Director's Report to the National Advisory Council on Drug Abuse
May, 2001
Research Findings
Basic Research
Experiences During Drug Withdrawal Alter Brain Chemistry
Researchers from Texas Southwestern Medical Center recently showed significant changes in the brain enzyme tyrosine hydroxylase (TH) in rats following withdrawal from cocaine. TH is the rate-limiting enzyme for the synthesis of the brain neurotransmitter dopamine. In the experiment, rats pressed a response lever to self-administer cocaine over twelve consecutive days. Following this phase of "acquisition," one group of rats went through cocaine withdrawal for one week in their home cage. A second group of rats was returned to the chamber where they previously received cocaine each day for one week, but this time lever presses did not result in cocaine infusions (a procedure known as "extinction"). When TH was measured in these two groups, the rats that went through withdrawal in the home cage had lower TH levels in the nucleus accumbens and ventral tegmental area, two brain regions implicated in drug addiction, than rats who experienced "extinction" training. Importantly, these data suggest that experiences during cocaine withdrawal can result in differential neuroadaptations in neurotransmitters affected by drugs of abuse. The investigators hypothesized that the experience of seeking cocaine and not obtaining it increased TH levels, and presumably dopamine levels, leading to accelerated recovery from the negative effects associated with cocaine withdrawal. These results have important implications for treating cocaine addiction and understanding the role of experiential factors in reversing drug-induced brain changes. Schmidt, E.F., Sutton, M.A., Schad, C.A., Karanian, D.A., Brodkin, E.S., and Self, D.W. Extinction Training Regulates Tyrosine Hydroxylase during Withdrawal from Cocaine Self-Administration, The Journal of Neuroscience, 21, pp. 1-5. 2001.
Neurotoxic Effects of Nicotine
The laboratory group of Gaylord Ellison has previously shown that a variety of addicting drugs (e.g. cocaine, methamphetamine, MDMA), when given continuously to rats so as to mimic "binging" in people, all induce degeneration of axons. These axons run from the lateral habenula through the sheath of the fasciculus retroflexus to midbrain nuclei such as the ventral tegmental area, substantia nigra, and raphe nuclei. With some of these drugs, such as cocaine, this is virtually the only degeneration induced in all of the brain. Dr. Ellison's group very recently found that nicotine given continuously for five days at relatively low doses which induce plasma levels of nicotine comparable to heavy smokers, also selectively induces degeneration in fasciculus retroflexus. However, with the nicotine, this damage is in the other half of the fasciculus retroflexus tract - the cholinergic axons running from medial habenula in the core of the tract to the interpeduncular nucleus.
Humans generally begin their smoking habits by smoking only a few cigarettes each day, and thus probably rapidly induce a great deal of tolerance to this potential neurotoxicity. For this and other reasons, it is uncertain to what degree these observations in rats can be applied to humans. However, it is now apparent that, at least, the fasciculus retroflexus may be a "weak link" in the brain for diverse stimulant drugs of addiction. Damage to this tract may be involved in the induction of various behaviors associated with progressive addiction and relapse. Since fasciculus retroflexus provides negative feedback from the limbic forebrain back onto the midbrain monoaminergic "reward" cells that innervate it, it is predicted that its degeneration may be linked to a loss of higher brain control over behavior. Carlson, J., Armstrong, B., Switzer, R.C. 3rd, and Ellison, G. Selective Neurotoxic Effects of Nicotine on Axons in Fasciculus Retroflexus Further Support Evidence That This is a Weak Link in Brain Across Multiple Drugs of Abuse. Neuropharmacology, 39(13), pp. 2792-2798, 2000. In addition to the NIDA grant, this study was also funded in part by the Tobacco Related Disease Research Program, which is funded by California's tobacco tax.
