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National Institute on Drug Abuse

Director's Report to the National Advisory Council on Drug Abuse

May, 2000

Research Findings

Basic Research

Repeated Exposure to Psychostimulants and Opiates Produces Morphological Changes in Dendrites of Nucleus Accumbens and Prefrontal Cortex

Changes in behavior and brain function that outlast the pharmacological actions of drugs are hallmarks of repeated drug use. Most studies of long-lasting drug effects have focused on changes in the biochemical or neurophysiological properties of neurons. Dr. Terry Robinson and Dr. Bryan Kolb have been investigating morphological changes in dendrites as another marker of long-lasting alterations of synaptic function in response to drug treatment. Repeated injections in rats of either amphetamine or cocaine produced an increased number of dendritic branches and a higher density of dendritic spines on medium spiny neurons in the shell of the nucleus accumbens and on apical dendrites of layer V pyramidal cells in the prefrontal cortex, measured 25 days after drug exposure. Both of these neuron types are targets of dopaminergic input from the VTA. Interestingly, a similar study with repeated doses of morphine showed changes in dendritic morphology in the opposite direction: the complexity of dendritic branching and the number of dendritic spines was reduced. These morphological changes are likely to alter the patterns of synaptic connectivity in NAc and prefrontal cortex and may account for some of the persistent neurobehavioral consequences of repeated exposure to psychostimulant and opioid drugs. The opposite results from the two classes of drugs may underlie some of the differences in their long-term consequences, such as differential effects on cognitive processes mediated by the prefrontal cortex. Robinson, T.E. and Kolb, B. Alterations in the Morphology of Dendrites and Dendritic Spines in the Nucleus Accumbens and Prefrontal Cortex Following Repeated Treatment with Amphetamine or Cocaine. European Journal of Neuroscience, 11, pp. 1598-604, 1999; Robinson, T.E. and Kolb, B. Morphine Alters the Structure of Neurons in the Nucleus Accumbens and Neocortex of Rats. Synapse, 33, pp. 160-162, 1999.

Cellular Machinery for the Endocytosis of the AMPA Receptor Plays an Important Role in Synaptic Plasticity

Glutamate is the major excitatory neurotransmitter in the central nervous system. One important class of glutamate receptors is the ionotropic receptors or AMPA receptors. This class of receptors has been shown to be important in synaptic plasticity and has been shown to be regulated by chronic treatment with either cocaine or morphine. Potential mechanisms by which the responsiveness to glutamate can be altered at a synapse is by modifying the number of receptors, the sensitivity of each receptor, or the distribution of the receptors. The number of receptors at a synapse can be changed by increasing synthesis and the rate of insertion of the receptor, or decreasing the rate in which the receptors are removed from the membrane and degraded. The removal of the AMPA receptor from the membrane takes place via endocytosis. Another mechanism by which sensitivity can be changed is by altering the distribution of receptors so that receptor concentration can be more or less concentrated at a synapse. Work by NIDA grantees, Drs. Robert Malenka at Stanford University, Roger Nicoll, and Mark Von Zastrow at UCSF, have shown that the removal of the AMPA receptor from the postsynaptic membrane of a neuron by endocytosis is induced by glutamate attaching or binding to the AMPA receptor. They demonstrate that this form of endocytosis is mediated by a dynamin-dependent process. In a subsequent paper, they show that post synaptic blockade of endocytosis enhances the amplitudes of the AMPA receptor response and blocks long term depression, a special form of synaptic plasticity. This result shows that the composition of the post synaptic membrane like the presynaptic membrane is dynamically regulated by endocytosis and that the cellular machinery for the endocytosis of the AMPA receptor plays an important role in synaptic plasticity. Luscher, C., Xia, H., Beattie, E.C., Carroll, R.C., von Zastrow, M., Malenka, R.C., and Nicoll, R.A. Role of AMPA Receptor Cycling in Synaptic Transmission and Plasticity. Neuron, 24(3), pp. 649-58, 1999. Carroll, R.C., Beattie, E.C., Xia, H., Luscher, C., Altschuler, Y., Nicoll, R.A., Malenka, R.C., and von Zastrow, M. Dynamin-dependent Endocytosis of Ionotropic Glutamate Receptors. Proc. Natl. Acad. Sci. U.S.A., 96(24), pp. 14112-14117, 1999.

