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

May, 1998

Research Findings

Basic Research

Acetylcholine Receptors Containing the Beta-2 Subunit are Involved in the Reinforcing Properties of Nicotine

Using knockout mice lacking the beta2 subunit of the neuronal nicotinic acetylcholine receptor (nAChR), but preserving expression of all other subunits of the neuronal nAChR, NIDA grantee Dr. Marina Picciotto and her colleagues at Yale University have uncovered evidence that the beta2 subunit is a necessary component of the receptor that mediates the reinforcing properties of nicotine in the brain. Each of the steps in the pathway leading to nicotine addiction has been examined in these animals: high affinity nicotine binding is completely absent in the mesolimbic system of mice lacking the beta2 subunit; both the electrophysiological response to nicotine of dopaminergic neurons, as well as nicotine-induced dopamine release, were absent in beta2 mutant mice; and beta2 mutant mice will self-administer cocaine, but extinguish self-administration behavior when nicotine is substituted for cocaine, implying that these animals cannot experience the reinforcing properties of nicotine in this paradigm. These experiments identify binding of nicotine to nAChRs containing the beta2 subunit as the first step in the pathway toward nicotine addiction. Future experiments using mice deficient in various other subunits of the nAChR will allow identification of the other subunits that make up this receptor. Picciotto, M.R., Zoli, M., Rimondini, R., Lena, C., Marubio, L.M., Merl, E., Pich, K. Fuxe, Changeux, J.P. Nature. 391(173), 1998.

An Ultra Sensitive Method for the Detection of Vesicular Components

One of the principal way cells communicate with each other is through the secretion of chemical signals known as hormones and neurotransmitters. These hormones and neurotransmitters are stored in vesicles that have volumes as small as a zeptoliter (10-12 liter), and are released in response to physiological stimuli. Dr. Richard Zare and his colleague at Stanford University have developed and perfected a technique that permits the chemical characterization of the contents of a single vesicle. In the February 20, 1998 issue of Science Dr. Zare reported that secretory vesicles isolated from the atrial gland of the sea snail Aplysia Californica were chemically analyzed individually. A single vesicle, having a volume of an attoliter (10-18 liter) was introduced into the tapered inlet of a separation capillary using a technique called optical trapping and lysed. The components of the vesicles were fluorescently labeled with napthalene-2, 3 dicarboxaldehye. The fluorescently labeled contents of the synaptic vesicle were then separated with capillary electrophoresis and analyzed with a laser-induced florescence technique. The amino acid, taurine, was detected in some vesicles but not in others. Standard biochemical techniques that examine the contents of a population of a vesicle would have failed to reveal the variations in vesicle content. This breakthrough will enable scientists to elucidate the cellular mechanisms used to package and sort chemical messengers into vesicles, and provides a precision previously unattainable for identifying types of secreted bioactive products from single vesicles. Chiu, D.T., Lillard, S.J., Scheller, R.H., Zare, R.N., Rodriguez-Cruz, S.E., Williams, E.R., Orwar, O., Sandberg, M., Lundqvist, J.A. Probing Single Secretory Vesicles with Capillary Electrophoresis. Science. 279(5354), pp. 1190-1193, Feb. 20, 1998.

Amphetamine Release of Dopamine by Reversal of Transport and Displacement

Mark Wightman, of the University of North Carolina at Chapel Hill, and his colleagues at Duke recently published data on the intraneuronal regulation of monoaminergic transmitters. The intracellular and extracellular concentrations of monoaminergic neurotransmitters are regulated by two types of transporters. Na+/Cl- -dependent plasma membrane transporters terminate neurotransmission by removing transmitters from the extracellular space, while vesicular transporters move the monoamines from the cytoplasm into vesicles, a process critical for stimulated release of transmitter. The plasma membrane transporters are the initial cellular targets of cocaine, and both transporters are targets of amphetamine. Wightman and his colleagues used fast-scan voltammetry to measure extracellular dopamine in the striata of mice lacking the plasmalemmal dopamine transporter to elucidate the mechanism of action of amphetamine. They demonstrated that amphetamine releases dopamine by two separate mechanisms, through reversal of transport (movement of dopamine out of the cell via the dopamine transporter) and by displacement of vesicular dopamine into the cytoplasm of the terminal. Mice lacking the dopamine transporter do not show the typical increase in locomotor activity to an injection of cocaine. Mice that lack the vesicular transporter (VMAT2), in contrast, are not viable, but heterozygotes, which show a 50 percent decrease in the VMAT2, respond vigorously with increased motor activity in response to amphetamine. However, they do not appear to develop behavioral sensitization to repeated injections of amphetamine. These mice also have lower tissue content of dopamine and decreased basal dopamine extracellularly relative to control animals. The significance of this observation is to suggest that one role of the VMAT2 may be to calibrate the capacity of neuron to store the appropriate monoamine intracellularly. Accordingly, either the size of the releasable pool or transmitter or the amount of transmitter stored in each vesicle may be regulated by VMAT2. Jones, S.R., Gainetdinov, R.R., Wightman, R.M., Caron, M.G.. Mechanisms of Amphetamine Action Revealed in Mice Lacking the Dopamine Transporter. J. Neuroscience. 18, pp. 1979-1986, 1998. Wang, Y-M., Gainetdinov, R.R., Fumagalli, F., Xu, F., Jones, S.R., Bock, C.B., Miller, G.W., Wightman, R.M., Caron, M.G. Knockout of the Vesicular Monoamine Transporter 2 Gene Results in Neonatal Death and Supersensitivity to Cocaine and Amphetamine. Neuron, 19, pp. 1285-1296, 1997.