Endomorphins and CNS Sites
Endomorphins (EM-1 and EM-2) have been shown to act as endogenous agonists for the mu-opioid receptor. However to date, the sites within the CNS that are activated by these two peptides have not been elucidated. Recently, Dr. Sulie Chang and her co-workers published a study in Brain Research that examined the sites of action of these EM in the rat brain using EM-induced FOS immunoreactivity (FOSir) as an anatomic marker of neuronal activation. They observed dose-dependent EM-1 and EM-2 activation of FOSir in various nuclei through out the rostral-caudal axis of the rat brain and the activation was correlated with mu-opioid receptor location in brain areas except in caudate putamen and the accumbens nucleus. The differential activation of structures by these two novel peptides, EM-1 and EM-2, suggests that these molecules may be involved either in separate physiological functions or may play different roles within the same physiological processes. Jiang, Y., Klodesky, C.M., and Chang, S.L. Endomorphine-1 and Endomorphine-2 Induce the Expression of c-FOS Immunoreactivity in the Rat Brain. Brain Research, 873, pp. 291-296, 2000.
Anti-Inflammatory and Analgesic Action of a Cannabinoid Compound
A number of studies have shown that THC and structurally related synthetic cannabinoids are capable of modulating pain sensitivity in animal models at the level of brain or spinal cord CB1 receptor activation. This, in part, has generated an interest in finding cannabinoids that may activate peripheral CB1 receptors, resulting in both anti-inflammatory and antihyperalgesic effects. Of particular interest has been to search for a metabolite of THC that might have the indicated actions, but without psychotropic effects, and which could be delivered topically or orally. One of the first such compounds studied was the 11-nor-delta-9-THC-9-carboxylic acid (also known as THC-11-oic acid), which is a major metabolite of THC. This has been shown to inhibit prostaglandin synthesis by inhibiting cyclo-oxygenase activity, and to have antinociceptive activity in mice at 20-40 mg/kg orally. The metabolite was found to bind only weakly to the CB1 receptor. More recently, a synthetic analog of THC-11-oic acid, known as ajulemic acid, or CT-3, has been similarly investigated by Dr. Sumner Burstein. The compound is also under patent to Atlantic Pharmaceuticals Inc. In mice, CT-3 showed approximately equal potency with morphine in the hot plate test when administered intragastrically, and similar potency to morphine over several hours observation in the mouse tail clip test administered intragastrically or intraperitoneally. Dose-dependent analgesia was also observed in the rat tail clip procedure. The compound also demonstrated anti-inflammatory effects in an adjuvant-induced rat model for arthritis, with a lower ulcerogenic potential than shown by other anti-inflammatory agents, including indomethacin. Additionally, CT-3 reduced the accumulation of leukocytes induced by injection of pro-inflammatory cytokines into subcutaneous air pouches on the backs of mice, as a model of acute inflammation. In preliminary data, CT-3 did not produce signs of strong dependency in rats tested over fourteen days at oral doses of 10-40mg/kg, administered in an oil-based formulation. Burstein, S.H. Current Pharmaceutical Design, 6, pp. 1339-1345, 2000.
Design, Synthesis, and Monoamine Transporter Binding Site Affinities of Methoxy Derivatives of Indatraline
In this study, a series of methoxy-containing derivatives of indatraline, a nonselective monoamine reuptake inhibitor capable of antagonizing methamphetamine-induced neurotransmitter release, were synthesized. The structure of some intermediate compounds such as indanone, and trans-1-azido-3-(3,4-dichlorophenyl)-5-methoxyindan was confirmed by single crystal X-ray analysis. The binding affinity of these compounds for dopamine, serotonin, and norepinephrine transporter binding sites was determined. Out of several methoxy group-containing derivatives, 6-methoxy displayed the highest affinity for both serotonin and norepinephrine transporters, however this compound also retained reasonable affinity for the dopamine transporter. This high affinity compound might eliminate methamphetamine's reinforcing effects by blocking the transporters that are thought to contribute or modulate the effects of stimulant-like drugs. The authors concluded that this compound would be a promising template for the development of a long acting inhibitor of monoamine transporters. Such inhibitors have high potential as medications for treatment, as a substitution medication, or for prevention of the abuse of methamphetamine-like stimulants. Gu, X-H, Yu, H., Jacobson, A.E., Rothman, R.B., Dersch, C.M., George, C., Flippen-Anderson, J.L. and Rice, K.C. Design, Synthesis, and Monoamine Transporter Binding Site Affinities of Methoxy Derivatives of Indatraline, J. Med. Chem., 43, pp. 4868-4876, 2000.
