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NIDA Home > Publications > Director's Reports > September, 2007 Index    

Director's Report to the National Advisory Council on Drug Abuse - September, 2007

Research Findings - Basic Neuroscience Research

CB2 Receptor Analysis

Understanding the structure and function of the peripheral CB2 cannabinoid receptor requires that it be available in adequate and purified quantities and its various helical and loop amino acid sequences be accurately analyzable by modern techniques. Of particular importance is the precise identification of an amino acid at which a ligand may bind covalently, possibly affecting the function and conformation of the receptor. In a collaborative effort, Dr. Alexandros Makriyannis and Dr. Barry Karger have recently completed a proteomic characterization of the human CB2 receptor (hCB2R), in which refinements in expression, purification, and tryptic digestion gave rise to full "coverage" of the known sequence of this receptor, based on mass spectral analysis of its fragments. In the present example, a membrane-bound hCB2 protein was obtained by expression in Sf-21 insect cells, which showed suitable equilibrium binding of the tritiated cannabinoid agonist CP 55,940. The results described have demonstrated the production of purified human CB2 receptor, the use of complementary proteolytic digestion techniques, the separation of resulting long hydrophobic and short sequence peptides, their analysis by electro spray mass spectrometry, and confirmation of fragments with tandem MS/MS. Zvonok, N., Yaddanapudi, S., Williams, J., Dai, S., Dong, K., Retjar, T., Karger, B. L., and Makriyannis, A., Journal of Proteome Research 6(6), pp. 2068-2079, 2007.

Prenatal Cocaine Exposure and the Developing Brain

Children exposed to cocaine during gestation have a higher incidence of neurobehavioral deficits. The neurochemical bases of these deficits have not been determined but the pharmacology of cocaine and the nature of abnormalities suggest that disruptions in catecolaminergic systems may be involved. In a recent study, NIDA-supported researchers report that prenatal cocaine exposure resulted in lasting changes to the regulation and responsivity of rat locus coeruleus norepinephrine (NE) neurons. From these findings, the researchers speculate that a similar dysregulation of locus coeruleus NE neurons may occur in children exposed to cocaine during gestation, and this may explain, at least partly, the increased incidence of cognitive deficits that have been observed in these subjects. This study was conducted in pregnant rats that received intravenous injection of cocaine twice daily between gestational days 10 and 20 and the progeny was tested as juveniles. The researchers also observed that the locus coeruleus NE system was more responsive to stimuli, such as exposure to a mild stressor, in rats exposed to prenatal cocaine compared to rats exposed to saline solution. Elsworth, J.D., Morrow, B.A., Nguyen, V.T., Mitra, J., Picciotto, M.R., and Roth, R.H. Prenatal Cocaine Exposure Enhances Responsivity of Locus Coeruleus Norepinephrine Neurons: Role of Autoreceptors. Neuroscience 147, pp. 419-427, 2007.

A Functional Proteomic Strategy to Discover Inhibitors for Uncharacterized Hydrolases

Hydrolytic enzymes constitute one of the largest and most diverse protein classes in nature and play key roles in nearly all physiological and pathological processes. The mammalian serine hydrolase superfamily contains a remarkable number of uncharacterized members, with at least 40-50% of these enzymes lacking experimentally verified endogenous substrates and products. Assignment of metabolic and cellular functions to these enzymes requires the development of pharmacological tools to selectively perturb their activity. Cravatt et al. provided a functional proteomic strategy to systematically develop potent and selective inhibitors for uncharacterized serine hydrolases and its application to the brain-enriched enzyme alpha/beta-hydrolase-6. The methods described herein will facilitate the development of selective chemical probes to annotate the metabolic and (patho) physiological functions of many of the uncharacterized serine hydrolases that currently populate eukaryotic and prokaryotic proteomes. Li, W., Blankman, J.L., and Cravatt, B.F. A Functional Proteomic Strategy to Discover Inhibitors for Uncharacterized Hydrolases. July 13, 2007 [Epub ahead of print].

