Research Findings - Basic Neuroscience Research

Paternal Cocaine Exposure & Its Consequences

In a recent study, NIDA supported researchers, Dr. Michael Lidow and his associates report that paternal cocaine abuse may have a significant negative impact on offspring development and that such paternal impact could be brought about by long-term cocaine exposure of males prior to coitus. This study conducted using a novel drug inhalation model revealed that male mice were capable of learning self-administration of cocaine via inhalation route as well as achieving and maintaining blood levels of this drug during daily inhalation sessions comparable to that reported for females. Authors also noted a reduction in biparietal head diameter in newborn pups sired by cocaine-inhaling males suggesting a decreased cerebral volume. Most importantly, they observed a greater negative impact in female offspring compared to males with respect to working memory and light stimulus duration. This study also showed that chronic cocaine exposure in male mice did not result in substantial breakage of spermatozoal DNA, but significantly altered expression of DNA methyltransferase 1 and 3a in the germ cell-rich seminiferous tubules of the testis. Since these enzymes are essential for generating and maintaining parental gene imprinting in germ cells, the authors' observations point to an intriguing possibility that cocaine may cause paternally induced neuroteratological effects by interfering with gene-imprinting patterns in male gametes. He, F., Lidow, I.A., and Lidow, M.S. Consequences of Paternal Cocaine Exposure in Mice, Neurotoxicology and Teratology, 28, pp. 198-209, 2006.

Supraspinal Brain-derived Neurotrophic Factor Acts at the TrkB Receptor to Produce

Pain Sensitivity Brain-derived neurotrophic factor, or BDNF, plays a critical role in learning and memory by actions at the TrkB receptor. NIDA grantees Drs. Ronald Dubner and Ke Ren (University of Maryland, Baltimore) and colleagues now report that BDNF-TrkB signaling also plays a critical role within the brain to produce pain hypersensitivity. They found that after tissue injury, the BDNF-TrkB complex in the brain stem triggers pain facilitating signals to the spinal cord. This facilitating signal causes the amplification and spreads the pain. This discovery not only helps us understand the mechanisms of chronic pain, but suggests the possibility of targeting the BDNF-TrkB complex in the development of novel pain therapies. Guo, W., Robbins, M.T., Wei, F., Zou, S., Dubner, R., and Ren, K. Supraspinal Brain-Derived Neurotrophic Factor Signaling: A Novel Mechanism for Descending Pain Facilitation, Journal of Neuroscience, 26(1), pp. 126-137, 2006.

Buprenorphine Pharmacokinetics

Buprenorphine, a partial mu agonist, was approved by the Food and Drug Administration in 2001, in part for the treatment of dependence on opioids such as heroin, morphine, and oxycontin. It is formulated under the trade name Suboxone, consisting of a sublingual tablet of 2 or 8 mg buprenorphine, together with 0.5 or 2 mg naloxone as a mu antagonist. In work supported by NIDA awards to the University of Utah and the Virginia Commonwealth University, Dr. David Moody and Dr. Elinore McCance-Katz have recently collaborated to determine the in-vivo pharmacokinetics of this formulation in human plasma and urine samples. Five opioid-dependent subjects were treated with sixteen mg daily doses of buprenorphine for twenty-one days, with collection of blood and urine (twenty-four hour) samples obtained. These clinical samples were treated with appropriate deuterated internal standards, centrifuged in the case of blood samples, and the supernatants applied to solid phase extraction cartridges for extraction, solvent removal, and reconstitution. Separation of four particular analytes, buprenorphine, norbuprenorphine, and the three position glucuronides of both buprenorphine and norbuprenorphine, was carried out. The pharmacokinetic data indicated that norbuprenorphine-3-glucuronide was the predominant metabolite found in both urine and plasma, and that buprenorphine was converted to its glucuronide and to norbuprenorphine in approximately the same time frame (i.e, one hour after administration). Huang, W., Moody D.E., and McCance-Katz, E.F. The In-vivo Glucuronidation of Buprenorphine and Norbuprenorphine Determined by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry, Therapeutic Drug Monitoring, 28(2), pp. 245-251, 2006.

