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National Institute on Drug Abuse

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

May, 1997


Research Findings


Basic Research


9-THC Stimulates Dopamine Neurons Via Their Afferent Monoaminergic Neurons

9-Tetrahydrocannabinol (9-THC), the major active constituent of marijuana, is believed to stimulate the brain's dopamine systems, as do other drugs of abuse. Dr. Edward D. French's group at the University of Arizona College of Medicine is determining the mechanisms of actions of 9-THC on dopamine neurons using electrophysiological techniques. First, these investigators confirmed that acute iv administration of 9-THC increases the firing rate of substantia nigra and ventral tegmental area dopamine neurons (both about a 50% maximum increase over baseline) (E.D. French, X. Wu & K. Dillon, 1997, NeuroReport 8, in press). Pretreatment with the selective cannabinoid CB1 receptor antagonist SR141716A blocked this effect. Last year, the availability of active and inactive enantiomers of the synthetic cannabinoid CB1 receptor agonist WIN-55,212 provided an additional approach. The active enantiomer (WIN-55,212-2) increased dopamine neuron firing rates in these brain areas, while the inactive isomer (WIN-55,212-3) was ineffective. Thus, they confirmed that the dopamine neuronal changes induced by 9-THC are mediated through the CB1 cannabinoid receptor. Also last year, they demonstrated that reserpine or alpha-methyl-p-tyrosine pretreatment in vivo each reduced the response of the dopamine neurons to 9-THC. Thus, it would appear that catecholamine or serotonin afferents to the dopamine neurons are needed for the cannabinoid stimulation. This was confirmed by further experiments on dopamine neurons in midbrain slice preparations, where the afferents are cut; neither 9-THC nor WIN-55,212-2 stimulated cell firing in the slices.


Nitric Oxide Synthase Inhibitor Blocks Sensitization to Cocaine

The development of sensitization to cocaine and methamphetamine is thought to underlie the processes of

1) addiction, 2) neurotoxicity and 3) psychopathology. In rodents, sensitization is manifested by increased responsiveness to the locomotor-stimulating, stereotypy- and seizure-inducing (kindling) effects of cocaine. It has been established by the groups of Karler, White, and Wolf that glutamatergic neurotransmission and the NMDA subtype of glutamate receptor are the primary mediators of the induction of sensitization. Dr. Yossef Itzhak from the University of Miami School of Medicine found an increase in the number of NMDA receptors in the cortex of cocaine kindled mice, and further demonstrated that activation of the NMDA receptor's intracellular messengers, Ca2+ and nitric oxide (NO), mediates the development of sensitization to the convulsive and lethal effects of cocaine. Recently, he reported that neuronal selective NO synthase (NOS) inhibitors prevent the induction and expression of sensitization (Itzhak, Neuropharmacology 35, pp. 1065-1073, 1996). Further, a neuronal NOS inhibitor protects against methamphetamine neurotoxicity in mice (Itzhak and S. Ali, J. Neurochem. 67, pp.1770 1773, 1996). Elucidation of these mechanisms may advance the development of medications for the management of psychostimulant abuse.


Cannabinoids and Excitotoxicity

Because many drugs of abuse are known to affect glutamatergic synaptic transmission, Dr. Stanley Thayer and coworkers are testing the hypothesis that such drugs might influence the progression of excitotoxicity. These researchers found that cannabinoids significantly inhibit glutamatergic synaptic transmission. Their report identifying a synaptic mechanism of the cannabinoids is of considerable importance to understanding the cellular effects of marijuana. Thus, focus was directed toward the study of cannabinoids on glutamatergic synaptic transmission and excitotoxicity. In hippocampal neurons, activation of cannabinoid receptors inhibits the N and P/Q subtypes of Ca++ channels suggesting a likely molecular mechanism for the synaptic inhibition produced by cannabinoids. Cannabinoids were found to protect from excitotoxicity in vitro, suggesting that if compounds could be developed in which the abuse properties were separated from this potentially therapeutic effect, certain cannabimimetic drugs might be useful neuroprotective agents. A key feature of many abused drugs is the development of tolerance upon repeated exposure. Experimenters have found that the effects of full cannabinoid agonists on synaptic transmission desensitize during prolonged exposure. In contrast, the effects of one of the synthetic compounds CP55940, a compound that acts as a partial agonist, does not desensitize, suggesting that this drug may be effective in preventing the neurodegeneration that results from glutamate release. Thayer, S.A., and Shen, M. Cannabinoid Receptor Agonists Inhibit Glutamatergic Synaptic Transmission in Rat Hippocampal Cultures. Symposium on Cannabis and the Cannabinoids (Int. Cannabinoid Res. Soc.) p. 45, 1996. Shen, M. and Thayer, S.A. Desensitization of Cannabinoid-Mediated Inhibition of Glutamatergic Synaptic Transmission between Cultured Rat Hippocampal Neurons. Soc. Neurosci. Abst. 22: 82, 1996.