Coupled Plasmon-Wavelengh Resonance (CPWR) Technology for Receptor
Structural changes accompanying the binding of ligands to the cloned human sigma-opioid receptor immobilized in a solid-supported lipid bilayer have been investigated using coupled plasmon-waveguide resonance(CPWR). This highly sensitive technique directly monitors mass density, conformation, and molecular orientation changes occurring in anisotropic thin films and allows direct determination of binding constants. Although both agonist binding and antagonist binding to the receptor cause increases in molecular ordering within the proteolipid membrane, only agonist binding induces an increase in thickness and molecular packing density of the membrane. This is a consequence of mass movements perpendicular to the plane of the bilayer occurring within the lipid and receptor components. These results are consistent with models of receptor function that involve changes in the orientation of transmembrane helices. This methodology has the unique capability of independently examining the real-time changes in the structure of the receptor both parallel and perpendicular to the lipid membrane in response to receptor-ligand interactions. CPWR also provides greatly enhanced sensitivity and spectral resolution as compared to conventional surface plasmon resonance (SPR). In addition, the method should be readily adaptable to high-throughput screening, in view of the minute amounts of receptor and ligand needed for complete dose-response binding assay and for evaluation of receptor structural changes. In the present paper, the highly selective sigma-opioid receptor agonist DPDPE was incorporated into the preformed lipid layer and studied by CPWR. Salamon, Z., Cowell, S., Varga, E., Yamamura, H.I., Hruby, V.J. and Tollin, G. Biophysical Journal, 79, pp. 2463-2474, 2000.
Role of Positive Charge on the N-Terminal Amino Group of Opioid Peptides
To investigate the role of positive charge on the N-terminal amino group of opioid peptides in the interaction with their receptors, Schiller and colleagues undertook the synthesis and pharmacological characterization of enkephalin analogs in which the amino group is replaced with the neutral methyl group. As the parent compound, the potent enkephalin analog H-Dmt-D-Ala-Gly-Phe-Leu-NH2 was selected as it exhibits potent mu and delta agonist activities. The replacement of amino group in this analog with a methyl group required the development of a stereospecific synthesis of (2s)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)-propionic acid (Mdp). Following the synthesis of Mdp, the pentapetide, (2S)-Mdp-D-Ala-Gly-Phe-Leu-NH2, was prepared by the solid-phase method and the purity and structural identity were established by analytical HPLC and FAB-MS. The enkephalin analog was pharmacologically characterized. The enkephalin analog turned out to be a quite potent delta opioid antagonist and somewhat less potent mu antagonist, indicating that a positively charged N-terminal group is not a condition sine qua non for the binding of opioid peptides to delta and mu receptors. Based on the results, Schiller and colleagues proposed that the elimination of the positive charge through substitution of the N-terminal group with a methyl group, leads to a peptide that is no longer capable of binding to and stabilizing an active conformation of the mu or delta receptor. In the future, this concept might lead to the synthesis of new opioid peptide antagonists that are very much needed as research probes. Lu, Y., Weltroska, G., Lemieux, C., Chung, N.N. and Schiller, P.W. Bioorganic and Medicinal Chemistry Letters, 11, pp. 323-325, 2001.