Cocaine & Female Reproductive Function

Analgesic Activity of Orphanin FQ2, Murine Prepro-Orphanin FQ141-157 in Mice

Orphanin FQ/Nociceptin (OFQ/N) is generated from a larger precursor peptide, prepro-orphanin FQ (ppOFQ). Dr. Gavril W. Pasternak and his research team at the Memorial Sloan-Kettering Cancer Center have discovered another putative heptadecapeptide within the sequence of murine ppOFQ, orphanin FQ2 (OFQ2), corresponding to murine ppOFQ141-157. OFQ2 was a potent analgesic given either supraspinally (ED50 0.5 g, i.c.v.) or spinally (ED50 0.7 g, i.t.). As with opioids and OFQ/N, OFQ2 analgesia was enhanced by blockade of sigma receptors with haloperidol, which increased the potency of the peptide over 10-fold. Supraspinal OFQ2 analgesia was readily reversed by the opioid antagonist naloxone, implying that OFQ2 activated opioid systems. Spinal OFQ2 analgesia was insensitive to naloxone. OFQ2 also inhibited gastrointestinal transit. Together, these studies suggest that OFQ2 may be a useful neuropeptide with important physiological actions. Rossi, G.C., Mathis, J.P., Pasternak, G.W. NeuroReport, In Press.

High Concentrations of Nicotine Inactivate Nicotinic Receptor Function in Vitro

Acetylcholine receptors (nAChRs) exposed to brief pulses of nicotine results in the release of dopamine, whereas prolonged exposure with low concentrations of nicotine (approximately 10 nM) produces a reversible blockade of a subsequent nicotine challenge (i.e., nAChRs desensitization). Dr. Peter Rowell of the University of Louisville School of Medicine and others have observed that, following prolonged exposure with a stimulating (M) concentration of nicotine, there is incomplete recovery from desensitization. In a recent study, Dr. Rowell and his research team investigated this nonrecoverable phenomenon by characterizing the ability of nicotine to stimulate [3H]dopamine release from rat striatal synaptosomes following recovery from nicotine-induced desensitization. Brief (12 seconds) exposure to 30 M nicotine, or longer exposure (Æ5 minutes) to 0.3 M nicotine, produced a long-lasting decrease in nAChR function with an apparent IC50 of 0.7 M. The maximum inactivation achieved was approximately 50 percent. Recovery of nAChR function did not return even after five hours, whereas recovery from desensitization occurred within 20 minutes. Determinations of the con-centration of nicotine in the superfusate indicated that residual nicotine could not account for the observed decrease in response as a consequence of desensitization. These results indicate that high concentrations of nicotine can produce a long-lasting nAChR inactivation which can be distinguished from reversible nAChR desensitization. The phenomenon may have important implications for the use of nicotinic agonists as therapeutic agents or in smoking cessation programs. Rowell, P.P., Duggan, D.S. Neuropharmacology. 37(1), pp. 103-111, 1998.

Highly Delta-Receptor Selective Peptides Useful as Chemical Probes

A recent report has described the preparation and pharmacological properties of several cyclic enkephalin peptide analogs, having the structure Tyr-c[D-Pen-Gly-Phe(p-X)-Pen]-Phe-OH, which show high potency at the delta receptor in the rat brain. In addition, when part of the chemical structure (the 4-X-phenylalanine (p-X)) is substituted for iodine, chlorine, or fluorine, very high selectivity ratios of 10000-45000 for delta opioid versus mu opioid binding were found. H-Tyr-c[D-Pen-Gly-Phe(p-F-Pen]-OH, exhibited an exceptionally low IC50 value of 0.016 nM. These peptides may serve as potentially useful probes in opioid research. Hruby, et al. J. Medicinal Chemistry. 40, pp. 3957-3962, 1997.

Extraordinary Potency of a Novel Delta Opioid Receptor Agonist

A new cyclic opioid peptide Tyr-D-Pen-Gly-Phe-Cys-Phe (HBP2) was evaluated for its delta-receptor interaction. HBP2 was approximately 160 times as potent as a standard delta-opioid ligand, DPDPE. Estimation of the affinity and efficacy of the peptide revealed that the higher potency of HBP2 was due to a 5.3-fold increase in efficacy and 37-fold increase in affinity. This compound may serve as a good receptor probe. Kramer, et al. Life Sciences. 61(2), pp. 129-135, 1997.