Tropane Derivative Holds Promise as a Methadone-Like Approach for Treating Cocaine Addiction As reported in the February 1997 issue of the Journal of Pharmacology and Experimental Therapeutics, Dr. Michael Nader and his co-workers from the Drug Abuse Center Neuroscience Program at the Bowman Gray School of Medicine in Winston Salem, NC tested PTT, a tropane derivative, in nonhuman primates. These researchers found that the drug produces internal cues similar to cocaine (that is, it is perceived as having properties like cocaine), but it will not substitute for cocaine in monkeys trained to self-administer cocaine. Also, PTT itself is not self-administered. Further, a single pretreatment dose of PTT to monkeys trained to lever press for intravenous cocaine prevented cocaine self-administration for 4 hours or more. The findings that the compound is not self-administered, prevents cocaine self-administration, and has subjective effects similar to cocaine, suggest that this tropane derivative, or a drug like it, would be useful in preventing cocaine relapse without compliance problems.


Biosensors

A recent analytical report describes a fiber optic system, known as an immunobiosensor, capable of determining the cocaine content of coca leaf samples in the field. It requires optical fibers which have been coated with a monoclonal antibody, in this case, one generated against benzoyl ecgonine-fluorescein as a sensor, and a portable fluorometer. Since cocaine competes with benzoyl ecgonine-fluorescein in binding to the antibody, its concentration is proportional to the decrease in fluorescence of the sensor when both are present in a sample. The method is rapid, of reasonable precision, and can be performed directly on acid extracts of coca leaves. Mohyee Eldefrawi, Charles Helling et al., Biosensors and Bioelectronics, 12(2), pp.113 124, 1997.


Blood Barrier and Narcotic Peptides

The tritiated mu-selective antagonist CTAP has been shown in a rat brain perfusion study to cross the blood brain barrier and the cerebral spinal fluid barrier in amounts approximating those of tritiated morphine. CTAP was bound to albumin in the perfusion medium and to rat serum protein, and remained 63% "intact" in the brain after a 20 minute perfusion, based on radioactive counts for the major HPLC peak in the detection system used. It is suggested that the blood brain barrier transport is based on passive diffusion rather than saturation transport, since the degree of transport is unaffected by the presence of unlabeled CTAP. The compound may have a potential role in narcotic addiction treatment. T. Abbruscato, S. Thomas, V. Hruby, T. Davis, J. Pharmacology and Experimental Therapeutics, 280, pp. 402 409, 1997.


Homer, a PDZ-domain Protein Selectively Binds Metabotropic Glutamate Receptors

Using differential cloning strategies, NIDA Grantee Paul Worley of Johns Hopkins University discovered Homer, a novel, brain-specific, small protein of 186 amino acids that differs from the conventional immediate early genes in that it can directly modify cellular function.

Recently a new protein motif called PDZ domain has been shown to be important in the targeting of a variety of membrane proteins to cell-cell junctions including synapses. The most widely recognized member of this family of proteins containing a PDZ domain is PSD95 which was originally identified as a component of the postsynaptic density and recently shown to interact with the C-terminus of the ionotropic glutamate receptor, N-methyl-D-aspartate receptor (NMDA receptor). By virtue of the physical interaction between PSD95 and the NMDA receptor, it is hypothesized that PSD95 functions to restrict the spatial distribution of the NMDA receptor.

While sequence comparisons fail to demonstrate a significant homology between Homer and PSD95, there were a number of functional similarities that suggested they may possess a similar domain for interaction. First, Dr. Worley and his coworkers demonstrated that Homer interacts with the C-terminal 4 amino acids of metabotropic glutamate receptor, mGluR5. This is the same C-terminal dependency for the interaction between PSD95 and NMDA receptor. Second, Homer possesses a sequence GLGF that is present in all PDZ family members and deletion of this region destroys the interaction between Homer and mGluR5. Third, the crystal structure of PSD95 demonstrated that the GLGF sequence forms the critical interaction site. Researchers also demonstrated that Homer is remarkably selective for mGluR receptors that mediate turnover of the phosphoinositide pathway (mGluR1 and mGluR5). Immuno- localization of Homer showed that it is concentrated at synaptic spines; this selective expression at neuronal spines suggests that Homer may be targeted to the spines.