Glial-Cell Inhibitor, AV411, Reduces Pain and Attenuates Morphine Analgesia in a Rat Model of Neuropathic Pain

AV411 (ibudilast) is a relatively nonselective phosphodiesterase inhibitor that also suppresses glial-cell activation. Recent data strongly implicates activated glial cells in the spinal cord in the development and maintenance of neuropathic pain. NIDA grantee Dr. Linda Watkins of the University of Colorado, Boulder and colleagues recently tested the efficacy of AV411 as an analgesic in several neuropathic pain models in rats. AV411 was found to be very effective in reducing pain in these models. She also found that AV411 reduced tolerance to morphine in nerve-injured rats. Safety pharmacology, pharmacokinetic and initial mechanistic analyses indicated that that AV411 is safe and effective in diverse models of neuropathic pain, suggesting usefulness of this agent as a treatment for pain, either alone, or in combination with opioids. Ledeboer, A.M., Liu, T., Shumilla, J.A., Mahoney, J.H., Vijay, S., Gross, M.I., Vargas, J.A., Sultzbaugh, L., Claypool, M.D., Sanftner, L.M., Watkins, L.R., and Johnson, K.W. The Glial Modulatory Drug AV411 Attenuates Mechanical Allodynia in Rat Models of Neuropathic Pain. Neuron Glia Biology, (2), pp. 279-291, 2006.

Improved Procedure for the Synthesis of DAMGO

DAMGO (Tyr-D-Ala-Gly-Na-Me-Phe-Gly-ol), a selective u-opioid receptor ligand is a widely used peptide in investigations of u-receptor-mediated pharmacology. Dr. Ivy Carroll and his coworkers previously developed a solution methodology suitable for the preparation of gram amounts of highly pure DAMGO. The synthetic scheme consisted of a (3+1+1) approach in which synthetic Boc-Tyr-D-Ala-Gly-Na-Me-Phe-OH was prepared starting from Boc-Na-Me-Phe-OH in three steps to give TFA*DAMGO in 6% overall yield. They have just reported a new synthetic methodology for introduction of the Gly-ol residue. Specifically, they determined that heating the tetrapeptide, Boc-Tyr-D-Ala-Gly-Na-Me-Phe-OR, in excess ethanolamine led to amidation of the Na-methylpenylalanine methyl ester residue cleanly and quantitatively. Thus, the protected tetrapeptide, Boc-Tyr-D-Ala-Gly-Na-Me-Phe-OR was prepared in three steps (68% overall yield), following the previously described methodology. Condensation with ethanolamine quantitatively afforded the protected penta-peptide, Boc-Tyr-D-Ala-Gly-Na-Me-Phe-Gly-ol. Deprotection of the Boc group using 50% TFA in CH2Cl2 afforded DAMGO trifluoroacetate in 58% overall yield after purification by preparative high performance liquid chromatography (HPLC). The significantly higher yield resulted primarily from the improved yield obtained in the preparation of precursor tetrapeptide, Boc-Tyr-D-Ala-Gly-Na-Me-Phe-OR (68%) vs. the precursor Boc-Tyr-D-Ala-Gly-Na-Me-Phe-OR (25%) and in the introduction of the fifth Gly-ol residue (85% vs. 24%). In summary, the PI and his associates have developed a racemization-free synthetic protocol that is relatively shorter and affords the product with high overall yield. Reddy, A.P., Lewin, A. and Carroll, I.F. Improved Procedure for the Synthesis of DAMGO Synthetic Communications, 37 (14), pp. 2345-2348, 2007.

Endocannabinoid-Mediated Long-Term Depression Requires cAMP/PKA Signaling

Endocannabinoids (eCBs) have emerged as key activity-dependent signals and their neuropharmacology is of interest to NIDA. The binding of eCBs to presynaptic cannabinoid receptors (i.e., CB1) causes a reduction in neurotransmitter release and neuronal excitability that can be transient (short-term depression) or that can persist long after CB1 receptor activation (long-term synaptic depression, LTD) has occurred. The conundrum is how activation of the same neurotransmitter receptor can produce both transient and long-lasting depression. Previous studies have found that eCB-dependent short-term depression is mediated through CB1-dependent inhibition of voltage -gated calcium channels. However, the molecular events linking CB1 receptors to LTD have been unknown. This paper now reports that in the hippocampus, long-term eCB-dependent depression requires presynaptic cAMP/PKA signaling. Providing additional support for the involvement of cAMP signal transduction in eCB-dependent LTD, it was further shown that the active zone protein RIM1alpha, which is a PKA substrate protein that regulates neurotransmitter release, is a key mediator of both CB1 receptor effects on neurotransmitter release and eCB-dependent LTD. Taken together, these findings show that eCB-dependent short-term and long-term depression occur through distinct mechanisms. Whether CB1 receptor activation by eCBs results in transient or long-term depression should distinctly impact neural circuit processing and behavior. Chevaleyre, V., Heifets, B.D., Kaeser, P.S., Suedhof, T.C., Purpura, D.P., and Castillo, P.E. Endocannabinoid-mediated Long-term Plasticity Requires cAMP/PKA Signaling and RIM1alpha. Neuron, 54(5), pp. 801-812, 2007.