Cannabinoids' Role in Infection
Some health consequences of smoking marijuana can be explained through analysis of basic studies on the role of endogenous cannabimimetic ligands in immune regulation. Two major cannabinoid receptor subtypes exist; subtype 1 (CB1) is expressed primarily in the brain whereas subtype 2 (CB2) is expressed primarily in the periphery, including the immune system. Most immune studies therefore focus on CB2, which is primarily expressed in T cells. However, a recent report identifies a role for CB1 in B cells. When B-cells are stimulated, the CB1 receptor is upregulated; this may show an importance of this receptor in antigen processing or to secondary immune responses to infection. In this study authors show that splenic B cells express more CB1 mRNA than T cells. Furthermore, splenocytes stimulated with the T cell mitogens, PMA/Io and anti-CD3, showed a decrease in CB1 message while cultures stimulated with the B cell mitogen, anti-CD40 antibody, showed an increase in message. In addition, co-treatment with mitogens and IL-2 uniformly caused an increase in CB1 mRNA. It is suggested that signaling pathways activated by T cell mitogens lead to decreased CB1 gene activation while pathways activated by B cell mitogens and IL-2 lead to increased CB1. Noe, S.N., Newton, C., Widen, R., Friedman, H. and Klein, T.W. Anti-CD40, Anti-CD3, and IL-2 Stimulation Induced Contrasting Changes in CB1 mRNA Expression in Mouse Splenocytes. J. Neuroimmun., 110, pp. 161-167, 2000.
Opioid Receptors and Immunity
Researchers from the University of Tennessee have investigated the role of delta type opioid receptors on immune cells. Most investigators study the regulation of cells by measuring actions in vitro in cell cultures. However, a recent paper has shown that these receptors are increased in number following an immune stimulant administered to whole animals. Thus, these receptors are increased as a result of a foreign substance and may be important in infections. Delta opioid receptors (DORs) are known to modulate multiple T-cell responses. However, little is known about the expression of these receptors. These studies evaluated the expression of DOR mRNA and protein after a single in vivo exposure to staphylococcal enterotoxin B (SEE). SEE enhanced splenocyte DOR mRNA expression 8 and 24 h after injection. SEE also increased the fractions of the total splenocyte and T-cell populations expressing DOR protein. Thus, SEE significantly increased DOR expression in vivo, affecting both mRNA and protein levels primarily within the T-cell population. To determine whether T-cell DORs modulate the activity of extracellular-regulated kinases (ERKs), the phosphorylation of ERKs 1 and 2 was studied using splenocytes from SEE-treated mice. D-AlaD-leu-enkephalin, a selective DOR agonist, significantly inhibited anti-CD3-epsilon-induced phosphorylation of the ERKs. Therefore, the DORs expressed by activated T-cells are capable of attenuating T-cell activation that depends on ERK phosphorylation. Shahabi, N.A., McAllen, K., Matta, S.G., and Sharp, B.M. Expression of Delta Opioid Receptors by Splenocytes from SEB-Treated Mice and Effects on Phosphorylation of MAP Kinase. Cell Immunol., 205, pp. 84-93, 2000.
Receptor Clustering on the Presynaptic Membrane
Glutamate receptors mediate most excitatory neurotransmission in the central nervous system, and the metabotropic class of glutamate receptors specifically regulate many aspects of neuronal function, including synaptic plasticity and memory formation. One theme of increasing importance in neurobiology is that clustering of neurotransmitter receptors in synaptic regions is crucial for efficient neuronal communication. In a recent paper, Dr. Ann Marie Craig and NIDA grantee Dr. Paul Worley and their colleagues demonstrate that the PICK1 protein is required for aggregating the mGluR7a metabotropic glutamate receptor at presynaptic sites. They showed that the PDZ domain of PICK1 interacts with the C-terminal region of mGluR7a to mediate this aggregation. While clustering and assembly of receptors and signaling molecules at excitatory post-synaptic sites has been well studied, this is one of the first demonstrations of the role of a clustering protein in the distribution of a receptor expressed on presynaptic membranes. The authors hypothesize that PICK1 may act as a scaffolding molecule at presynaptic sites organizing mGluR7a receptors with specific signal transduction complexes. Boudin, H., et al. Presynaptic Clustering of mGluR7a Requires the PICK1 PDZ Domain Binding Site. Neuron, 28, pp. 485-497, 2000.
The Human Genome and Drug Abuse Research
The publication of the sequence of the entire human genome was one of the most significant events in biological research in recent years. In a short article that appeared in the human genome issue of Nature, NIDA grantee Dr. Eric Nestler and Dr. David Landsman discussed the implications of genome sequencing for understanding drug addiction. They point out that the sequence will further understanding of the biology of addiction by allowing researchers to identify genes that contribute to individual risk for addiction and genes that mediate the addictive response to drugs. To illustrate their point, the authors scanned the human genome and found several new potential genes related to those known to be involved in the cellular response to drugs. Nestler, E.J. and Landsman, D. Learning about Addiction from the Human Genome. Nature, 409, pp. 834-835, 2000.