Tritiation of the Cannabinoid Receptor Antagonist SR144528 Involving Lithium Aluminum Tritide Reduction; Assessment of the Kinetic Isotope by 3H-NMR

The pyrazole caboxamide SR144528 is a selective antagonist at the peripheral cannabinoid CB2 receptor. The potential value of this compound to researchers for pharmacological studies of the CB2 receptor and its potential role in immune function prompted the synthesis of the tritiated compound. In this paper the authors reported the synthesis of this compound and used an approach that enabled the incorporation of highly specific activity tritium label while circumventing the liability of the target compound to catalytic hydrogenation. Lithium aluminum tritide of less than maximum specific activity was employed to introduce tritium, resulting in hydride/tritide incorporation indicative of non kinetic isotope effect for the hydride/tritide reduction of a methyl benzoate. Seltzman, H.H., Foster, M.C., Wyrick, C.D., Burgess, J.P. and Carroll, F.I. Tritiation of the Cannabinoid Receptor Antagonist SR144528 Involving Lithium Aluminum Tritide Reduction; Assessment of the Kinetic Isotope Effect by 3H-NMR, Journal of Labeled Compounds and Radiopharmaceuticals, 48, pp. 589-596, 2005.

Marijuana Smoke and Rimonabant Precipitated Withdrawal

The goals of the present study were to assess whether the CB1 antagonist SR 141716 (rimonabant) precipitates withdrawal in mice that had been repeatedly exposed to marijuana smoke, and to compare these precipitated withdrawal effects to those elicited following intravenous administration of its chief psychoactive component _9-tetrahydrocannabinol _9-THC. SR 141716 elicited a significant increase in paw tremors in mice that were repeatedly dosed with either marijuana or _9-THC. Unexpectedly, the blood and brain concentrations of _9-THC following marijuana exposure were considerably lower than those found following _9-THC injection when comparing an equivalent magnitude of paw tremors in both conditions. Finally, _9-THC dose-dependently alleviated SR 141716-induced paw tremors in marijuana-dependent mice, but marijuana itself failed to reverse the precipitated withdrawal effect. It is likely that marijuana exposure generated insufficient _9-THC brain levels to reverse the withdrawal signs compared with the brain levels following intravenous injection. These findings taken together indicate that mice exposed repeatedly to marijuana smoke exhibit similar precipitated withdrawal effects as _9-THC-injected mice. Wilson, D.M., Varvel, S.A., Haroe, J.P., Martin, B.R., and Lichtman, A.H. SR 141716 (Rimonabant) Precipitates Withdrawal in Marijuana-Dependent Mice, Pharmacology Biochemistry and Behavior, 85(1), pp. 105-113, 2006.

AMPA Receptor Subunits in the Shell of the Nucleus Accumbens Regulate Brain Reward

Although it is known that drugs of abuse alter the expression of AMPA-type glutamate receptor subunits (GluRs) in the nucleus accumbens (NAc), the impact of this regulation on general motivational states is unclear. Dr. Carlezon and his colleagues used herpes simplex virus vectors to examine how transient increases in the expression of GluR1 or GluR2 protein in the shell of the NAc affect the rewarding impact of electrical stimulation of the medial forebrain bundle, as reflected by intracranial self-stimulation (ICSS) thresholds in rats. After they raised the densities of GluR1 in NAc shell, they saw increased ICSS thresholds, an effect that was similar to what is seen after treatments that cause anhedonia and dysphoria (prodepressive effects) in rats and humans (e.g., drug withdrawal). In contrast, elevated GluR2 decreased the ICSS thresholds, an effect similar to that seen after rewarding treatments (e.g., drugs of abuse). To confirm that viral vector-mediated elevations of GluR1 in the NAc shell produced molecular consequences that were different from those produced by elevated GluR2, they used quantitative PCR to examine the expression of a set of drug-regulated genes 3 days after treatment. Elevated GluR1 was accompanied by sustained increases in the gene for GluR1, whereas elevated GluR2 was accompanied by decreases in prodynorphin. These data suggest that GluR1 and GluR2 in the NAc shell play opposing roles in the regulation of motivated behavior. Todtenkopf, M.S., Parsegian, A., Naydenov, A., Neve, R.L., Konradi, C., and Carlezon Jr., W.A. Brain Reward Regulated by AMPA Receptor Subunits in Nucleus Accumbens Shell. The Journal of Neuroscience, 26(45), pp. 11665-11669, 2006.