An Opioid Agonist That Does Not Induce Micro-Opioid Receptor-Arrestin Interactions or Receptor Internalization

G protein-coupled receptor desensitization and trafficking are important regulators of opioid receptor signaling that can dictate overall drug responsiveness Furthermore, different mu-opioid receptor (MOR) ligands can lead to varying degrees of receptor regulation, presumably because of distinct structural conformations conferred by agonist binding. For example, morphine binding produces a MOR with low affinity for beta-arrestin proteins and limited "trafficking" and receptor internalization, whereas enkephalin analogs promote robust trafficking of both beta-arrestins and the receptors. Here, Dr. Laura Bohn and her research team at the Ohio State University evaluate MOR trafficking in response to activation by a novel mu-selective agonist derived from the naturally occurring plant product, salvinorin A. It is interesting that this compound, termed "herkinorin," does not promote the recruitment of beta-arrestin-2 to the MOR and does not lead to receptor internalization. Moreover, whereas G protein-coupled receptor kinase over-expression can promote morphine-induced beta-arrestin interactions and MOR internalization, such manipulations do not promote herkinorin-induced trafficking. Studies in mice have shown that beta-arrestin-2 plays an important role in the development of morphine-induced tolerance, constipation, and respiratory depression. Therefore, drugs that can activate the receptor without recruiting the arrestins may be a promising step in the development of opiate analgesics that distinguish between agonist activity and receptor regulation and may ultimately lead to therapeutics designed to provide pain relief without the adverse side effects normally associated with the opiate narcotics. Groer, C.E., Tidgewell, K., Moyer, R.A., Harding, W.W., Rothman, R.B., Prisinzano, T.E., and Bohn, L.M. An Opioid Agonist that Does Not Induce Micro-opioid Receptor-arrestin Interactions or Receptor internalization. Mol. Pharmacol, Feb 71(2), pp. 549-557, 2007. Epub 2006 Nov 7.

Role of Akt-GSK-3_ Signaling and Synaptic Strength in PCP-Induced Neurodegeneration

N-Methyl-D-aspartate (NMDA) receptor antagonists such as phencyclidine (PCP) and ketamine are abused drugs with powerful effects on behavior. They can replicate positive and negative symptoms of schizophrenia in humans and induce related effects in rodents. In addition, PCP treatment of developing rats induces apoptotic neurodegeneration. Later in life, without further exposure to PCP, these rats exhibit behavioral deficits that mimic some symptoms of schizophrenia. The mechanism of PCP-induced neural degeneration is unknown. Dr. Lei and colleagues investigated the role of the Akt-glycogen synthase kinase 3_ (GSK-3_) pathway in PCP-induced neuronal apoptosis in both neuronal culture and postnatal day 7 rats. Akt-GSK-3_ signaling is thought to be impaired in schizophrenia. Further, the Akt1 gene is thought to be a potential susceptibility gene for schizophrenia, and antipsychotic medications have been reported to enhance Akt-GSK-3_ signaling. Akt is a serine/threonine protein kinase involved in diverse cellular processes. GSK-3_ is one of the downstream substrates for Akt. Normally, Akt decreases GSK-3_ activity by enhancing GSK-3_ phosphorylation, promoting cell survival. PCP administration in vivo and in vitro reduced the phosphorylation of AktSer427 and GSK-3_Ser9, decreasing Akt activity and increasing GSK-3_ activity. Altered Akt-GSK-3_ signaling paralleled the temporal profile of caspase-3 activation by PCP, which has a key role in the final stages of the apoptotic cascade. Reducing GSK-3_ activity by application of selective inhibitors, or depleting GSK-3_ by siRNA, attenuated caspase-3 activity and blocked PCP-induced neurotoxicity. Moreover, increasing synaptic strength by either activation of L-type calcium channels or potentiation of synaptic NMDA receptors completely blocked PCP-induced cell death by increasing Akt phosphorylation. Overall, these data suggest that PCP-induced hypo-function of synaptic NMDA receptors impairs the Akt-GSK-3_ cascade, which is necessary for neuronal survival during development, and that interference with this cascade by PCP or natural factors may contribute to neural pathologies, perhaps including schizophrenia. Lei, G., Xia, Y., and Johnson, K.M. The Role of Akt-GSK-3_ Signaling and Synaptic Strength in Phencyclidine-Induced Neurodegeneration. Neuropsychopharmacology, 2007. [Epub ahead of print]

