutamate, which are the sources of its stimulant effects. Some drugs mimic neurotransmitters. Opioid drugs such as Because a neurotransmitter often stimulates or inhibits a cell heroin and OxyContin, for example, chemically resemble the that produces a different neurotransmitter, a drug that alters one brain’s natural opioids sufficiently to engage and stimulate their can have secondary impacts on another. In fact, the key effect that specialized receptors. Since heroin stimulates many more recep- all abused drugs appear to have in common—a dramatic increase tors than the brain uses in the normal cycle of endorphin and in dopamine signaling in the nucleus accumbens (NAc), leading enkephalin release and uptake, the result is a massive amplifica- to euphoria and a desire to repeat the experience—is in many cases tion of opioid activity. Marijuana and hashish mimic cannabinoid an indirect one. For example, nicotine stimulates dopamine- neurotransmitters, the most important of which is anandamide. releasing cells directly by stimulating their acetylcholine recep- Nicotine attaches to receptors for acetylcholine, the neurotrans- tors, and also indirectly by triggering higher levels of glutamate, a mitter for the cholinergic system. neurotransmitter that acts as an accelerator for neuron activity Some drugs alter neurotransmission by interacting with molec- throughout the brain.1 ular components of the sending and receiving process other than receptors. Cocaine, for example, attaches to the dopamine trans-

12 NIDA Notes | Volume 21, Number 4 REFERENCE SERIES: NEUROTRANSMISSION ■ porter, the molecular conduit that draws free-floating dopamine out of the synapse and back into the sending cell. As long as cocaine Getting the Message Across occupies the transporter, dopamine cannot reenter the cell by this route. It builds up in the synapse, stimulating receiving cell recep- tors more copiously and producing much greater dopamine impact on the receiving cells than occurs naturally. “Cocaine’s Dopamine Connections” enumerates some of cocaine’s interactions with the mechanisms of dopamine signaling, and how they motivate abuse and contribute to dependence and addiction. Finally, some drugs alter neurotransmission by means other than increasing or decreasing the quantity of receptors stimulated. , such as or , enhance receiv- ing cells’ responses when the neurotransmitter gamma-aminobu- tyric acid (GABA) attaches to their receptors. Benzodiazepines’ relaxation effects result from this increased sensitivity to GABA’s inhibitory impact on cellular activity.

What Changes Occur With Chronic Drug Abuse? The task in neurotransmission is to convey a signal from a sending During the early phase of an individual’s drug experimentation, cell to a receiving cell across an open space known as a synapse. All neurotransmission normalizes as intoxication wears off and the brain cells accomplish this in approximately the same way. substance leaves the brain. Eventually, however, drugs wreak The sending cell manufactures neurotransmitter molecules and changes in cellular structure and function that lead to long-lasting stores them in packets called vesicles. When stimulated appropriately, or permanent neurotransmission abnormalities. These alterations the cell generates an electric signal and causes some vesicles to underlie , addiction, withdrawal, and other persist- migrate to the cell membrane, merge with it, open up, and release ent consequences. their contents into the synapse. Some molecules drift across the Some longer term changes begin as adjustments to compensate synapse and link up, lock-and-key fashion, with molecules called for drug-induced increases in neurotransmitter signaling intensities. receptors on the surface of the receiving cell. Receptors bridge the For example, drug tolerance typically develops because sending cells receiving cell’s membrane; they have one facet on the outside and reduce the amount of neurotransmitter they produce and release, or one on the inside of the cell. When the neurotransmitter links up with receiving cells withdraw receptors or otherwise dampen their respon- the exterior facet, the interior facet precipitates an electrical response siveness. Scientists have shown, for example, that cells withdraw in the cell membrane or inside the cell. The result may be increased opioid receptors into their interiors (where they cannot be stimulat- production of some cell product or—often—a repeat of the process ed) when exposed to some opioid drugs; when exposed to morphine, just described, so that the message gets relayed in turn to the next however, cells appear instead to make internal adjustments that pro- cell in the circuit. duce the same effect—reduced responsiveness to opiate drugs and At this point, cell-to-cell communication is complete. The natural opioids.2 Over time, this and related changes recalibrate the neurotransmitter molecules drop off the receptors. Loose again in the brain’s responsiveness to opioid stimulation downward to a level synapse, they meet three fates: where the organ needs the extra stimulation of the drug to function ■ Some attach to another receptor; normally; without the drug, withdrawal occurs. ■ Some encounter an enzyme, a chemical that breaks them apart; The drug-related mechanisms producing cumulative changes and in neurotransmission sometimes are genetic in nature. While a ■ Some reenter the sending cell via a special pathway through the drug cannot change a person’s genes, drugs can prod some genes to axon membrane, called a transporter. Once back inside the cell, increase their production of proteins, leading to changes in cell they are available for re-release in future neurotransmission function or even actual reshaping of the physical structure of cells. episodes. For example, in rats, cocaine and amphetamine stimulate genes Normally, when drugs are not present, the cycle of release, that produce the proteins used to build dendrites, branch-like cell breakup, and cell re-entry maintains the amount of neurotransmitter structures that contain neurotransmitter receptors.3 Brains in the synapse, and hence neurotransmission, within certain limits. In normally sprout new dendrites as they register new learning; the most cases, when an abused drug enters the brain, it causes neuro- accelerated dendrite formation stimulants induce may partially transmission to increase or decrease dramatically beyond these limits. account for these drugs’ unusual hold on an abuser’s attention.