Tuned for Reconfiguration: New Forms of Intrinsic, Negative-Feedback Regulation of Activity-and Experience-Dependent Neural Plasticity Stabilize Synaptic Strengths and are Present During Neural Development

Signal transmission between neurons and within neural networks occurs at synapses where efferent signals from one presynaptic neuron are integrated and coded into firing discharges of the postsynaptic responding neurons. The inputs received by the postsynaptic neuron may change dramatically in response to specific patterns of correlated synaptic activity that occur during learning and development, and these patterns depend on the strength and flexibility of the synapses. Long-lasting changes in synaptic strength, also known as LTP and LTD, reflect a persistent reinforcement of the synaptic activities. It is one major way to store information in neural circuits, promoting learning and memory formation. However, these are very dynamic states, and even in the face of dramatic changes in activity, the neurons and circuits must maintain a degree of stability in their firing properties to prevent the synapse from firing at increasingly higher rates with every instance of excitatory input. NIDA grantee Dr. Gina Turrigiano has discovered that the neurons achieve this self-regulation by using homeostatic synaptic plasticity, which includes new forms of intrinsic negative-feedback regulation of activity- and experience-dependent neural plasticity. She found that activity of a neuron could be tuned up and down according to the strength of synaptic connections, a phenomenon called synaptic scaling, and showed that between cortical pyramidal neurons, increased activity would decrease synaptic strengths, and vice versa. Her experiments indicate that the negative feedback regulation of activity/experience-dependent neural plasticity is an event intrinsic to the postsynaptic neuron. Such 'synaptic scaling' has been observed early in development using cultured neurons, but she found that these properties were retained even after the neuron became more mature. In addition to the synaptic scaling, blockade of activity blockade with TTX induced a large increase in the frequency of mEPSCs, and this was associated with an increased density of excitatory synapses due to elevation of presynaptic vesicle trafficking and increased presynaptic vesicle release in response to electrical stimulation. These results raise the intriguing possibility that the expression mechanism of homeostatic plasticity can be tailored to the needs of the network during different stages of development or in response to different challenges to network function. Wierenga, C.J., Walsh, M.F., and Turrigiano, G.G. Temporal Regulation of the Expression Locus of Homeostatic Plasticity. Journal of Neurophysiology, 96, pp. 2127-2133, 2006. and Maffei, A., Nataraj, K., Nelson, S.B., and Turrigiano, G.G. Potentiation of Cortical Inhibition by Visual Deprivation. Nature, 443, pp. 81-84, 2006.

Endocannabinoid-Mediated Synaptic Plasticity in the CNS

Changes in synaptic efficacy are essential for neuronal development, learning, and memory formation. Dr. Pablo Castillo studies how synapses modify their efficacy as well as the functional impact of such changes in a neural network. One of the main goals of his work is to elucidate both the specific molecular events that underlie various forms of synaptic plasticity and the exact modifications in synaptic proteins that are responsible for the observed, short- and long-lasting changes in synaptic efficacy. While most of the current knowledge on long-term synaptic plasticity is derived from studies at excitatory synapses, he has recently reported that endogenous cannabinoids mediate long-term plasticity at inhibitory synapses in the hippocampus. He demonstrated a temporal and spatial relationship at hippocampal synapses between the depolarization-induced suppression of inhibition (DSI) and the long-term synaptic inhibition, which can be generated by electric brain stimulation that mimics physiologically relevant brain theta activity. In addition, the DSI facilitated the subsequent induction of long-term potentiation (LTP) at nearby excitatory inputs. This study provides functional evidence that the synaptic integration at focal points of brain circuitry between excitatory and inhibitory inputs and the endocannabinoid signaling are important modulators of activity-dependent synaptic plasticity. A single exposure to _9THC can disrupt functional plasticity mediated by endocannabinoid in the hippocampus and suggests that endogenous cannabinoid and their receptors in the hippocampus and other brain areas may be important in tuning the synaptic activity and plasticity of neurons. Chevaleyre, V., Takahashi, K.A., and Castillo, P.E. Endocannabinoid-Mediated Synaptic Plasticity in the CNS. Annual Review of Neuroscience, 29, pp. 37-76, 2006. and Castillo, P.E., and Khodakhah, K. Biochemical Confinements without Walls in Aspiny Neurons. Nature Neuroscience, 6, pp. 719-720, 2006.

Increased Susceptibility to Methamphetamine-Induced Dopamine Neurotoxicity by HIV-Tat and Tumor Necrosis Factor-alpha