Cannabinoid/Dopamine Agonist Combinations

A recent study by Drs. Bertha Madras and Allyn Howlett has indicated that intramuscular injection of a synthetic THC derivative, levonantradol, at a non-sedating dose of 0.03mg/kg in monkeys, followed by a course of successive injections of increasing concentrations of the D2 dopamine agonist pergolide, produced a significant decrease in locomotor activity and an increase in eyelid closure (sedation), rather than the expected promotion of hyperactivity. The same trend was found to a lesser extent for the selective D2 agonist quinelorane, but not for the D1 agonist SKF81297. The choice of the agents used was based on data showing that levonantradol by itself can produce sedation and reduced activity in monkeys, pergolide is of interest in potentially treating cocaine addiction, and quinelorane is an agent for use in improving activity in animal models of Parkinson's disease. If these results are found to extend further to other cannabinoids and THC itself interacting with dopamine agonists, they may have implications in the management of neuropsychiatric diseases and in the co-administration of cocaine or amphetamines, together with marijuana. J. Pharmacology and Experimental Therapeutics, 292, pp. 952-959, 2000.

Regulation of Immune Responses by the Cannabinoid Receptor

The regulation of immune responses by a cytokine (IL2) has been modulated through the administration of cannabinoids. This was shown to occur through the regulation of transcription processes in the same immune-type cell. A strong interaction between the cannabinoid and cytokine systems was displayed and it was also revealed that the immune system can use the cannabinoid system to regulate host susceptibility. NIDA-supported investigators previously reported that immunosuppressive cannabinoids inhibited interleukin (IL)-2 steady-state mRNA expression and secretion by phorbol-12-myristate-13-acetate plus ionomycin-activated mouse splenocytes and EL4 murine T-cells. In this study these researchers show that inhibition of IL-2 production by cannabinol, a modest central nervous system-active cannabinoid, is mediated through the inhibition of IL-2 gene transcription. Moreover, electrophoretic mobility shift assays demonstrated that cannabinol markedly inhibited the DNA binding activity of nuclear factor of activated T-cells (NF-AT) and activator protein-1 (AP-1) in a time- and concentration-dependent manner in activated EL4 cells. The inhibitory effects produced by cannabinol on AP-1 DNA binding were quite transient, showing partial recovery by 240 min after cell activation and no effect on the activity of a reporter gene under the control of AP-1. Conversely, cannabinol-mediated inhibition of NF-AT was robust and sustained as demonstrated by an NF-AT-regulated reporter gene. Collectively, these results suggest that decreased IL-2 production by cannabinol in EL4 cells is due to the inhibition of transcriptional activation of the IL-2 gene and is mediated, at least in part, through a transient inhibition of AP-1 and a sustained inhibition of NF-AT. Yea, S.S., Yang, K.H., and Kaminski, N.E. Role of Nuclear Factor of Activated T-Cells and activator Protein-1 in the Inhibition of Interleukin-2 Gene Transcription by Cannabinol in EL4 T-cells. J. Pharm. Exper. Ther., 292, pp. 597-605, 2000.