A Serotonin-Depleting Regimen of MDMA Prevents Mating-Induced Conditioned Place Preference in Rats

Although MDMA ("ecstasy") appears to increase sexual desire and satisfaction shortly after its consumption, human self-reports suggest that repeated use of MDMA results in long-lasting loss of sex interest or pleasure. In the present study, a regimen of MDMA that produced long-lasting depletion of brain serotonin in sexually naive male rats prevented the formation of a mating-induced conditioned place preference (CPP) two weeks following drug treatment. This regimen of MDMA produced no change in parameters of sexual performance 12-40 days following MDMA administration. The failure of MDMA-treated rats to form a CPP response to sex may be due to MDMA-induced long-lasting impairment in brain circuits mediating sexual reward. Straiko, M.M.W., Gudelsky, G.A., and Coolen, L.M. Treatment with a Serotonin-Depleting Regimen of MDMA Prevents Conditioned Place Preference to Sex in Male Rats. Behavioral Neuroscience, 121, pp. 586-593, 2007.

Over-expression of Serotonin-6 Receptors in the Nucleus Accumbens Blocks the Rewarding But Not Psychomotor Activating Properties of Cocaine

Repeated exposure to cocaine produces enduring forms of drug experience-dependent behavioral plasticity, including conditioned place preference (CPP) and psychomotor sensitization, a progressive and persistent increase in cocaine's locomotor activating effects. Although serotonin-6 receptors (5-HT6Rs) are abundantly expressed in the brain regions thought to underlie these phenomena, such as the nucleus accumbens (NAc) shell, little is known about the role of 5-HT6Rs in the rewarding and psychomotor activating effects of cocaine. Viral-mediated gene transfer was used to selectively increase 5-HT6R expression in the NAc shell of rats. Increased expression of 5-HT6Rs in the NAc shell blocked CPP to cocaine but had no effect on either the acute locomotor response to cocaine or on the development of cocaine-induced locomotor sensitization. Further, antagonism of 5-HT6Rs (by systemic administration of Ro4368554) facilitated acquisition of CPP to cocaine but had no effect on cocaine-induced stereotypy. These results demonstrate that 5-HT6Rs in the NAc shell can selectively modulate drug reward, possibly through facilitation of reward learning. Ferguson, S.M., Mitchell, E.S., and Neumaier, J.F. Increased Expression of 5-HT6 Receptors in the Nucleus Accumbens Blocks the Rewarding But Not Psychomotor Activating Properties of Cocaine. Biological Psychiatry, 2007. [Epub ahead of print].

IRS2-Akt Pathway in Midbrain Dopamine Neurons Regulates Behavioral and Cellular Responses to Opiates