Methamphetamine abuse is a major risk factor for HIV transmission due to sharing of contaminated needles and increased high risk sexual activity. Previous studies have shown synergistic increases in neurotoxicity, particularly in dopamine neurons, by the combination of HIV infection (or HIV proteins) in the brain and MA exposure. Possible mechanisms of this effect include oxidative stress, microglial and astrocyte activation, and release of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), all of which are correlated with neuropathology in the context of HIV infection. In a study published in Neurobiology of Disease, Dr. William Maragos and colleagues showed that TNF-alpha mediates the interaction between Tat and methamphetamine. In Sprague-Dawley rats, injections of Tat caused a small but significant increase in striatal TNF-alpha levels, whereas MA resulted in no change. The increase in TNF-alpha induced by Tat and methamphetamine was not significantly different from that induced by Tat alone. Temporal analysis of TNF-alpha levels revealed a 50-fold increase 4 h after Tat administration. In C57BL/6 mice, Tat and methamphetamine induced a 50% decline in striatal dopamine levels, which was significantly attenuated in mice lacking both receptors for TNF-alpha. TNF-alpha synthesis inhibitors significantly attenuated Tat and methamphetamine neurotoxicity in hippocampal neuronal culture. The results suggest that Tat-induced elevation of TNF-alpha may predispose the dopaminergic terminals to subsequent damage by methamphetamine. Theodore, S., Cass, W.A., Nath, A., Steiner, J., Young, K., and Maragos, W.F. Inhibition of Tumor Necrosis Factor-alpha Signaling Prevents Human Immunodeficiency Virus-1 Protein Tat and Methamphetamine Interaction. Neurobiology of Disease, 23(3), pp. 663-668, 2006.

Unlimited Access to Heroin Self-Administration as a Rat Model of Opiate Dependence

A major goal in addiction research is to develop an animal model that can be used to study the mechanisms of opiate dependence. Dr. George Koob and colleagues at The Scripps Research Institute addressed this issue using a paradigm of unlimited access to intravenous heroin self-administration combined with responding for food and water to characterize the transition from exposure to drug dependence. Male Wistar rats were allowed to lever press for heroin and nose-poke for food and water in consecutive, daily 23-h sessions. Daily heroin intake increased over days, reaching significance by Day 14. Drug-taking increased across the circadian cycle, reflected as increases in both the nocturnal peak and diurnal nadir of heroin intake. Changes in the circadian pattern of food intake and meal patterning preceded and paralleled the changes in heroin intake. By Day 7, the circadian amplitude of feeding was blunted. Nocturnal food intake decreased because rats consumed smaller and briefer meals. Diurnal intake increased due to increased meal frequency, whereas total daily food intake decreased. To control for time or experience in the self-administration boxes, rats with saline (no drug) tethers were tested and did not show significant changes in food intake pattern. Body weight gain slowed slightly in rats taking heroin relative to saline controls. Separate groups of rats revealed that significant physical dependence as measured by physical signs of opiate withdrawal following a naloxone injection was reached by Day 14. Significant increases in heroin intake could be produced using low doses of naloxone on days 28-31 of heroin access. After 6 weeks of heroin self-administration, rats injected with buprenorphine, an FDA approved medication for opiate addiction, showed a dose-dependent reduction in heroin intake. Changes in the pattern of drug and food intake in the present unlimited heroin access model may serve as independent motivational markers for the transition to a drug-dependent state. Chen, S.A., O'Dell, L.E., Hoefer, M.E., Greenwell, T.N., Zorrilla, E.P., and Koob, G.F. Unlimited Access to Heroin Self-Administration: Independent Motivational Markers of Opiate Dependence. Neuropsychopharmacology, 31, pp. 2692-2707, 2006.

Planaria Model Demonstrates Reduced Withdrawal Effects from Poly-Drug Exposure

Many drug abusers engage in 'polydrug' abuse, the co-incident use of more than one controlled substance. Specific combinations of substances are used, possibly because they maximize the euphoric effect. An alternative view is that the combination might result in less intense withdrawal during periods of abstinence. In a recent issue of Brain Research, Dr. Robert Raffa of Temple University measured responses to withdrawal from fixed ratio combinations of cocaine and a _-opioid agonist in Planaria, which are useful for studying drug interactions because of their permeable exteriors and their relevant neurotransmitter systems (e.g., dopaminergic, opioid, and serotonergic). In this study, the investigators used "joint-action" analysis as a mathematical method to quantify interactions between drugs. The D₅₀ (concentration producing half-maximal effect) for cocaine and U-50,488H was 10.3 and 1.02 _g, respectively. The D50 for 19:1 or 1:19 combinations did not differ significantly (p > 0.05) from expected additive values (11.6 ± 3.0 vs. 9.9 ± 1.4 and 1.1 ± 0.2 vs. 1.5 ± 0.1, respectively), but the 3:1, 1:1, and 1:3 ratios did (34.5 ± 6.9 vs. 7.7 ± 1.1; 55.1 ± 10.0 vs. 5.7 ± 0.7; and 40.8 ± 8.9 vs. 3.3 ± 0.4, respectively), indicating subadditive interaction at these ratios. The finding of "subadditivity" in this model suggests that abstinence-induced withdrawal from the combination is less intense than that predicted from the individual drug potencies. The concept that certain combinations of drugs lead to attenuated withdrawal might generalize to humans. Raffa, R.B., Stagliano, G.W., and Tallarida, R.J. Subadditive Withdrawal from Cocaine/_-opioid Agonist Combinations in Planaria. Brain Research, 1114(1), pp. 31-35, 2006.