NIDA Notes | Volume 21, Number 4 13 ■ REFERENCE SERIES: NEUROTRANSMISSION

Some drugs are toxic to nerve cells, and the effect accumulates tuations at tenth-of-a-second intervals by measuring electrical with repeated exposures. For example, the club drug methylene- changes related to neurotransmitter concentrations.7 dioxymethamphetamine (MDMA, ecstasy) damages axons that release serotonin; the result is disruption of serotonin neurotrans- Live Studies mission that likely underlies the long-lasting memory problems Studies with living animals or people are essential for tying drugs’ experienced by abusers.4 Similarly, methamphetamine, over time, effects on neurotransmitters to behaviors or symptoms. A com- damages enough dopamine-sending cells to cause significant mon design for experiments with either animals or people is to defects in thinking and motor skills; with abstinence, dopamine give study subjects a chemical that has a known effect on a particu- function can partially recover, but it is unclear whether cognitive lar neurotransmitter, and then observe the impact on their behav- and motor capabilities come back as well.5 ior. Typically, the chemical is either an agonist (promoter) or antagonist (blocker) of signaling by the neurotransmitter. EXPERIMENTAL METHODS In a recent experiment, for example, a research team adminis- To determine whether or how a drug affects a particular neuro- tered a glutamate agonist to rats and showed that the resulting transmitter, researchers typically will compare individuals who increased levels of the neurotransmitter correlated with a reduc- have a history of drug exposure with others who do not. If tion in the animals’ cocaine seeking.8 Another team using the same researchers are investigating links between a drug’s impact on neu- strategy implicated glutamate in nicotine withdrawal.9 Such studies rotransmission and a drug-related behavior or symptom, they may are a staple of testing compounds to identify medication classes compare individuals who exhibit the behavior or symptom with with potential for treating abuse or addiction. others who do not. The subjects in these experiments may be ani- Researchers also genetically alter animals to have special char- mals or people. In the case of animals, drug exposure often takes acteristics, such as producing less or more than the normal place under laboratory conditions designed to mimic human drug amounts of a particular neurotransmitter, or lacking receptors for consumption. Studies can be divided into those in which measure- a neurotransmitter. Researchers expose such animals to a drug and ments are made in living animals or people and those in which observe whether the animals’ display of some particular animal brain tissue is removed and examined. drug-related behavior—for example, pacing restlessly after being given a stimulant—increases or decreases. Brain Tissue Assays With removed tissue, scientists may perform chemical assays to Brain Scans quantify the presence of a neurotransmitter, receptor, or other Brain imaging techniques enable neuroscientists to directly assess structure of interest. In a recent experiment, scientists assayed neurotransmission in people and living animals. With positron brain tissue from 35-day-old rat pups and found that those that had emission tomography (PET), researchers can compare groups of been exposed to nicotine in utero had fewer nicotine receptors in drug-abusing and nonabusing individuals, quantifying differences the reward system than unexposed rats.6 in their levels of a particular neurotransmitter molecule (e.g., A second experimental method using removed brain tissue—in dopamine) or neurotransmission component (e.g., a receptor or vitro, literally, in glass, a historical term referring to the containers transporter). With PET, researchers also can correlate a drug’s for the tissue and solution—enables researchers to view a drug’s transit through the brain with fluctuations in a target neurotrans- effects on neurotransmission in action. Scientists place the tissue mitter. They can elicit a drug-related behavior or symptom (e.g., in a laboratory solution of nutrients that enables the cells to con- craving) and relate neurotransmitter fluctuations to the rise and tinue to carry out some of their living functions. The researchers fall in its intensity. may then, for example, add the drug being investigated to the solu- One recent PET study, for example, showed that smokers have tion and monitor whether the cells respond by increasing their less of the neurotransmitter-degrading enzyme monoamine release of neurotransmitters. Alternatively, they may measure cell oxidase-B (MAO-B) throughout their bodies than nonsmokers.10 membrane or electrical properties that stimulate or inhibit the The relative deficit of MAO-B may help explain why smokers are at release of neurotransmitters. higher risk for hypertension and other chronic diseases. In both in vitro experiments and in living animals, the tech- Researchers use both PET and functional magnetic resonance niques for measuring neurotransmitter quantities and fluctuations imaging (fMRI) to monitor metabolic activity in selected regions of include microdialysis and fast-scan cyclic voltammetry (FSCV). the brain. Because each neurotransmitter has a unique distribu- Microdialysis involves taking a series of samples of the intercellu- tion among the regions of the brain, information on locations of lar fluid containing the neurotransmitter through a microscopic heightened or decreased activity provides clues to which neuro- tube inserted into the tissue or living brain. FSCV, recently devel- transmitter is affected under the conditions of the study. oped by NIDA-funded scientists, monitors neurotransmitter fluc-