A study from Dr. Nestler's lab reports that tolerance to morphine reward in rats is due to down-regulation of IRS2-Akt signaling in the ventral tegmental area (VTA), the cell body region of the mesolimbic dopamine reward system. Chronic morphine administration (via subcutaneous pellet) is known to decrease the size of dopamine neurons in the VTA, a key reward region in the brain, yet the molecular basis and functional consequences of this effect were unclear. In this study, the investigators used viral-mediated gene transfer in rat to show that chronic morphine-induced down-regulation of the insulin receptor substrate 2 (IRS2)-thymoma viral proto-oncogene (Akt) signaling pathway in the VTA mediated the decrease in dopamine cell size seen after morphine exposure and that this down-regulation diminished morphine reward, as measured by reduced conditioned place preference. The reduction in size of VTA dopamine neurons persisted for up to 2 weeks after morphine withdrawal, which parallels the tolerance to morphine's rewarding effects caused by previous chronic morphine exposure. These findings directly implicate the IRS2-Akt signaling pathway as a critical regulator of dopamine cell morphology and opiate reward, and may be part of the molecular basis for the observation that addicts report that opiate drugs lose their rewarding effects over time. Russo, S.J., Bolanos, C.A., Theobald, D.E., DeCarolis, N.A., Renthal, W., Kumar, A., Winstanley, C.A., Renthal, N.E., Wiley, M.D., Self, D.W., Russell, D.S., Neve, R.L., Eisch, A.J., and Nestler, E.J. IRS2-Akt Pathway in Midbrain Dopamine Neurons Regulates Behavioral and Cellular Responses to Opiates. Nat Neurosci, 10(1), pp. 93-99. 2007.

Opioid Receptor Expression and Glial Precursor Cell Susceptibility to Toxic Effects of Morphine and HIV-Tat

Previous work by Dr. Hauser and colleagues showed combined effects of morphine and HIV proteins on astrocyte function. To determine whether opiates and HIV-1 proteins are intrinsically toxic to glial precursors, mouse neural stem cells were isolated and a subset of glial-restricted precursors (GRPs) was tested for opioid receptor expression and effects of morphine and HIV-1 Tat exposure. These investigators found that exposure to either morphine or HIV-1 Tat protein alone significantly increased GRP death in vitro. GRP death was preceded by increases in caspase-3 enzyme activity, and cytotoxicity coincided with the onset of MOR and KOR expression and progressive glial differentiation in vitro. Opioid receptor expression by GRPs was dynamic and highly coordinated with glial maturation, and their findings suggested that opioid receptors are necessary, but not sufficient, in defining critical periods of vulnerability of GRPs and their progeny to opiates. If similar patterns of susceptibility occur in vivo, then the implication for these findings would be that the production and maintenance of glial oligodendroglial populations are preferentially vulnerable to chronic opiate exposure and HIV-1 infection. Buch, S.K., Khurdayan, V.K., Lutz, S.E., Knapp, P.E., El-Hage, N., and Hauser, K.F. Glial-Restricted Precursors: Patterns of Expression of Opioid Receptors and Relationship to Human Immunodeficiency Virus-1 Tat and Morphine Susceptibility In Vitro. Neuroscience, 146, pp. 1546-1554, 2007.

Dynamic BDNF Activity in Nucleus Accumbens with Cocaine use Increases Self-Administration and Relapse

Brain-derived neurotrophic factor (BDNF) is important in regulating synaptic plasticity in the brain areas that process reward information. A new study reports that BDNF in the nucleus accumbens, a brain area critical for the rewarding effects of cocaine, promotes persistent cocaine-seeking behaviors and heightens relapse vulnerability. Dr. David Self and his colleagues tested whether BDNF is necessary and sufficient to mediate cocaine reward. Self and colleagues report that cocaine self-administration produced an increase in the levels of BDNF, whereas natural rewards, such as food, did not. Self and colleagues reported also that injections of BDNF directly into the nucleus accumbens increased cocaine self-administration in rats while an antibody to BDNF decreased cocaine self-administration. Furthermore, rats receiving repeated injections of BDNF worked harder for cocaine than rats working for cocaine without BDNF injections. Thus, BDNF appears to enhance the motivation to cocaine and possibly promote the transition to the addicted state. Self and colleagues report also that cocaine self-administration in response to stress or to cocaine associated cues was enhanced by prior treatment with BDNF, while infusion of the BDNF antibody blocked enhanced responding. Finally, using a genetically engineered mouse Self and his colleagues report that turning off the BDNF gene in the nucleus accumbens decreased BDNF by 40% and decreased the rewarding properties of cocaine. These results suggest that BDNF synthesized in the nucleus accumbens in response to cocaine mediates increases in self-administration and reinstatement of drug seeking behavior. Thus, BDNF may play an important role in the development of addiction. Graham, D.L., Edwards, S., Bachtell, R.K., DiLeone, R.J., and Rios, M. Dynamic BDNF Activity in Nucleus Accumbens with Cocaine Use Increases Self-administration and Relapse. Nature Neuroscience, 10, pp. 1029 - 1037, 2007.