In studies of neuroAIDS, brain levels of the chemokine CCL2 (or MCP-1) are highly elevated compared to serum levels as well as levels from brains of HIV-negative subjects. CCL2 is believed to stimulate inflammatory responses and/or recruit infected monocytes to the brain in neuroAIDS, as well as in Alzheimer's disease, Multiple Sclerosis, and possibly other neurodegenerative diseases. Illicit drug use is a common cofactor in HIV transmission, and there is evidence that opioids and stimulants exacerbate the neurological damage associated with HIV infection. Previous work by Dr. Kurt Hauser and colleagues at the University of Kentucky has demonstrated that opioids and HIV-Tat protein synergistically increase neuronal dysfunction and glial activation in a rodent model system. In a recent study published in the Journal of Neuroimmunology, Dr. Hauser's group found that CCR2, the primary receptor for CCL2, is important for the glial activation resulting from Tat and opioid exposure. The effects of systemic morphine and intrastriatal HIV-1 Tat on macrophage/microglial and astroglial activation were assessed in wild type and CCR2 null mice. Tat and/or morphine additively increased the proportion of CCL2 immunoreactive astroglia. The effects of morphine were prevented by the opioid antagonist naltrexone. Glial activation was significantly reduced in CCR2(_/_) versus wild-type mice following Tat or morphine plus Tat exposure. Thus, CCR2 contributes to local glial activation caused by Tat alone or in the presence of opiates, implicating CCR2 signaling in HIV-1 neuropathogenesis in drug abusers and non-abusers. El-Hage, N., Wu, G., Ambati, J., Bruce-Keller, A.J., Knapp, P.E., and Hauser, K.F. CCR2 Mediates Increases in Glial Activation Caused by Exposure to HIV-1 Tat and Opiates. Journal of Neuroimmunology, 178, pp. 9-16, 2006.

Combined Chronic Stress and MDMA Enhances Mesoaccumbens Dopamine

MDMA acutely releases serotonin and dopamine, and produces long-term damage to serotonin terminals in animals. It is also known that MDMA-induced dopamine release is dampened by serotonin. Since stress activates the mesolimbic dopamine pathway, Bryan Yamamoto's group hypothesized that chronic stress after exposure to neurotoxic doses of MDMA may enhance the effect of a subsequent challenge dose of MDMA on dopamine release in the nucleus accumbens shell (NAcc(sh)). The present study used in vivo microdialysis to assess changes in mesolimbic responses to MDMA in rats with prior exposure to a neurotoxic regimen of MDMA and/or chronic unpredictable stress (CUS). Rats were pretreated with a neurotoxic regimen of MDMA. Seven days later, the rats were subjected to 10 days of CUS and the dopamine release in the NAcc(sh) and serotonin in the VTA were measured after a challenge injection of MDMA. Pretreatment with MDMA or CUS alone blunted MDMA-induced serotonin release in the VTA. As predicted, pretreatment with MDMA + CUS enhanced MDMA-stimulated dopamine release in the NAcc(sh). The augmentation of MDMA-induced dopamine release in rats pretreated with MDMA + CUS was attenuated by perfusion of the 5HT1B antagonist, GR127935, into the VTA prior to the MDMA challenge injection. This is the first demonstration that CUS after previous exposure to neurotoxic doses of MDMA alters mesolimbic dopamine and serotonin release, suggesting that stress can alter the neuronal mechanisms associated with MDMA reward. Pre-exposure to MDMA and stress is not simply additive but synergizes to augment mesolimbic dopamine neurotransmission in a manner that is both quantitatively and qualitatively different from either the effects of MDMA or stress alone. Further, stress exposure after a neurotoxic regimen of MDMA did not affect the neurotoxicity of MDMA, as indicated by unaltered long-term depletion of serotonin tissue content. Thus, the effects of stress exposure after MDMA are not the result of added damage to serotonergic neurons. Finally, the serotonin receptor-1B receptor is newly implicated as a mediator of chronic stress-induced changes in the effects of MDMA. These results suggest that prior exposure to neurotoxic doses of MDMA can augment the reactivity of the dopamine-mediated reward system to chronic stress via a serotonin receptor-1B mechanism that might increase the vulnerability to drug abuse in the future. Amato, J.L., Bankson, M.G., and Yamamoto, B.K. Prior Exposure to Chronic Stress and MDMA Potentiates Mesoaccumbens Dopamine Release Mediated by the Serotonin receptor-1B. Neuropsychopharmacology, 2 August 2006 (Epub ahead of print).