14 NIDA Notes | Volume 21, Number 4 REFERENCE SERIES: NEUROTRANSMISSION ■

SUMMARY Cocaine’s Dopamine Connections By altering neurotransmission, addictive drugs produce effects that make people want to continue to abuse them and Research on cocaine illustrates that many dimensions may be involved in a single drug’s inter- induce health problems that can be long- action with the activity of a single neurotransmitter. Studies show that cocaine alters lasting and profound. The effects are drug- dopamine neurotransmission with effects on: specific: Each drug disrupts particular neurotransmitters in particular ways. Reward Some important effects, however, are

■ Cocaine causes the pleasurable feelings that motivate drug abuse by raising dopamine shared by all: initial pleasurable feelings, concentrations in the synapses of the reward system.a and subsequent dependence and addic-

■ Besides keeping dopamine in the synapses by blocking the transporters, cocaine can tion, resulting from disruption of the indirectly promote release of additional dopamine into the synapses by mobilizing a sup- dopamine neurotransmitter system. ply that the sending cells normally hold in reserve.b Scientists use a wide variety of experi-

■ Cocaine’s yield of pleasurable feelings arises largely through the activity of one mental tools and techniques to study particular set of dopamine receptors, called D3 receptors.c drugs’ effects on neurotransmission, and their consequences, in both animals and Addiction people. Their findings enhance our under-

■ Some studies indicate that the transition from casual cocaine abuse to addiction begins standing of the experiences of drug with the abuser’s very first doses. For example, a single exposure to cocaine causes some abusers and the plight of addicts, point the cells in the brain’s reward system to increase their responsiveness to subsequent way to new behavioral and medication stimulations.d treatments, and provide potential bases

■ In living animals with minimal exposure to cocaine, the drug alters the dopamine for prevention strategies and monitoring