In an accompanying paper in the same issue of Nature, another team at Johns Hopkins University led by Richard Huganir described GRIP, a larger synaptic protein possessing seven PDZ domains that interacts with another ionotropic glutamate receptor, a-amino-3-hydroxy- 5-methyl-4-isoxazole propionic acid receptor (AMPA receptor). Because glutamate is the major excitatory neurotransmitter in the mammalian brain, the identification of these two proteins, Homer and GRIP, will undoubtedly advance our understanding of the molecular mechanism of synaptic clustering of receptors.

Cocaine produces long lasting changes in the responsiveness of neurons in the basal ganglia and neuronal circuits in the frontal cortex and midbrain. These changes may play a role in the addictive potential of cocaine. Dr. Worley and his coworkers discovered that Homer is markedly induced in neurons of the basal ganglia in response to acute administration of cocaine. Homer is also persistently upregulated in the frontal cortex following chronic administration of cocaine. Metabotropic receptors have recently been demonstrated to play an important role in motor and behavioral responses of the nucleus accumbens and striatum and to be regulated by dopamine signaling from the midbrain. Homer may be critical in regulating this interaction. Brakeman, P.R., Lanahan, A.A., O'Brien, R., Roche, K., Barnes, C.A., Huganir, R.L. and Worley, P.F., Nature, p. 284, March 20, 1997.


Opioid Receptor, Hematopoiesis, and Reproduction

Using a knockout mouse deficient in mu opioid receptor, NIDA grantee Dr. Lei Yu and his colleagues at the Indiana University School of Medicine recently have discovered that the mu receptor gene disruption affected a number of aspects of the mouse physiology. Of particular interest is the observation that a lack of the functional mu receptor resulted in changes in both the host defense system and the reproductive system. They found increased proliferation of hematopoietic progenitor cells in both bone marrow and spleen, indicating a link between hematopoiesis and the opioid system, both of which are stress-responsive systems. They also detected changes in sexual function in male homozygous mice, including reduced mating activity, decrease in sperm count and motility, and smaller offspring litter size. These results suggest a novel role of the mu opioid receptor in hematopoiesis and reproductive physiology, in addition to its known involvement in pain relief. Journal of Experimental Medicine, April 21, 1997.


Gender-Related Differences

A recent article published by NIDA supported researchers demonstrates pronounced gender-related differences in the antinociceptive effects of morphine. These differences appear to reflect markedly enhanced CNS sensitivity to morphine in males compared with females, as opposed to any intrinsic differences in the bioavailability of morphine. Furthermore, these gender-related differences appear to exist at both spinal and supraspinal levels. Their findings also suggested that the acute effects of steroids play little role in the gender-related differences observed; rather, it appears more probable that the organizational effects of steroids, which occur in the late prenatal and early postnatal stages and in large part determine gender-related distinctions in males and females, may be more significant. Although the clinical significance and the underlying mechanisms of these findings are unknown at this point, these results may provide a means to begin examining gender-related differences in abuse liability of psychoactive drugs. Cicero, T.J., Nock, B. and Meyer, E.R. J Pharmacol. Exp. Therap. 279, pp. 767-773, 1996.


Gender-Related Differences

Dr. Marlene Wilson and her co-workers have examined gender related differences and regional variations in the ability of neuroactive steroid derivatives of progesterone, testosterone and glucocorticoids to alter physiological gama-aminobutyric acid (GABA) responses in brain regions of male and female rats. All four steroids examined increased GABA-activated chloride influx; however, the maximal enhancement in GABA responses differed significantly among brain regions. Limbic areas, such as hippocampus, amygdala and cortex, displayed greater maximal responses to these steroids than hypothalamic or cerebellar preparations. Since gender-related differences in neuroactive steroid modulation of GABA responses were observed with the glucocorticoid derivative, tetrahydrodeoxy-corticosterone (THDOC) but not with the progesterone derivative, 3-alpha 5-alpha tetrahydro-progesterone (THP), it would further suggest that the ability of THDOC and THP to potentiate GABA responses are affected differentially by the hormonal milieu. Wilson, M.A. and Biscardi, R. Life Sciences, In press.