MDMA Increases Cocaine Reward in Adolescent Rats and Decreases It in Adults

Adolescent rats at postnatal day (PND) 33 and adult rats at PND 60 were exposed to MDMA or saline for 7 days. Exposing adolescent rats to MDMA resulted in cocaine becoming reinforcing, but not for adolescent rats exposed to saline. In contrast, the reinforcing effects of cocaine were diminished after MDMA exposure in adult rats. These findings suggest that exposure to MDMA during adolescence may carry a greater risk for subsequent stimulant abuse. Åberg, M., Wade, D., Wall, E., and Izenwasser, S. Effect of MDMA (Ecstasy) on Activity and Cocaine Conditioned Place Preference in Adult and Adolescent Rats. Neurotoxicoly and Teratology, 14 September 2006 (Epub ahead of print).

Opiate Actions on Immunity via the Dopa System

Morphine is known to modulate immune function and interacts with neurotransmitter systems to modulate specific immune parameters. To approach an understanding of the basis of the interaction of these two systems, this study investigated whether dopaminergic projections to the nucleus accumbens are involved in morphine-induced suppression of splenic natural killer (NK) cell activity. Administration of the dopamine D-1 antagonist SCH-23390 into the nucleus accumbens shell blocked morphine's suppressive effect on NK activity in male Lewis rats. In addition, morphine's effects were also prevented by intra-accumbens microinfusions of the dopaminergic immunotoxin anti-DAT-saporin or the administration of the dopamine D-1 agonist SKF-38393. These results indicate a critical role for dopamine D-1 receptors in the modulation of NK activity and that dopaminergic inputs to the nucleus accumbens are involved in opioid-induced immunosuppression. Thus opioid-induced increases in dopamine D-1 receptor activation may have adverse consequences on immune status. Saurer, T.B., Carrigan, K.A., Ijames, S.G., and Lysle, D.T. Suppression of Natural Killer Cell Activity by Morphine Is Mediated by the Nucleus Accumbens Shell. Journal of Neuroimmunology, 173, pp. 3-11, 2006.

From the Genome to the Proteome: Uncovering Peptides in the Apis Brain

The honey bee is an excellent model system for studying biological systems including social behavior and gene environment interactions. As a model system, the honey bee is relatively inexpensive when compared to a rodent model, yet still possesses some of the complex attributes such as a social structure and the ability of individuals to learn and communicate with other members of its society. These characteristics make it particularly attractive as a model for studying neurobiology. While possessing these complex attributes, at the same time, the honey bee is a simpler biological model than a higher order animal and as such, offers an increased likelihood for identifying biological molecules or collections of molecules involved in the condition of interest. This can be extremely valuable for dissecting the underlying causes for health and disease when those molecules or collections of molecules have human orthologs. In October of this year, the first draft of the honey bee genome was published in the journal Nature. As with other species, sequencing of the genome is the first and critical step toward more advanced genetic and proteomic studies such as identifying gene networks, biomarkers, candidate genes of diseases, etc. One of the more challenging classes of molecules to identify is biologically significant peptides including neuropeptides. The challenge is a result of the way in which neuropeptides are generated by the cell. Neuropeptides are generally produced by targeted cleavage of a larger protein into smaller parts, the neuropeptides. In this Science article, researchers report development of a computer algorithm that looks for repetitive sequences in the genome. Using this algorithm in conjunction with homology searching, they are able to infer neuropeptides even in an unannotated genome. They then use mass spectrometry to analyze biological samples from the animal to see if they are able to detect the predicted peptides. Using this approach, they have inferred more than 200 peptides and confirmed the presence of 100 peptides from 20 precursor genes in the honey bee. The researchers have made the neuropeptide prediction tool freely available at http://neuroproteomics.scs.uiuc.edu/cgi-bin/neuropred.py. This tool, used in conjunction with homology searching and verification techniques like mass spectrometry should aid researchers who are looking for new neuropeptides or neuropeptides in model organisms that are not as well annotated as mouse and human for example. This should greatly facilitate research on these otherwise elusive but highly important biomolecules. Hummon, A.B., Richmond, T.A., Verleyen, P., Baggerman, G., Huybrechts, J., Ewing, M.S., Vierstraete, E., Rodriquez-Zas, S.L., Schoofs, L., Robinson, G.E., and Sweedler, J.V. From the Genome to the Proteome: Uncovering Peptides in the Apis Brain. Science, 314, pp. 647-649, 2006.