Neuroimmune Regulation by Cannabinoids

Two different cannabinoid receptors have been cloned, CB1 and CB2. CB1 is more common to the central nervous system and CB2 to the peripheral systems such as lymphocytes. The cannabinoid modulation of glia, the macrophages of the brain, was studied by Puffenbarger, Boothe, and Cabral. Little is known about cannabinoid's action on glial cell function. Using several cannabinoid agonists and antagonists, they found that neither CB1 nor CB2 receptors were involved in glial cell regulation. The usual cannabinoid antagonists were not functioning as antagonists. These results suggest the possibility of the involvement of other systems and/or the functioning of unique heretofore unrecognized properties of one of the two known cannabinoid receptors in these cells. In this study the effect of cannabinoids on the induction of cytokine mRNA by rat microglial cells was examined. Exposure of neonatal rat cortical microglial cells to the exogenous cannabinoid delta(9)-tetrahydrocannabinol (THC) resulted in reduced amounts of lipopolysaccharide (LPS)-induced mRNAs for IL-1-alpha, IL-1 beta, IL-6, and TNF-alpha. Of these cytokine mRNAs, the response of that for IL-6 was exquisitely sensitive to THC. Similarly, exposure of microglial cells to the putative endogenous cannabinoid anandamide before LPS treatment resulted in a decrease in cytokine mRNA levels, but not to the same extent as that caused by THC; however, when methanandamide, the nonhydrolyzable analog of anandamide was tested, its ability to inhibit cytokine mRNA expression was comparable to that of THC. Exposure of microglial cells to either of the paired enantiomers CP55,940 or CP56,667 resulted in similar inhibition of LPS-induced cytokine mRNA expression. A comparable inhibitory outcome was obtained when the paired enantiomers levonantradol and dextronantradol were employed. Neither the CB1-selective antagonist SR141716A nor the CB2-selective antagonist SR144528 was able to reverse the inhibition of cytokine mRNA expression by levonantradol. The CB2 antagonist, however, when administered alone, augmented the production of cytokine mRNAs. Collectively, these studies demonstrate that cannabinoids can modulate levels of cytokine mRNA in rat microglial cells; however, the inhibition of cytokine mRNA expression is apparently not mediated through either of the CB1 or CB2 cannabinoid receptors. Puffenbarger, R.A., Boothe, A.C., and Cabral, G.A. Cannabinoids Inhibit LPS-Inducible Cytokine mRNA Expression in Rat Microglial Cells GLIA, 29, pp. 58-65, 2000.

Opioids and Cell Division in Embryonic Brain

Recently Dr. Kurt Hauser and his collaborators designed experiments to assess whether opioids can modulate cell proliferation in the embryonic brain through the endogenous opioid systems of the embryonic and maternal organisms. Their findings demonstrated that the endogenous embryonic and maternal opioid systems are involved in the regulation of cell division in the ventricular zone of the embryonic cortex. Their data also showed that small numbers of neural precursors in the neocortical germinal zone possessed mu, delta and kappa opioid receptor immunoreactivity in embryonic day 16 of the mouse. Furthermore, an acute exposure of mouse embryos to opioid agonist or antagonists modified labeled thymidine incorporation in the cells. In their view, these findings support the notion that endogenous opioids participate in prenatal cortical development. Reznikoz, K., Hauser, K. F., et al. Opioids Modulate Cell Division in the Germinal Zone of the Late Embryonic Neocortex. European J. Neuroscience, 11, pp. 2711-2719, 1999.

Dynorphin A and Neurodegeneration

Dynorphin A is an endogenous opioid peptide that preferentially activates kappa opioid receptors and is antinociceptive at physiological concentrations. However, growing evidence suggests that exposure to high concentrations of dynorphin can induce hyperalgesia and allodynia and may contribute to neural damage. A recent study undertaken by Dr. Kurt Hauser and his team to assess the role of dynorphin in neurodegeneration showed that dynorphin A could have paradoxical effects on neuronal viability through both opioid and non-opioid (glutamatergic) receptor mediated actions. These studies were carried out using isolated populations of neurons enriched in both kappa-opioid and NMDA receptors from embryonic mouse spinal cord. The researchers observed that at micromolar concentrations, dynorphin A elevated intracellular calcium levels and caused a significant loss of neurons. These excitotoxic effects were completely blocked by the NMDA antagonist MK-801, suggesting that dynorphin A was acting through NMDA receptors. In contrast, opioid antagonists exacerbated the toxic effects of dynorphin. These findings led the investigators to suggest that toxic effects of dynorphin are mediated by glutamatergic receptors and override the potential beneficial actions of opioid receptors. Hauser, K.F., Foldes, J.F., and Turbek, C.S. Dynorphin D (1-13) Neurotoxicity in Vitro: Opioid and Non-Opioid Mechanisms in Mouse Spinal Cord Neurons. Experimental Neurology, 106, pp. 361-375, 1999.