e responsiveness for at least a week. progress in treatment. ■

■ After chronic cocaine abuse dopamine ticks up in the reward system when the abuser encounters a cue associated with the drug.f SOURCES ■ Brains normally sprout new neurotransmitter receiving structures in the process of 1 “Nicotine's Multiple Effects on the Brain's Reward turning new experience into learning. Cocaine accelerates this process, which may help System Drive Addiction,” NIDA Notes 17-6. account for the drug’s unusual hold on an addicted individual’s attention.g 2 “Genetic Engineering Studies May Lead to Development of More Effective Pain Relievers,” NIDA Notes 15-3. Vulnerability to Abuse 3 “Stimulant Drugs Limit Rats' Brain Response to ■ A young person’s marked taste for novelty may be an indication that dopamine Experience,” NIDA Notes 19-3. activity in his or her brain’s reward system is especially sensitive to cocaine.h 4 "Ecstasy Damages the Brain and Impairs Memory ■ An individual’s attraction to cocaine’s dopamine-stimulating effects also may relate to in Humans,” NIDA Notes 14-4. i his or her social circumstances. 5 “Methamphetamine Abuse Linked to Impaired ______Cognitive and Motor Skills Despite Recovery of a “Brain Scans Open Window to View Cocaine’s Effects on the Brain,” NIDA Notes 13-2. Dopamine Transporters,” NIDA Notes 17-1. b Venton, B.J., et al. Cocaine increases dopamine release by mobilization of a synapsin-dependent reserve 6 “Nicotine Alters the Developing Rat Brain,” pool. The Journal of Neuroscience 26(12):3206-3209, 2006. Article will be featured in NIDA Notes 21-5. NIDA Notes 21-2. c “Animal Studies Suggest D3 Receptors Offer New Target for Treatment Medications,” NIDA Notes 18-4. 7 “Microscopic Probe Detects Changes in Brain Chemistry as They Occur,” NIDA Notes 19-1. d “Altered Cellular Activity May Be First Step in Progression to Cocaine Addiction,” NIDA Notes 16-5. 8 “Brain Glutamate Concentrations Affect Cocaine e “Even Modest Cocaine Use May Cause Brain Changes That Could Contribute to Addiction,” Seeking,” NIDA Notes 19-3. NIDA Notes 16-3. 9 “Nicotine Withdrawal Linked to Disrupted f “In Chronic Drug Abuse, Acute Dopamine Surge May Erode Resolve to Abstain,” NIDA Notes 19-1. Glutamate Signaling,” NIDA Notes 19-6. g “Stimulant Drugs Limit Rats’ Brain Response to Experience,” NIDA Notes 19-3. 10 “Smoking Decreases Key Enzyme Throughout h “Behavioral Response to Novelty Foreshadows Neurological Response to Cocaine,” NIDA Notes 21-2. Body,” NIDA Notes 19-1. i “Social Environment Appears Linked to Biological Changes in Dopamine System, May Influence Vulnerability to Cocaine Addiction,” NIDA Notes 17-5.

NIDA Notes | Volume 21, Number 4 15 ■ RESEARCH FINDINGS Study Links Anabolic Steroids to Brain Changes in Adolescent Female Mice

Exposure may promote aggression in girls.

BY CARL SHERMAN, anxiety, and aggression. They found that in DRUG TARGETS RECEPTORS NIDA Notes Contributing Writer female, but not male, animals the AAS The researchers injected mice with a interfered with GABA transmission in the solution of 17 -MeT in sesame oil, in doses nabolic androgenic steroid area. Theoretically, this effect would reduce (7.5 mg/kg/d) that would correspond to (AAS) abuse, once largely lim- GABA’s inhibitory influence and thus those taken by humans who are abusing the ited to elite athletes, has potentially contribute to the excessive drug heavily. They injected a control group A spread to a wider population emotions and behaviors seen in AAS abuse. of mice with the sesame oil vehicle alone. that includes adolescents along with adults, Various studies have linked increased anxi- The researchers examined brain tissue and girls as well as boys. While the psycho- ety and aggression, and both increased and from half the mice in each group after 3 logical and behavioral consequences of AAS decreased libido to AAS use. weeks of treatment and from the other half use presumably reflect its impact on a num- “The GABA system isn’t the only target after 6 weeks. They focused on the subunits ber of brain areas, a NIDA-funded study at for the effects of AAS, but it is likely an that make up GABA type A receptors

Dartmouth Medical School has identified important one,” Dr. Henderson says. (GABAA) in cells of the MPN and on the one neurobiological effect that has poten- “Going into the experiment, we assumed way that AAS exposure affected the func- tially important implications for the emo- we’d see an effect on the tion of these receptors. Each receptor con- tional stability and well-being of adolescent GABA system in the MPOA and expected tains five of these subunits, proteins that girls in particular. there would be differences between males determine the receptor’s sensitivity to Principal investigator Dr. Leslie Hender- and females.” This area of the brain, partic- drugs and hormones. son and colleagues studied the effect of the ularly the cluster of neurons within it called To test how the reduction in 2 subunit AAS, 17 -methyltestosterone (17 -MeT), the medial preoptic nucleus (MPN), is production might affect GABAA receptor on the activity of the neurotransmitter structurally different in the sexes. gamma-aminobutyric acid (GABA) in adoles- EFFECTS ON RELATIVE LEVELS OF GABA RECEPTOR mRNA The researchers measured the effects induced cent mice. Loosely by anabolic androgenic steroids (AAS) on GABAA receptor subunit mRNA levels for male and female mice treated with 17 -MeT for 3 (blue) or 6 (purple) weeks. Six weeks of AAS exposure decreased the amount of one of the subunits of the speaking, GABA acts as a receptor 2 only in female mice. No significant effects were observed in the treated male mice. calming agent through- out the nervous system: 3 weeks 6 weeks Males Females It dampens activity 1.8 1.8 of the neurons to 1.6 1.6 which they are connect- 1.4 1.4 s s l l e ed. Specifically, the e v 1.2 v 1.2 e e L L