Using Proteomics to Study the Mechanism of Nitric Oxide Signaling
Nitric oxide (NO) affects many physiological processes, including neurotransmitter release, and has been implicated in mediating responses to various drugs of abuse. NO is known to work via stimulation of the enzyme guanylyl cyclase, but this mechanism cannot explain all of NO's effects. Dr. Solomon Snyder and his colleagues hypothesized that NO can also act by forming nitrosothiol adducts at cysteine residues in proteins (S-nitrosylation). Using mass spectrometry to test whether proteins are endogenously nitrosylated, Dr. Snyder and his colleagues analyzed the mice lacking neuronal nitric oxide synthase. Endogeneous s-nitrosylation was observed for GAPDH, glycogen phosphorylase, creatine kinase, Rb, Hsp72, Na+/K+ ATPase alpha-2 subunit, the NMDA glutamate subunits (NR1 and NR2A), beta-tubulin, actin, and NF-H in wild type mice but not in mutant mice. Nitrosylation of these proteins may affect such physiological functions as membrane potential, cytoskeletal reorganization, neurite outgrowth, and cell growth. Thus, this paper establishes S-nitrosylation as a physiological signaling mechanism, and opens the way for studies of mechanisms that regulate S-nitrosylation signaling. Jaffrey, S.R. et al. Protein S-Nitrosylation: A Physiological Signal for Neuronal Nitric Oxide. Nature Cell Biol., 3, pp. 193-196, 2001.
Effects of Chronic Exposure to Cocaine are Regulated by the Neuronal Protein Cdk5
Cocaine acts by blocking uptake of dopamine released from presynaptic terminals that synapse onto medium spiny neurons in the striatum. Repeated exposure to cocaine causes long lasting changes in the nervous system that play a role in addiction. One of the changes observed following chronic cocaine administration is the induction of delta FosB, a transcription factor that persists in striatum long after the end of cocaine exposure. The induction of delta FosB has been shown to mediate increased behavioral sensitization to cocaine, characterized by augmentation of locomotor activity following repeated injections of cocaine. Behavioral sensitization is considered by some to be a model for the intensification of drug craving in humans that characterizes addiction and promotes relapse. In a recent paper in Nature, the Greengard and Nestler laboratories report that overexpression of delta FosB in mice or repeated exposure to cocaine results in increased expression of cyclin dependent kinase 5 (cdk5). This suggests that delta FosB regulates the expression of cdk5. To test the role of cdk5 in sensitization, the Nestler and Greengard laboratories treated animals with cdk5 inhibitors. These cdk5 inhibitors resulted in an even greater locomotor response following repeated exposure to cocaine. Thus, increases in cdk5 expression act to attenuate the responses to subsequent cocaine exposure and oppose other biochemical pathways induced by delta FosB that mediate behavioral sensitization. Cdk5 attenuates the response to cocaine by regulating the dopamine signaling pathway in the dendrites of medium spiny neurons in the striatum. Dopamine binding to the D1 receptor activates adenylate cyclase to increase cAMP production. Increased levels of cAMP lead to the phosphorylation of proteins by protein kinase A. One of the proteins that is phosphorylated by protein kinase A is DARPP-32. Phosphorylation of DARPP-32 at threonine 34 by protein kinase A causes DARPP-32 to inhibit the activity of protein phosphatase-1 and augments the phosphorylation of proteins by protein kinase A. In contrast, the activation of cdk5 leads to the phosphorylation of DARPP-32 at threonine75. Phosphorylation of this threonine in DARPP-32 prevents DARPP-32 from blocking the activity of protein phosphatase-1. Increased protein phosphatase-1 activity leads to a decrease in the amount of phosphorylation in medium spiny neurons in the striatum. One of the physiological consequences of decreased phosphorylation is diminished currents elicited by AMPA receptor agonists. The role that cdk5 plays as a homeostatic mechanism in attenuating the rewarding effects of cocaine and in drug seeking behavior remains to be determined. Bibb, J.A., et al. Effects of Chronic Exposure to Cocaine are Regulated by the Neuronal Protein Cdk5. Nature, 410, pp. 376 - 380, 2001.