POMC and Leptin Regulation

The mechanisms by which leptin influences energy homeostasis are not yet understood. Several observations indicate that proopiomelanocortin (POMC) is involved in the regulation of food intake and may be a mediator of leptin action. A recent study undertaken by Dr. Wardlaw and her team to assess this interaction in obese leptin receptor-deficient rats showed significant decreases in POMC gene expression and peptide levels in the medial basal hypothalamus (MBH) of obese as compared to lean rats. They also observed an acute increase in the levels of POMC primary transcript in the medial basal hypothalamus of non-obese animals after a single intracerebro-ventricular injection of leptin, which supports their notion that leptin plays a role in the regulation of POMC gene transcription. These findings led the investigators to conclude that POMC is an important mediator of the effects of leptin on food intake and energy expenditure. These findings may have applications for drug abuse and AIDS-associated complications such as weight loss and anorexia, and may have implications for understanding and managing opiates and withdrawal. Korner, J., Chua, S.C., Williams, J.A., Leibel, R.L. and Wardlaw, S.L. Regulation of Hypothalamic Proopiomelanocortin by Leptin in Lean and Obese Rats. Neuroendocrinology, 70, pp. 377-383, 1999.

Fundamental Mechanisms of Dopamine Neurotransmission

Amphetamines can dramatically stimulate locomotor activity by increasing dopamine release and by inhibiting its reuptake. Dr. David Sulzer of Columbia University, in collaboration with colleagues from Genentech, Univ. of California San Francisco, Stanford University, and the Universidad de Valencia, produced genetically altered mice lacking the alpha-synuclein protein and studied the response of the knockout mice to amphetamine. The mutant mice displayed a decreased locomotor response to amphetamines. In addition, the mutant mice had reduced levels of total striatal dopamine and the neurons exhibited increased dopamine release in response to paired electrical stimuli. The researchers concluded that alpha-synuclein is an essential presynaptic, activity-dependent negative regulator of dopamine neurotransmission; they attributed the attenuated response to amphetamine to either the altered pattern of dopamine neurotransmission or the reduced dopamine content. This finding is significant for basic neurobiology as well as for understanding why alpha-synuclein is associated with the etiology of Parkinson's Disease and Alzheimer's Disease. Abeliovich, A., Schmitz, Y., et al. Mice Lacking Alpha-Synuclein Display Functional Deficits in the Nigrostriatal Dopamine System. Neuron, 25, pp. 239-252, 2000.

Making the Connection Between Synaptic Vesicle Recycling and Cytoskeletal Organization

Drugs of abuse effect both synaptic transmission and synaptic plasticity, the growth of new synaptic connections. Synaptic vesicle recycling, an integral part of synaptic transmission, takes place via endocytosis. Actin-mediated cytoskeletal reorganization is an important component of neural plasticity and is required to guide axons to their target. Regis Kelly and Britta Qualmann at the University of California San Francisco determined that isoforms of the protein syndapin play a role in both endocytosis and actin dynamics. Syndapins localize to sites of high actin turnover in neuron-like cells and co-localizes with dynamin, a part of the endocytosis machinery, in neuroendocrine cells. Overexpression of full-length syndapin had a strong effect on cortical actin organization and induced extensions of the axon called filopodia, while overexpression of a portion of the syndapin protein inhibited endocytosis in vivo. The researchers concluded that syndapin isoforms link endocytosis and cytoskeletal dynamics in mature nerve terminals and other cell types. Thus this protein links two cellular functions, synaptic transmission and synaptic plasticity, that are affected by drugs of abuse. Qualmann, B. and Kelly, R.B. Syndapin Isoforms Participate in Receptor-Mediated Endocytosis and Actin Organization. J. Cell Biol., 148, pp. 1047-1061, 2000.