Opiate Withdrawal Increases ProTRH Gene Expression in the Ventrolateral Column of the Midbrain Periaqueductal Gray

The midbrain periaqueductal gray matter (PAG) has a critical role in the modulation of behavioral and autonomic manifestations of the opiate withdrawal syndrome. Utilizing multi disciplinary approaches including behavioral studies, in situ hybridization histochemistry, immunohistochemistry and radioimmunoassay, Dr. Ronald M. Lechan of the New England Medical Center and his research team have demonstrated a nearly 5 fold increase in proTRH gene expression in neurons of the ventrolateral column of the PAG following naltrexone precipitated morphine withdrawal. The accumulation of immunoreactive proTRH-derived peptides, but not the mature TRH tripeptide, was concomitantly observed in these cells. These observations along with the fact that the ventrolateral PAG mediates a hyporeactive pattern of behavioral and autonomic reactions indicate that proTRH-derived peptides synthesized in neurons of the ventro-lateral PAG may function as modifiers of opiate withdrawal responses. Currently Dr. Lechan and his colleagues are attempting to elucidate the anatomical connectivity of this unique population of opiate-responsive proTRH neurons and to determine how these neurons are integrated into the control system that responds to the hyperactive state of morphine withdrawal.

The observation that there is a nearly 5-fold increase in proTRH gene expression in neurons of the ventrolateral column of the PAG following naltrexone precipitated morphine withdrawal is a novel and exciting finding. It opens up an entirely new area of opiate research and offers opportunities to design new approaches for the treatment of opioid addiction and the withdrawal syndrome. The data generated from this work could have significant clinical relevance. Legradi,G., Rand, W.M., Hitz, S., Nillni, E.A., Jackson, I.M.D., and Lechan, R.M. Opiate Withdrawal Increases ProTRH Gene Expression in the Ventrolateral Column of the Midbrain Periaqueductal Gray. Brain Research, 729, pp.10-19, 1996.

Isozyme-Specific Opioid-Induced Adenylyl Cyclase Supersensitization Acute stimulation of opiate receptors inhibits adenylyl cyclase (AC) and reduces cAMP in the cell, while chronic activation has been shown to lead to a progressive increase in AC activity. This phenomenon, particularly manifest upon withdrawal of the opiate agonist, is referred to as AC superactivation. The mechanism of AC superactivation is not clear, although it seems to play an important role in opiate addiction.

Zvi Vogel of The Weizman Institute of Science, Rohovot, Israel has transfected AC of types I-VIII (currently ten AC isozymes are known) into COS cells and studied the regulations of these AC isozymes by acute and chronic opiate exposures. The results show that the various AC isozymes are differently regulated by opiates. AC types I, V, VI and VIII are inhibited by acute opiate exposure and super-activated following chronic exposure. AC II, IV and VII are activated by acute opiate exposure and do not show the superactivation. AC type III is not affected by the presence of opiates. AC-V yielded the largest superactivation. This information will enable identification of the particular AC isozymes which participate in opiate signaling , and the reward system, in vivo and hence may facilitate the development of effective treatment strategies for opioid abuse. Avidor-Reiss, T., Nevo, I., Saya, D., Bayewitch, M. and Vogel, Z. Opioid-Induced Adenylyl Cyclase Supersensitization is Isozyme-Specific. J. Biol. Chem. 272, pp. 5040-5047, 1997.

Evaluation of Discriminative Stimulus Anandamide was shown to produce behavioral effects in mice characteristic of psychoactive cannabinoids; however, differences have also been found between anandamide and delta-9 THC. Drs. Billy Martin and Raj Razdan, and their colleagues designed a study to examine the discriminative stimulus effects of anandamide in rhesus monkeys trained to discriminate delta-9 THC from vehicle. While anandamide failed to produce reliable substitution for delta-9 THC and did not reduce response rates, 2-methylarachidonyl-2 prime-fluoroethylamide (methylated fluoroanandamide, a stable analog of anandamide), produced full dose-dependent substitution for delta-9 THC at doses that caused no significant changes in response rates. The results suggest that systematically-administered anandamide may be metabolized in monkeys before behaviorally active concentrations could reach the brain and further suggest that the metabolically stable analog of anandamide, methylated fluoroanandamide, may aid in the discovery of functional properties of the endogenous cannabinoid system. Wiley, J.L., Golden, K.M., Ryan, W.J., Balster, R.L., Razdan, R.K., and Martin, B.R. Discriminative Stimulus Effects of Anandamide and Methylated Fluoroanadamide in delta-9-THC-Trained Rhesus Monkeys. Pharmacol. Biochem. Behav., In press.