Dopamine Transmission in the Nucleus Accumbens as a Function of Free-Choice Novelty

To assess dopamine efflux during novelty-seeking behavior in rats, fast-scan cyclic voltammetry in the nucleus accumbens was combined with free-choice entry into a novel environment. Cyclic voltammograms, confirmed by in vitro testing, revealed that entry into novel, but not familiar, surroundings increased dopamine efflux in a regionally and temporally distinct pattern. Whereas dopamine failed to change in the core region of the accumbens and overlying neostriatum, an abrupt increase occurred in accumbal shell, a limbic-related area implicated in goal-directed behavior. Although the dopamine response was confined to the brief period of entry into novelty (approximately 8 s duration), a less rapid and more persistent dopamine change (> 20 s duration) occurred in the shell-core transition zone. These results suggest that novelty mimics other positively reinforcing stimuli in enhancing dopamine transmission in the nucleus accumbens, but the regional and temporal heterogeneity of this effect may represent different aspects of accumbal dopamine function. Rebec, G.V., Christensen, J.R., Guerre, C. & Bardo, M.T. Regional and Temporal Differences in Real-Time Dopamine Efflux in the Nucleus Accumbens During Free-Choice Novelty. Brain Research. 776(1-2), pp. 61-67, 1997.

Repeated Amphetamine Produces Persistent Structural Changes in Nucleus Accumbens and Prefrontal Cortex Neurons

Terry E. Robinson and Bryan Kolb at the University of Michigan found that repeated administration of amphetamine produces morphologic changes lasting more than a month in neurons in the nucleus accumbens and prefrontal cortex in rats. The exposure to amphetamine produced an increase in the length of dendrites, in the density of dendritic spines, and in the number of branched spines on the medium spiny neurons of the accumbens, and similar effects on the apical, but not basilar, dendrites of layer III pyramidal neurons in the prefrontal cortex. The ability of amphetamine to alter patterns of synaptic connectivity in these brain structures may contribute to some of the long-term behavioral consequences of repeated amphetamine use, including amphetamine psychosis and addiction. Robinson, T.E., Kolb, B. J. Neurosci. 17, pp. 8491-8497, 1997.

Catecholaminergic Activity in the Forebrain During Exposure to Novelty

Voltammetric recordings with electrochemically modified carbon-fiber electrodes were obtained from specific regions of the forebrain in rats given free-choice access to a novel environment. Entry into novelty increased the catechol signal in the medial prefrontal cortex and shell of the nucleus accumbens by more than 100%, but had no consistent effect in either the neostriatum or accumbal core. In both the medial prefrontal cortex and accumbal shell, moreover, the novelty-induced increase in catecholaminergic activity was detectable only during the initial entry into the novel compartment and did not reappear when animals returned to the familiar environment. These results support increasing evidence for a functional distinction between the accumbal core and shell, with the latter having been linked to brain reward mechanisms. The results also indicate that novelty activates some of the same neurochemical systems believed to play a critical role in the reinforcing effects of certain drugs of abuse. Rebec, G.V., Grabner, C.P., Johnson, M., Pierce, R.C., & Bardo, M.T. Transient Increases in Catecholaminergic Activity in Medial Prefrontal Cortex and Nucleus Accumbens Shell During Novelty. Neuroscience. 76(3), pp. 707-714, 1997.

Reviewing the Neuropharmacologic Mechanisms of Drug Reward

Multiple lines of research have implicated the mesolimbic dopamine system in drug reward measured by either the drug self-administration or conditioned place preference paradigm. The present review summarizes recent work that examines the neuropharmacological mechanisms by which drugs impinge on this dopaminergic neural circuitry, as well as other systems that provide input and output circuits to the mesolimbic dopamine system. Studies examining the effect of selective agonist and antagonist drugs administered systemically have indicated that multiple neurotransmitters are involved, including dopamine, serotonin, acetylcholine, glutamate, GABA, and various peptides. Direct microinjection studies have also provided crucial evidence indicating that, in addition to the mesolimbic dopamine system, other structures play a role in drug reward, including the ventral pallidum, amygdala, hippocampus, hypothalamus, and pedunculopontine tegmental nucleus. GABAergic circuitry descending from the nucleus accumbens to the pedunculopontine tegmental nucleus via the ventral pallidum appears to be especially important in directing the behavioral sequelae associated with reward produced by various drugs of abuse. However, activation of the reward circuitry is achieved differently for various drugs of abuse. With amphetamine and cocaine, initiation of reward is controlled within the nucleus accumbens and prefrontal cortex, respectively. With opiates, initiation of reward involves the ventral tegmental area, nucleus accumbens, hippocampus, and hypothalamus. It is not clear presently if these multiple anatomical structures mediate opiate reward by converging on a single output system or multiple output systems. Bardo, M.T. Neuropharmacological Mechanisms of Drug Reward: Beyond Dopamine in the Nucleus Accumbens. Critical Reviews in Neurobiology. 12(1-2), pp. 37-67, 1998.

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