Region-Specific mRNA Processing of MOR-1 and MOR-1C in Rat Brain

Using rabbit antisera generated against the C-terminal peptide sequences of the mu opioid receptor splice variant MOR-1C, Dr. Pasternak and his team immunohistochemically examined the regional distribution of MOR-1C and compared it with MOR-1. They found that, overall, the distribution of MOR-1C-like immunoreactivity (-LI) differed from MOR-1-LI. Both MOR-1C-LI and MOR-1-LI were prominent in a few central nervous system regions, including the lateral parabrachial nucleus, the periaqueductal gray, and laminae I-II of the spinal trigeminal nuclei and the spinal cord. In the striatum, hippocampal formation, presubiculum and parasubiculum, amygdaloid nuclei, thalamic nuclei, locus coeruleus, and nucleus ambiguous MOR-1-LI predominated, whereas MOR-1C-LI was absent or sparse. Conversely, MOR-1C-LI exceeded MOR-1-LI in the lateral septum, the deep laminae of the spinal cord, and most hypothalamic nuclei such as the median eminence, periventricular, suprachiasmatic, supraoptic, arcuate, paraventricular, ventromedial, and dorsomedial nuclei. Double-labeling studies showed colocalization of the two receptors in neurons of the lateral septum, but not in the median eminence or in the arcuate nucleus, even though both MOR-1 isoforms were expressed. Because both MOR-1 and MOR-1C are derived from the same gene, these differences in regional distribution represent region-specific mRNA processing. Abbadie, C., Pan, Y.X., and Pasternak, G.W. Differential Distribution in Rat Brain of Mu Opioid Receptor Carboxy Terminal Splice Variants MOR-1C-Like and MOR-1-Like Immunoreactivity: Evidence for Region-Specific Processing. J. Comp. Neurol., 419, pp. 244-256, 2000.

Role of D1 Receptor in Cocaine- and Amphetamine-Induced Behavioral Changes

Cocaine and amphetamine can induce both short-term and long-term behavioral changes in rodents. The major target for these psychostimulants is thought to be the brain dopamine system. After cocaine and amphetamine treatments, Dr. Xu and his colleagues at the University of Cincinnati tested both the locomotor and stereotyped behaviors in mutant mice lacking the dopamine D1 receptor and wild-type control mice to determine whether the dopamine D1 receptor plays a crucial role in the behavioral effects of psychostimulants. They observed that the overall locomotor responses of D1 receptor mutant mice to repeated cocaine administration were significantly reduced compared to those of the wild-type mice. The responses of the D1 receptor mutant mice to cocaine injections were not significantly different from their responses to saline injections. D1 receptor mutant mice were less sensitive than the wild-type mice to acute amphetamine stimulation over a dose range although they exhibited similar behavioral responses to their wild-type cohorts. Immunostaining experiments indicated that there was no detectable neurotoxicity in the nucleus accumbens in both D1 receptor mutant and wild-type mice after repeated amphetamine administration. These data suggest that the D1 receptor plays an essential role in mediating cocaine- and amphetamine-induced behavioral changes in mice. Xu, M., Guo, Y., Vorhees, C.V., and Zhang, J. Behavioral Responses to Cocaine and Amphetamine Administration in Mice Lacking the Dopamine D1 Receptor. Brain Research, 852, pp. 198-207, 2000.

Pharmacogenetic Variability in Neuronal Nicotinic Receptor-Mediated Antinociception

The ability to predict inter-individual differences in drug efficacy or toxicity, based on genetic factors that influence drug disposition or drug action, is fast becoming a realistic goal. Dr. Christopher Flores of the University of Texas Health Science Center at San Antonio investigated epibatidine, a prototypical nicotinic analgesic drug, to see if it exhibits pharmacogenetic variability in antinociceptive activity. All strains of mice tested exhibited significant antinociception that peaked between 10 and 20 minutes following systemic injection of epibatidine. However, there was fourfold-variability in the magnitude of peak effect between mouse strains, with DBA/2 and A strains showing much greater sensitivity than all others tested. All mouse strains returned to baseline nociceptive threshold 30 minutes post-injection except for the A strain. In contrast, these mice exhibited significant antinociception for at least 3 hours following epibatidine administration. Thus, expressing the data as area under the time-latency curve to take into account both the magnitude and duration of effect, epibatidine displayed approximately 20-fold higher antinociceptive potency in the A strain as compared with the C3H/He strain. The effects of epibatidine in both the A and C3H/He strains were dose-dependent and sensitive to antagonism by the selective neuronal nicotinic channel blocker mecamylamine. Taken together, these data demonstrate the existence of pharmacogenetic variability in neuronal nicotinic receptor-mediated antinociception between inbred strains of mice and presage the potential for similar variability in analgesic response to nicotinic-based analgesics in humans. Future studies will seek to identify the chromosomal loci underlying the variability. Flores, C.M., Wilson, S.G., and Mogil, J.S. Pharmacogenetic Variability in Neuronal Nicotinic Receptor-Mediated Antinociception. Pharmacogenetics, 9, pp. 619-625, 1999.