Cannabinoid Effects on the Neural Encoding of Short-Term Memory in the Hippocampus
Cannabinoids, the principal psychoactive agents in marijuana, are known to have disruptive effects on memory processes in humans. In this study, the memory-disruptive effects of 9-tetrahydrocannabinol (9-THC) and the synthetic cannabinoid WIN 55,212-2 (WIN-2) were assessed in rats exposed to varying doses of each drug during performance of a spatial delayed non-match to sample (DNMS) task. In the DNMS task, the animal is first presented with a lever on either the right or left, which it is required to press (sample phase). After a variable delay interval, both levers are extended and the animal must press the lever opposite to the one extended in the sample phase. If the animal performs the correct non-match response, it is rewarded with water. Cannabinoids affected DNMS performance in a dose _ delay-dependent manner, and these effects on performance were eliminated if the cannabinoid CB1 receptor antagonist SR141617A was pre-administered. Cannabinoid receptors are particularly dense in the hippocampus, a brain structure that has been implicated in the performance of the DNMS task and other memory tasks that involve the encoding ("holding in mind") of specific information. The investigators have developed a method for recording the activity of ensembles of hippocampal neurons during the performance of the DNMS task. In previous studies, they found that hippocampal neurons increase their firing rate in specific patterns during various phases, or combinations of phases, of this task. In this study, both WIN-2 and 9-THC produced dose-dependent reductions in the amount of ensemble firing in the hippocampus during the sample phase of the task, but not during the non-match phase. This pattern of a selective decrease in firing during the sample phase, is similar to patterns seen when animals make natural (i.e., not drug-induced) mistakes in behavioral responses. These findings indicate that activation of CB1 receptors renders animals less able to retain item-specific information during a memory task. Hampson, R.E. and Deadwyler, S.A. Cannabinoids Reveal the Necessity of Hippocampal Neural Encoding for Short-Term Memory in Rats. Journal of Neuroscience, 20, pp. 8932-8942, 2000.
Individual Differences in Baseline Activity of Midbrain Dopamine Neurons are Correlated with Enhanced Vulnerability to Cocaine Self-Administration in Rats
In both humans and animals, there are considerable individual differences in sensitivity to the reinforcing effects of addictive drugs. Previous studies have shown that rats that respond with more locomotor activity when placed in a novel environment also tend to show greater behavioral and neurochemical responses to psychostimulant drugs. Specifically, rats with a high locomotor response to a novel environment (HRs) exhibit enhanced self-administration (SA) behavior, sensitization, and basal or drug-induced dopamine release in the nucleus accumbens as compared with rats with a low response to the novel context (LRs). In this study, the investigators asked whether such differences in vulnerability to drug addiction might be related to differences in dopamine (DA) neuron activity. Rats were divided into HRs and LRs according to their response to a novel environment and then tested for acquisition of cocaine self-administration. HRs rapidly acquired cocaine SA, whereas LRs did not. In a separate group of animals not exposed to any drugs, they recorded the activity of individual dopamine neurons with extracellular electrodes and found that HRs exhibit higher basal firing rates and bursting activity of DA neurons in the ventral tegmental area and, to a lesser extent, in the substantia nigra pars compacta. The greater activity of midbrain DA cells in HRs was accompanied by reduced sensitivity to the inhibitory effects of a DA D2-class receptor agonist, indicating possible sub-sensitivity of DA autoreceptors that are known to regulate the firing rate of these neurons. These results demonstrate that differences in the basal activity of DA neurons may be critically involved in determining individual vulnerability to drugs of abuse. Marinelli, M. and White, F.J. Enhanced Vulnerability to Cocaine Self-Administration is Associated with Elevated Impulse Activity of Midbrain Dopamine Neurons. Journal of Neuroscience, 20, pp. 8876-8885, 2000.