Hippocampus and GIRK1 Regulation of potassium channels by receptors coupled to heterotrimeric G proteins such as the different types of opioid receptors can have a profound effect on neuronal excitability by changing the duration of an electrical impulse or action potential, altering the membrane potential of neurons, and the number of action potentials fired by neurons. These changes can indirectly alter the amount of neurotransmitter released by a neuron which in turn can affect the excitability of neighboring neurons. One family of potassium channels modulated by opioid receptors, dopamine receptors, and other receptors coupled to G proteins that have been recently cloned is the G coupled inwardly rectifying potassium channels (GIRKs). To better understand the neuronal function of these GIRKS Dr. Charles Chavkin and coworkers at the University of Washington used immunohistochemistry and high resolution electron microscopy to define the subcellular localization and cell type that expresses GIRK1 in the hippocampus. Chavkin reports that GIRK1 immunoreactivity is regionalized within stratum lacunosum molecular and the superficial striatum radiatum in the hippocampus. At the cellular level electron microscopy revealed that GIRK immunoreactivity is found immediately adjacent to asymmetric (excitatory type) post synaptic densities along dendritic spines and shafts of pyramidal cells. Post synaptic densities are areas where receptors are located at synapses, the junctions where neurons communicate. Chavkin suggests that the localization of GIRK1 in dendritic shafts and spines could play a significant role in modulating the post synaptic responses at excitatory synapses by decreasing the likelihood of propagation of synaptic currents from distal dendrites. Drake, C.T., Bausch, S.B., Milner, T.A., Chavkin, C. GIRK1 Immunoreactivity is Present Predominantly in Dendrites, Dendritic Spines, and Somata in the CA1 Region of the Hippocampus. Proc. Natl. Acad. Sci. USA. 94, pp. 1007-1012, 1997.

µ3 opiate receptor and Morphine Morphine causes human monocytes, granulocytes, and endothelial cells as well as molluscan immunocytes and microglia to change from a flattened to a rounded shape. This change in morphology produced by morphine may play an important role in the ability of morphine to reduce inflammation by altering cell adhesion and cell migration through endothelial cells. Dr. Harold Magazine and his colleagues examined the role of nitric oxide in morphine-induced rounding of monocytes, granulocytes, and endothelial cells. Dr. Magazine and his colleagues showed that morphine induces the synthesis of nitric oxide. The synthesis of nitric oxide appears to play an important role in morphine-induced rounding because exposure to morphine elicits nitric oxide production, nitric oxide agonists induce cell rounding, and nitric oxide antagonists block morphine induced rounding. The synthesis of nitric oxide by morphine was blocked by naloxone; however, opioid peptides failed to elicit the production of nitric oxide. Magazine suggests that the morphine-induced NO release may be mediated by the activation of the opiate alkaloid-selective, opioid peptide insensitive m3 receptor and that the coupling of nitric oxide to the m3 opiate receptor has been conserved throughout evolution. Magazine, H.I., Liu, Y., Bilfinger, T.V., Fricchione, G.L., and Stefano, G.B. Morphine-Induced Conformational Changes in Human Monocytes, Granulocytes, and Endothelial Cells and in Invertebrate Immunocytes and Microglia are Mediated by Nitric Oxide. Journal of Immunology, 156, pp. 4845-4850, 1996.


Separation and Detection of Neurotransmitters and Neurochemicals in Ultra-Small Volumes

Richard N. Zare, Ph.D., Professor of Chemistry at Stanford University who co-authored a paper last summer suggesting possible fossil evidence of life on Mars is also a NIDA grantee who is devising methods to separate and detect chemical compounds in single synaptic vesicles that normally contain ultra small volumes of neurotransmitters. This is important because biological systems are partitioned into small compartments from single cells (pico-to femtoliters) to single mitochondria and vesicles (attoliters). Analysis of the composition of individual synaptic vesicles will provide insight into the basic mechanisms by which synaptic transmission is modified. In a paper published in Science he reports significant progress in separating neurotransmitters by capillary electrophoresis whose biological activity and presence are detected by patch-clamp electrophysiology. Capillary electrophoresis can be used to separate compounds dissolved in volumes as small as the low femtoliter range and patch clamp electrophysiology can detect the presence of a small number of molecules of agonist that can open a single ion channel. Orwar, O., Jardemark, K., Jacobson, I., Moscho, A., Fishman, H.A., Scheller, R.H., and Zare, R.N. Patch-Clamp Detection of Neurotransmitters in Capillary Electrophoresis. Science, 272, pp. 1779-1782, 1996.


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