Loss of MicroRNAs in Differentiated Neurons Leads to Neurodegeneration and Death

Our traditional concept of gene regulation has been that a gene made up of DNA is transcribed into mRNA, encoding instructions for making a protein, and sent out into the cytoplasm where the mRNA is then translated into a protein. Recent work now shows that in addition to coding mRNAs our genomes encode thousands of RNAs that do not code for proteins and the functions of some of these non-coding RNAs are just beginning to be elucidated. Do some of these molecules have special functions in the nervous system? There is evidence for non-coding RNA function in neural development, but little work has been done to investigate the function of non-coding RNAs in adult neurons. Dr. Anne Schaefer and her coworkers in the Greengard laboratory have identified a possible role for some of these non-coding RNAs in neurodegenerative disorders. These researchers eliminated a subset of non-coding RNAs, the ~22 base pair microRNAs, from Purkinje neurons. This was done using a genetic trick, in which Dr. Schaefer and colleagues specifically eliminated the Dicer protein (usually required for proper processing of microRNA precursor molecules into their mature functional forms) from the Purkinje neurons only after these neurons had reached adulthood. This manipulation leads to the reduction and eventual loss of many microRNAs in the Purkinje neurons over time. The normal function of the Purkinje neurons is to control motor output from the cerebellum, and impaired Purkinje neuron function is associated with locomotor function problems such as tremors and ataxia. The researchers found that Purkinje neurons lacking Dicer appeared normal, suggesting that the neurons had developed properly. However over time, these neurons exhibited reduced expression of many microRNA subtypes, altered dendritic spine morphology, neurodegeneration, and eventual cell death. Interestingly the mouse pups that lacked Dicer in the Purkinje cells initially had normal behavior, however these animals began to exhibit both tremor and ataxia, with the ataxia becoming more pronounced over time. This work suggests that microRNAs are required for the survival of Purkinje neurons, and possibly other neuronal subtypes, and that elimination of at least some as yet unidentified microRNAs from these neurons leads to neurodegeneration and eventually death. It is therefore possible that therapeutic agents that modulate microRNAs and their associated molecular pathways may be potentially useful in treating neurodegenerative disorders such as Alzheimer's or Parkinson's disease. The role of microRNAs and other non-coding RNAs in addictive processes is not known, but likely to be a fruitful area for future scientific investigation. Schaefer, A., O'Carroll, D., Tan, C.L., Hillman, D., Sugimori, M., Llinas, R., and Greengard, P. Cerebellar Neurodegeneration in the Absence of MicroRNAs. J. Exp. Med. 204, pp.1553-1558, 2007.

ERK May Play Roles in Addiction through mTOR Pathway Stimulation

Extracellularly regulated kinases (ERK) play important roles in drug addiction. For example, evidence from rodent studies suggest that ERK has roles in cocaine psychomotor sensitization, cocaine reward, consolidation and reconsolidation of memories for cocaine cues, and time-dependent increases in cocaine seeking after withdrawal. However the cellular mechanisms in drug-induced adaptations are not known. A group of NIDA funded researchers at Mount Sinai School of Medicine, New York recently reported that a link of ERK to mammalian target of rapamycin (mTOR) modulated protein translation in dendritic arbor in hippocampus neurons is found. Dendritic protein translations in neurons forming the CA3-CA1 synapses in the hippocampus are required for memory formation. Such protein translations are initiated when these synapses are stimulated, resulting in long term potentiation (LTP) of the stimulated synapses. In the present study, the team observed that ERK regulates mTOR through the stimulation of PI3K/Akt signaling pathway. First, ERK activity is required for the high frequency stimulation induced phosphorylation of p70S6K, an mTOR effector. Second, they discovered that ERK activates the PI3K-mTOR pathway by directly phosphorylating RSK and Akt, thereby activating PI3K upstream and at the same time suppressing the inhibitors of PI3K-mTOR downstream. Since ERK has been identified to mediate drug addiction and drug seeking behavior, elucidating these signaling events is important for the understanding and treatment of drug abuse and addiction. Tsokas, P., Ma, T., Iyengar, R., Landau, E.M., Blitzer, R.D. Mitogen-activated Protein Kinase Upregulates the Dendritic Translation Machinery in Long-term Potentiation by Controlling the Mammalian Target of Rapamycin Pathway. Journal of Neuroscience, 27, pp. 5885-5894, 2007.