A researchers focused on 1.0 A 1.0 N N R R m the steroid’s impact on m

0.8 0.8 e e v v i i t GABA functioning in the t a a * l 0.6 l 0.6 e e R R medial preoptic area 0.4 0.4 (MPOA) of the basal 0.2 0.2 forebrain, a region that 0.0 0.0 participates in the regu- ␣1 ␣2 ␣5 ␣1 ␣2 ␣5 lation of sexual behavior, Receptor Subunits Receptor Subunits *Statistically significant Relative levels measured in control animals

16 NIDA Notes | Volume 21, Number 4 function, the researchers measured the Chronic exposure to of the AAS aug- alter the brain’s sensitivity to these amplitude and frequency of inhibitory mented gender differences in both the chemicals.” postsynaptic currents (IPSCs)—a measure structure and function of certain GABA “Although this is a basic research study, of the receptor’s efficacy in inhibiting the receptors, Dr. Henderson says. “Overall, its potential translation to humans, even if activity of neurons—in the MPN. Here, too, the effect was to decrease GABA transmis- speculative, is striking,” says Dr. Pushpa they found sex-based differences that were sion in the MPN of female, but not male, Thadani, formerly of NIDA’s Division of magnified by AAS. In untreated mice, the adolescent mice. This would presumably Basic Neuroscience and Behavioral IPSCs were smaller in amplitude in females increase the level of activity or change the Research. “It demonstrates that AAS than in males. Female mice that received 3 pattern of activity in postsynaptic neurons exposure in the adolescent period produces to 4 weeks of AAS displayed smaller and of the female mice.” gender-specific changes at the molecular less frequent currents than controls, sug- level that may be correlated with known gesting that exposure to the drug had A CLOSER LOOK behavioral outcomes.” reduced GABAA receptor function, thereby How do these neurobiological changes Although applying findings from this widening the gender gap. There was no contribute to the behavioral manifestations and similar studies in actual interventions comparable change in males. of AAS abuse? “It could be that an AAS that remains a distant goal, “these studies The researchers concentrated on the 2 promotes aggression in males would pro- advance our understanding of the actions subunit family, which earlier studies had mote it more in females, or have different of AAS on the brain and behavior, which can shown that 17 -MeT alters. Before treat- effects on the expression of sexual behav- empower us to better educate the lay public ment, levels of messenger RNA (mRNA) iors, but this is something we are just begin- on the harmful effects associated with for the 2 subunit were lower in female ning to explore,” Dr. Henderson says. abuse,” Dr. Thadani says. “The research is than in male mice in cells of the MPN. After “What’s more, there are 60 to 100 AAS, and still in its infancy,” she observes. Other 6 weeks, 2 subunit mRNA—an indicator their neurobiological effects are unlikely to studies, now under way, are seeking to clar- of the quantity of the subunit being pro- be uniform. In time, we may be able to start ify the links between aggression in female duced—had declined by 37 percent in parsing out whether certain commonly mice and AAS-associated neurobiological female mice treated with the AAS com- abused steroids are likely to amplify aggres- changes. “When these findings are avail- pared with controls, but was essentially sion and libido in women or in men while able, we’ll probably be in a better position unchanged in males. When the researchers others affect both genders equally.” to translate this information into the measured the actual protein that makes up Dr. Henderson notes that alterations in human arena.” ■ the 2 subunit in female mice, they found a GABA receptor function could have other A SOURCE small but significant reduction (8 percent) important effects as well. “The GABAA Penatti, C.A.A., et al. Sex-specific effects of chronic in the number of neurons containing receptor is a major target of many drugs, anabolic androgenic steroid treatment on GABAA 2 protein. including alcohol and benzodiazepines. receptor expression and function in adolescent mice. Neuroscience 135(2):533-543, 2005. Changing the subunit composition could