Mutation of the RhoGAP18B Gene, a Likely Regulator of Actin Cytoskeletal Dynamics, Makes Flies Resistant to the Intoxicating Effects of Ethanol, Cocaine, and Nicotine

Individuals that are resistant to the intoxicating effects of a drug have an increased risk of developing drug dependence. What are the genes that regulate intoxication, and how are these genes involved in addiction? The Drosophila model system is particularly useful for gene identification because: 1)fruit fly genetics is rapid and powerful, 2) no prior assumptions need to be made about the identities of the genes involved, and 3) genes and processes frequently are evolutionarily conserved between species. To identify genes involved in drug intoxication, Dr. Ulrike Heberlein and coworkers used a mutagen to disrupt the genes of a large number of flies. The animals were then sorted to identify the very rare individuals that were resistant to the intoxicating effects of ethanol. The gene responsible for the mutant phenotype of these rare animals was identified using a molecular genetic strategy. In this case, the mutated gene identified was completely unexpected: RhoGAP18B, a putative regulator of actin cytoskeletal dynamics. Dr. Heberlein found that RhoGAP18B encodes several mRNA varieties or isoforms (A, B, C, and D). Interestingly, the A isoform regulates the locomotor stimulating effects of ethanol, while the C isoform regulates the sedating effects of ethanol. Furthermore, mutants in RhoGAP18B were also resistant to the intoxicating effects of nicotine and cocaine, revealing a role for RhoGAP18B in regulation of the intoxicating effects of multiple drugs. Although it is unclear exactly how RhoGAP18B modulates resistance to acute drug exposure, Dr. Heberlein and co-workers hypothesize that RhoGAP18B regulates actin cytoskeletal dynamics critical for reorganization of neuronal dendrites and axons which in turn leads to altered synaptic plasticity in response to drug exposure. In a companion paper strengthening Dr. Heberlein's hypothesis, Dr. Di Fiore and colleagues found that mutation of the mouse Eps8 gene (which normally regulates neuronal actin dynamics in response to NMDA glutamate receptor activation) increased ethanol resistance as well as ethanol consumption (Cell, 127:213-226, 2006). Identification of a role for RhoGAP18B in drug resistance has several potential outcomes: 1) the human homolog of RhoGAP18B could be studied to see if it plays a role in human drug resistance or dependence, 2) the role of RhoGAP18B and neuronal actin cytoskeletal remodeling can be further investigated to see if this process is an important biological underpinning of addiction, and 3) modulation of neuronal actin cytoskeletal remodeling by novel medicinal agents might be a useful therapeutic approach to treat drug addiction. Rothenfluh, A., Threlkeld, R.J., Bainton, R.J., Tsai, L.T-Y., Lasek, A.W. and Heberlein, U. Distinct Behavioral Responses to Ethanol are Regulated by Alternate RhoGAP18B Isoforms. Cell 127, pp. 199-211, 2006.

A Worm Model for Genetic Analysis of Nicotine-Dependent Behavior: Regulation by TRPC Family Calcium Channels