Cyclin-Dependent Kinase 5 Governs Learning and Synaptic Plasticity Via Control of NMDA Receptor Degradation

Since drug addiction seems to involve learning and memory processes, understanding the mechanisms of learning and memory is relevant to understanding drug addiction. Current evidence suggests that learning and the storage of memories occur by strengthening the connections between neurons. The mechanisms by which synapses, the connections between neurons are strengthened, is not entirely understood. A model for memory storage in the hippocampus is long-term potentiation (LTP) in which a brief repetitive electrical stimulation of a neuronal pathway subsequently enhances the strength of the synaptic connection between the pre-synaptic and post-synaptic neuron. In this study, Paul Greengard and colleagues report that conditional knockout of cyclin-dependent kinase 5, Cdk5, in the adult mouse brain improved performance in spatial learning tasks and enhanced hippocampal long-term potentiation and NMDA receptor (NMDAR)-mediated excitatory postsynaptic currents. Enhanced synaptic plasticity in Cdk5 knockout mice was attributed to reduced NMDAR subunit 2B, NR2B, degradation rather than increased expression. The reduced degradation caused elevations in total, surface and synaptic NR2B subunit levels and current through NR2B-containing NMDARs. The next question is how is Cdk5 mediating degradation of NR2B? In this study, Cdk5 and its cofactor p35, were extracted from cells as a complex with NR2B and calpain, a calcium-dependent protease, using co-immunoprecipitation. These data suggest that Cdk5 facilitates the degradation of NR2B by directly interacting with both it and its protease, calpain. The data also imply that Cdk5 is a critical mediator of calpain degradation of NR2B. These findings reveal a previously unknown mechanism by which Cdk5 facilitates calpain-mediated proteolysis of NR2B and may control synaptic plasticity and ultimately learning. Hawasli, A.H., Benavides, D.R., Nguyen, C., Kansy, J.W., Hayashi, K., Chambon, P., Greengard, P., Powell, C.M., Cooper,. D.C, and Bibb, J.A. Cyclin-dependent Kinase 5 Governs Learning and Synaptic Plasticity Via Control of NMDAR Degradation. Nat Neurosci. 10(7), pp. 880-886, 2007. Epub 2007 May 27.

Chromosomal Loci That Influence Oral Nicotine Consumption in C57BL/6J _ C3H/HeJ F2 Intercross Mice

Previous work has suggested that oral consumption of nicotine in mice is under genetic control. Stitzel and his colleagues have begun to map the regions of the mouse genome associated with the oral consumption of nicotine. These regions associated with a quantitative trait such as the amount of nicotine are defined as quantitative trait loci (QTL). To map the QTLs for oral nicotine administration C57BL/6 mice consuming large amounts of nicotine were crossed with C3H mice that consume very little nicotine. The mice were bred for two generations. In the first generation, F1, the offspring has two copies of each gene - one from the C57BL/6 parent and one from the C3H parent, located on two homologous chromosomes. Variants in some of these genes in the two different strains are associated with difference in the amount of oral nicotine consumed. In the course of making gametes, the homologous chromosomes in the F1 offspring pair and recombine resulting in shuffling of the chomosomes. Each offspring of the F1, the second generation (F2), are then born with different amounts of shuffling that has taken place. Because there are genetic markers in the DNA associated with the trait being mapped, it is possible to locate a region of the chromosome for a trait such as nicotine consumption. Stitzel and his colleagues report the identification of 4 major QTLs accounting for 62% of the variance in the amount of nicotine consumed. These chromosomal loci that influence the amount of nicotine consumed per day are on mouse Chromosome 1, chromosome 4, chromosome 7, and chromosome 15. Loci on chromosomes 1, 4, and 15 were associated with increased consumption while loci on chromosome 7 appeared to be protective. Chromosome 1, which accounts for most of the variance for amount of nicotine consumed, maps to its synthetic regions that have previously been reported to be associated with nicotine consumption in humans Wang et al., 2005; Bergen and Caporaso, 1999; Goode et al., 2003; XC Li, Karadsheh, M.S., Jenkins, P.N., Brooks, J.C., Drapeau, J.A., Shah, M.S., Lautner, M.A., and Stitzel, J.A. Chromosomal Loci that Influence Oral Nicotine Consumption in C57BL/6J _ C3H/HeJ F2 Intercross Mice. Genes, Brain and Behavior 6(5), pp. 401-410, 2007.