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■ Map locations and contacts for each network site ■ Network descriptions and missions ■ Links to network Web sites ■ Links to scientific protocols and papers ■ Links to procedural policies and manuals ■ NIDA news and events of interest to networks ■ NNP Colleagues Directory, a searchable database of members’ expertise and research focus nnp.drugabuse.gov

NIDA Notes | Volume 21, Number 4 17 ■ BULLETIN BOARD

Meeting Reviews Roles of Drug can reduce risky behaviors among outpatients addicted to heroin Abuse and Risky Behavior in HIV and cocaine. NIDA-funded research on the link between drug abuse and HIV infection extends to the international community. Dr. Steffanie A. Strathdee, an infectious disease epidemiologist at the University of California, San Diego, reported that HIV infection among injection drug users has risen sharply in many countries—including the Ukraine, the Russian Federation, Vietnam, Iran, and China—with emerging epidemics in other nations. Interventions that reduce risky behaviors among populations that interact with both high- and low-risk individuals (for example, prisoners, non-injecting drug users, and sex workers who inject drugs) are important More than 400 researchers and clinicians gathered in Bethesda, opportunities for HIV prevention. Maryland, May 8-9 to discuss the impact of drug abuse on the spread Participants also discussed the disproportionate burden of of HIV. NIDA sponsored the meeting in collaboration with other HIV/AIDS among people in prisons and jails and the importance National Institutes of Health agencies and the Centers for Disease of interventions in such settings. Improved technology now allows Control and Prevention. for faster HIV testing, and expanded testing and counseling are key Drug and can prompt poor judgment and actions components of prevention strategies for drug abusers and other that people might not engage in otherwise, for example, risky sexual groups. behaviors with infected partners. Drug abuse also may increase HIV transmission when abusers trade sex for drugs or money. The Adolescent Inhalant Use Is Stable increased risk of HIV related to drug abuse extends beyond the shar- Overall, but Rising Among Girls ing of drug injection equipment to include such risky behaviors. Dr. Igor Grant of the University of California, San Diego, Almost 5 percent of girls between the ages of 12 and 17 used an addressed methamphetamine abuse and HIV. The drug and the inhalant to get high in 2005, an increase from 4.1 percent in 2002, infection each cause degenerative brain disease. They may damage according to a new report. Overall, inhalant use by boys and girls in neural tissue through a common biological pathway—for example, this age group remained stable over the 4-year period, at an average enhanced inflammatory responses—but also seem to generate annual rate of 4.5 percent, or an estimated 1.1 million adolescents. distinct pathologies that may combine to exacerbate each other. The report, “Patterns and Trends in Inhalant Use by Adolescent The resulting cognitive impairments may explain why metham- Males and Females: 2002-2005,” is based on an analysis of the 2002- phetamine abuse reduces adherence to HIV medications, that, in 2005 National Household Survey on Drug Use and Health, an turn, may facilitate HIV transmission. annual survey sponsored by the Substance Abuse and Mental Dr. Jonathan M. Ellen of the Johns Hopkins University School Health Services Administration (SAMHSA). SAMHSA, NIDA, and of Medicine linked marijuana abuse with risky sexual behavior other federal agencies released the report at a March 15 press confer- among African-American youth in San Francisco. Another study ence to kick off the 15th National and Poisons Awareness indicated that HIV-infected young women who abuse marijuana are Week, March 18-25. less likely to keep medical appointments. “Young people who turn to inhalants may be completely Dr. Richard A. Rawson of the University of California, Los unaware of the serious health risks,” said NIDA Deputy Director Dr. Angeles, reported preliminary results suggesting that addiction Timothy P. Condon. “We know that inhalant abuse can start early, treatment reduced risky sexual behaviors among metham- with research suggesting that even preadolescent children seek phetamine abusers—adding to similar findings among them out because they are easy to obtain. NIDA research also methamphetamine-abusing men who have sex with men (see indicates that those who begin using inhalants at an early age are “Treatment Curbs Methamphetamine Abuse Among Gay and more likely to become dependent on them—and long-term inhalant Bisexual Men,” NIDA Notes, Vol. 20, No. 4). Similarly, Dr. Kenzie L. abusers are among the most difficult drug abuse patients to treat.” Preston of NIDA’s Intramural Research Program reported that The report is available at www.drugabusestatistics.samhsa.gov/ adding behavioral therapy to treatment 2k7/inhalants/inhalants.pdf.