Direct Protein-Protein Coupling Enables Cross-Talk Between Dopamine D5 and Gamma-aminobutyric Acid A Receptors

GABAA (gamma-aminobutyric-acid A) and dopamine D1 and D5 receptors represent two structurally and functionally divergent families of neurotransmitter receptors. The former comprises a class of multi-subunit ligand-gated channels mediating fast interneuronal synaptic transmission, whereas the latter belongs to the seven-transmembrane-domain single-polypeptide superfamily of receptors that exert their biological effects, including the modulation of GABAA receptor function, through the activation of second-messenger signaling cascades by G proteins. NIDA grantee Dr. Niznik and his coworkers at the University of Toronto show that GABAA-ligand-gated channels selectively form a complex with D5 receptors through the direct binding of the D5 carboxyl-terminal domain with the second intracellular loop of the GABAA gamma2(short) receptor subunit. This physical association enables mutually inhibitory functional interactions between these receptor systems. For the first time it also shows a mechanistic basis allowing for the functional differentiation of D1 and D5 receptors. The data highlight a previously unknown signal transduction mechanism whereby subtype-selective G-protein-coupled receptors dynamically regulate synaptic strength independently of classically defined second-messenger systems, and provide a heuristic framework in which to view these receptor systems in the maintenance of psychomotor disease states. Liu, F., Wan, Q., Pristupa, Z.B., YU, X-M, Wang, Y.T., and Niznik, H.B. Nature, 403, pp. 274-280, 2000.

Prefrontal Cortex is Necessary for the Induction, but not the Expression of Cocaine Sensitization

Behavioral sensitization is considered a useful animal model for studying the development of craving in humans. In collaborative studies, the laboratories of Drs. Marina Wolf and Frank White at the Chicago Medical School have been investigating the role of prefrontal cortex and its glutamatergic inputs to the VTA in cocaine sensitization. Previously, they found that behavioral sensitization to repeated doses of cocaine could be prevented by lesions of prefrontal cortex and by blockade of either the NMDA or AMPA classes of glutamate receptors. These treatments also prevented the changes in dopamine receptor sensitivity in the VTA and NAc that are likely cellular correlates of behavioral sensitization. A more recent study showed that sensitization was expressed with no decrement when these same treatments - prefrontal lesions or NMDA-receptor blockade - were carried out only on the day of testing for sensitization. These results are consistent with a model of sensitization that requires NMDA receptor activation in the VTA via glutamate release from axons originating in the prefrontal cortex. An implication of this model is that inputs to the prefrontal cortex from other events associated with drug taking could increase or decrease a drug's ability to modify midbrain function. Conversely, since neither prefrontal cortex nor NMDA-receptor activation is necessary for the expression of sensitization, it may not be possible to reverse some drug effects by engaging the cognitive and executive functions of prefrontal cortex. Li, Y., Hu, X.T., Berney, T.G., Vartanian, A.J., Stine, C.D., Wolf, M.E., and White, F.J. Both Glutamate Receptor Antagonists and Prefrontal Cortex Lesions Prevent Induction of Cocaine Sensitization and Associated Neuroadaptations. Synapse, 34, pp. 169-180, 1999; Li, Y., Wolf, M.E., White, F.J. The Expression of Cocaine Sensitization is not Prevented by MK-801 or Ibotenic Acid Lesions of the Medial Prefrontal Cortex. Behavioral Brain Research, 104, pp. 119-125 1999.

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