Genetically tractable organisms such as the roundworm or the fruit fly can facilitate the experimental examination of addictive processes, as well as allow the identification and characterization of new genes involved in these processes. In work initiated in NIDA grantee Dr. Paul Sternberg's lab, Dr. Shawn Xu developed a worm model for nicotine-dependent behavior. Using a worm locomotion tracking system, Dr. Xu and co-workers quantified worm movement in response to nicotine exposure and found that worms, like vertebrates, show acute behavioral response to nicotine as well as nicotine tolerance, withdrawal, and sensitization. In mammals, nicotinic acetylcholine receptors (ACRs) are required for nicotine behavioral responses so Dr. Xu tested all available mutants in worm ACRs (20 out of 28 total), and found that two of them, ACR-15 and ACR-16, were required for acute behavioral response to nicotine. Dr. Xu found that the mouse 4 2 ACR can functionally replace worm ACR-16 when it is expressed in a particular subset of worm interneurons. Dr. Xu's lab then decided to try to identify new genes involved in nicotine-dependent behaviors. He found that two members of the TRPC family of calcium channel, TRP-1 and TRP-2, were required for behavioral response to acute nicotine, while mutants in several other TRP channels had normal responses to nicotine. Again, Dr. Xu found that TRP-1 and TRP-2 were required in a particular subset of interneurons within the worm, and that the human TRPC3 channel can functionally replace TRP-2 when expressed in this subset of neurons. Where are the nicotinic acetylcholine receptors functioning with the TRPC channels to regulate acute behavioral response to nicotine? To narrow down precisely where the ACR and TRP genes were required, Dr. Xu used a laser to kill pairs of interneurons, and then tested the animals for acute response to nicotine. He found that killing a single pair of interneurons (the AVA neuron pair) abolished the acute nicotine response, indicating that these two neurons are required for acute behavioral response to nicotine. How do the nicotinic acetylcholine receptors function with the TRPC calcium channels to regulate acute behavioral response to nicotine? Dr. Xu and coworkers expressed a genetically encoded calcium sensor to look at AVA interneuron function in living animals. Acute nicotine exposure caused a robust increase in calcium levels in the AVA neurons, chronic nicotine exposure reduced calcium levels in the AVA neurons, but animals that were previously sensitized to nicotine had very strong calcium responses. Animals lacking ACR-15 had little or no calcium response in the AVA neurons, as did animals lacking TRP-1 or TRP-2. These data suggest that acute behavioral response to nicotine requires specific ACRs and TRPC calcium channels, and that these proteins function together to regulate neuronal calcium levels in response to nicotine. This research is important for several reasons: 1) it shows that nicotine-dependent behavior and the molecular apparatus necessary for this response is evolutionarily conserved, 2) this model system provides a rapid and inexpensive means for functional testing of genes and gene variants implicated in nicotine responses in humans and other organisms, and 3) it provides an elegant way to experimentally characterize the molecular genetic basis of nicotine behavioral responses and, perhaps ultimately, addiction itself. Feng, Z., Li, W.,Ward, A., Piggot, B.J., Larkspur, E.R., Sternberg, P.W. and Xu, X.Z.S. A. C. elegans Model of Nicotine-dependent Behavior: Regulation by TRP-family Channels. Cell, 127, pp. 621-633, 2006.

Novel Genes Identified for Nicotine Dependence: A Hybrid Design Using a High Density Genome Wide Association Scan and a Candidate Gene Approach

Dr. Laura Bierut and Dr. Scott Saccone have completed a hybrid study to identify nicotine dependence loci through a whole genome association scan using 2.4 million single nucleotide polymorphisms (SNPs) along with a candidate gene approach targeting 3,713 SNPs in 348 genes. The study was carried out in a two-stage design. In the first stage, genotyping was completed in case (smokers) and control pools. In the second stage, SNPs were selected for individual genotyping of based on the most significant allele frequency differences between cases and controls pooled results. Individual genotypes were performed in 1050 cases (smokers) and 879 controls using 31,960 selected SNPs. For the candidate gene study, 3,713 SNPs were added to the 31,960 SNPs selected from the whole genome scan and screened together. The whole genome study nominates several novel genes, such as Neurexin 1, while also identifying a known gene, the Β3 nicotinic receptor. Overall, there were 35 SNPs that were more commonly observed in the smokers than the "non-smokers", some of which highlighted several genes not previously implicated in the development of nicotine dependence. The candidate gene study showed several cholinergic nicotinic receptor genes among the top signals, namely CHRNB3, CHRNA5, KCNJ6, and GABRA4. Overall there were 39 top SNPs, five of which were SNPs within nicotinic receptor genes. Most significant was the CHRNA5 SNP( rs16969968). Compared to having no copies, the odds ratio for having 1 copy and 2 copies of the A allele was 1.1 (95% CI 0.9-1.4) and 1.9 (95% CI 1.4-2.6), respectively. Thus, individuals with the AA genotype were nearly twice as likely to be nicotine dependent as those with 1 or no copies of this A allele. The study was performed in collaboration with Perlegen Sciences, Inc. and the results are published in back-to-back papers. Bierut, L.J., Madden, P.A.F., Breslau, N., Johnson, E.O., Hatsukami, D., Pomerleau, O.F., Swan, G.E., Rutter, J., Bertelsen, S., Fox, L., Fugman, D., Goate, A.M., Hinrichs, A.L., Konvicka, K., Martin, N.G., Montgomery, G.W., Saccone, N.L., Saccone, S.F., Wang, J.C., Chase, G.A., Rice, J.P., Ballinger, D.G. Novel Genes Identified in a High Density Genome Wide Association Study for Nicotine Dependence. Human Molecular Genetics (epub ahead of publication), 2006.