18 NIDA Notes | Volume 21, Number 4 ■ TEAROFF Information to clip, save, and share

■ The relationships between needle-sharing practices and HIV Journal Highlights infection among heroin abusers in Dar es Salaam, Tanzania; The prevalence of HIV and hepatitis C infections among injec- Global Nexus of Drug ■ tion drug users receiving substance abuse treatment in two Abuse and HIV/AIDS large hospitals in Barcelona, Spain; ■ HIV prevalence among heroin-addicted individuals in Muar, Malaysia;

■ Sexual risk behaviors among injection drug users in Shanghai, ne of the most urgent public health goals of addiction China;

researchers is to curb drug abuse behaviors that con- ■ The impact of drug abuse on adherence to highly active anti- Otribute to the spread of HIV/AIDS, says a special supple- retroviral therapy (HAART) among HIV-positive outpatients ment to Drug and sponsored by the NIDA in France;

International Program. ■ HIV/AIDS risk factors along the U.S.-Mexico border; Dr. Steven Gust, director of the program and an editor of the ■ Regional differences in the characteristics of injection drug supplement, observes that about 5 percent of the world’s popula- users in New South Wales, Australia; and tion aged 15 to 64 abuses drugs and that this behavior is a major ■ The acceptability of audio computer-assisted self-interview factor in the transmission of HIV/AIDS and other illnesses. Two among substance abusers in Rio de Janeiro, Brazil. key routes of HIV infection are through the sharing of needles and other drug injection paraphernalia and high-risk sexual contact, Since 1990, the NIDA International Program has fostered coop- with the latter being one of the fastest-growing routes among erative research and the exchange of scientific information by drug women. abuse researchers worldwide. The program’s objectives include NIDA-supported research has shown that preventive interven- promoting international research and collaboration, communicat- tions and substance abuse treatment can reduce the transmission ing and disseminating science-based drug abuse information, and of HIV in drug-abusing populations. By promoting collaborative supporting research training and exchange opportunities. research across the globe, the NIDA International Program hopes to improve treatments and outreach programs to reduce high-risk More information on the link between drug abuse and drug use and sexual behaviors, Dr. Gust says. HIV/AIDS is available at www.hiv.drugabuse.gov. The supplement compiles studies—several of them funded by NIDA—on drug-related HIV transmission in 16 different localities SOURCE across the globe. Most of the studies focus on injection drug use, Gust, S.W., Strathdee, S.A., and Winstanley, E.L., eds.. Drug Abuse and HIV/AIDS: a primary pathway for HIV transmission. Some of the topics International Research Lessons and Imperatives. Drug and Alcohol Dependence covered are: 82(S1), 2006.

NIDA at Your www.drugabuse.gov Fingertips ■ Information on Drugs of Abuse ■ Publications (including NIDA Notes)

■ Calendar of Events

News and information about NIDA ■ Links to NIDA Organizational Units research, programs, and events is ■ Funding Information quickly and easily accessible through NIDA’s home page: ■ Internal Activities ■ Links to Related Web Sites

NIDA Notes | Volume 21, Number 4 19 ■ WHAT THE NUMBERS SAY

90 Among Young Attendees at STD Clinics, Substance Abusers

80 Report More Risky Sexual Behavior

70 Always Used Condoms Multiple Sex Partners Patients aged 15 to 24 of a public sexually transmitted 60 STD Diagnosed disease clinic who had a substance use disorder (SUD) s t

n were two to three times as likely as those without an e

i 48.8 t 50 SUD to report multiple sexual partners and inconsistent a

P condom use during the past year. Overall, 43 percent

f

o of the 448 patients who participated in the Pittsburgh 40 e 35

g clinic study had an alcohol or marijuana use disorder, a t 28.8 and these young people also were 70 percent more n 30 e 26.7 likely to be diagnosed with a sexually transmitted c r

e disease during their visit. P 20 14.4 10 6.1

0 No SUD With SUD

SOURCE: Cook R.L., et al. Alcohol and drug use and related disorders: An underrecognized health issue among adolescents and young adults attending sexually transmitted disease clinics. Sexually Transmitted Diseases 33(9):565-570, 2006.

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