Elevated Vulnerability of Male Adult to Drug Abuse after Prenatal Exposure to Cocaine Seen in Animal Model

It is estimated that for children born in the US 20% have been exposed to drugs of abuse in utero. Young adults who had in-utero drug exposure are more likely to abuse substances and show persistent affective and behavioral deficits; however, the biological bases of such deficits are not well understood. A team of NIDA supported researchers at Harvard University and Cornell University show that in animal models cocaine exposure during brain development changed dopaminergic neuron responses to cocaine in mice and increased their drug seeking behavior. Using brain stimulation-reward (BSR) method, Barry Kosofsky and colleagues report that the reinforcing effect of cocaine is greatly increased in adult male mice who were exposed to cocaine in utero. In pharmacological studies, these researchers demonstrate that a dopamine D1-receptor agonist has a similar effect as cocaine on these mice, indicating the involvement of limbic forebrain dopaminergic neural circuit in the BSR shift. Furthermore, they observed that a dopamine D2-receptor agonist had a biphasic effect on the threshold of BSR. Thus, the low doses increased the BSR threshold, but higher doses decrease the threshold. Similarly, they observed enhanced potency of the D2 agonist in the male adult mice that showed enhanced cocaine potency, which suggests adaptations in dopamine receptors may, in part, account for the changes in the rewarding potency of cocaine in adulthood after prenatal cocaine exposure. These results also provide biological evidence of developmental changes in the brain as consequences of early exposure to drugs of abuse. Malanga, C.J., Riday, T.T., Carlezon, W.A., and Kosofsky, B.E. Prenatal Exposure to Cocaine Increases the Rewarding Potency of Cocaine and Selective Dopaminergic Agonists in Adult Mice. Biological Psychiatry, June 21, 2007. Epub (ahead of print).

Opiate Actions on Neural Cell Movement and Functions

Most chemokines are produced and primarily used by glia for movement of these cells in inflammatory reactions to compensate for insults from environmental changes, including invasion by foreign substances. More recently these peptides have been observed in neurons and are utilized by neural cells for different activities or as a cooperative function with glia in compensating for these insults to the nervous system. Two recent articles by different laboratories correlate these systems in neurons with opioid actions. The first study focuses on the chemokine, CXCL12. Its receptor, CXCR4, and morphine's receptor (MOR) are coexpressed in numerous neurons. In this study, the administration of a MOR ligand, DAMGO, was observed to negate the neuroprotective effects of the chemokine and alter neuron survival.The second study focuses on the chemokine CCL2, which plays a pivotal role in the recruitment of inflammatory cells in the nervous system and is one of only a few chemokines produced by neurons. Opiates, have a variety of immuno-modulatory properties that may influence both neuroinflammatory and neurodegenerative disease processes. The effects of opiates on this chemokine in neurons was studied and compared those in glia. Morphine upregulated CCL2 mRNA and protein in neuronal cultures in a concentration- and time-dependent fashion, but had no effect on CCL2 production in astrocyte or microglial cell cultures. The stimulatory effect of morphine was abrogated by an opiate antagonist indicating a morphine-mediated mechanism. The authors conclude that morphine stimulates CCL2 production in neurons via a morphine-related mechanism. This finding provides another mechanism whereby opiates would affect neuroinflammatory responses. Morphine stimulates CCL2 production by human neurons. Rock, RB., Hu, S.X., Sheng, W.S., and Peterson, P.K. Opiate Actions on Neural Cell Movement and Functions. Neuroinflammation 3, p. 32, 2006.


Research Findings

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