Altered Gating of Opiate Receptor-Modulated K+ Channels on Amygdala Neurons of Morphine-Dependent Rats
The molecular mechanism of tolerance to opiate drugs is poorly understood. Dr. Jonathan E. Freedman of the Northeastern University and his research team have used single-channel patch-clamp recordings to study opiate receptor effects on dissociated neurons from rat amygdala, a limbic region implicated in addiction processes. A 130-pS inwardly rectifying K+-preferring cation channel was activated by mu opioid receptors in a membrane-delimited manner. After chronic treatment with morphine, channel gating changed markedly, with an approximately 100-fold decrease in open probability at a given morphine concentration. The change in channel gating correlated both in time course and in dose of morphine treatment with the development of functional opiate dependence and appeared to arise at a step after G-protein activation and before channel permeation by K+. This decreased receptor-channel coupling appears to be large enough to account quantitatively for opiate tolerance and may represent one of the mechanisms through which tolerance occurs. Chen, X., Marrero, H.G., Murphy, R., Lin, Y.J., and Freedman, J.E. Proc. Natl. Acad. Sci., USA, 97(26), pp. 14692-14696, 2000.
Cannabinoid Receptors Activation Produces Coupling to Multiple G Protein Alpha-Subunits with Different Potencies
Previously it was shown that the amplification factors for cannabinoid receptors, defined as the number of total G proteins activated per occupied receptor, differs between several rat brain regions. Dr. Paul Prather at the University of Arkansas for Medical Sciences and his colleagues sought to determine which specific Gi/Goa subunits were activated by CB1 receptors in several rat brain regions and if this coupling might explain the regional differences in receptor/G protein amplification factors. Furthermore, they examined whether cannabinoid agonists might activate different subtypes of Ga subunits with varying degrees of efficacy and/or potency. Activation of specific G proteins by cannabinoid receptors was evaluated by the ability of the agonist WIN 55212-2 to stimulate incorporation of [alpha-32P]azidoanilido-GTP into Ga subunits in membranes. Photolabeled G proteins were either directly resolved using urea/SDS-polyacrylamide gel electrophoresis or first immunoprecipitated with specific antisera for different Ga subunits before electrophoresis. Individual Ga subunits were separated into distinct bands on a single gel and the amount of agonist-induced increase in radioactivity was quantified by densitometry. Stimulation of CB1 receptors by WIN 55212-2 resulted in the activation of a distinct pattern of at least five different Gia/Goa subunits in several brain regions. Furthermore, although the pattern of G proteins activated by WIN 55212-2 appeared to be similar across brain regions, slight differences were observed in both the percentage of increase and the amount of the individual Ga subunits activated. Most importantly, the amount of WIN 55212-2 required to half-maximally activate individual G proteins in the cerebellum varied over a 30-fold range for different Ga subunits. These results suggest that cannabinoid receptors activate multiple G proteins simultaneously in several brain regions and both the efficacy and potency of cannabinoid agonists to activate individual Ga subunits may vary considerably. Prather, P.L., Martin, N.A., Breivogel, C.S. and Childers, S.R. Mol. Pharmacol., 57(5), pp. 1000-1010, 2000.
Cocaine Relevant Stimuli Activate the Limbic Brain Regions
It is well accepted that environmental cues are a major factor in drug relapse. Recent work has implicated the extended amygdala in the development of salient stimuli to drug use. In this report, Dr. Friedbert Weiss and co-workers at Scripps Research Institute showed that the presentation of stimuli previously paired with cocaine availability elicited strong recovery of responding in rats that had been extinguished on cocaine self-administration behavior. The stimuli were effective even after 4 months confinement to the home cage. In addition, exposure of the rats to the cocaine-salient cues produced a strong neural activation (fos immunoreactivity) in the basolateral amygdala and medial prefrontal cortex. Treatment with the D1 dopamine antagonist SCH 23390 dose-dependently reversed the effects of the cocaine cue. These data show that the ability of cocaine cues to elicit drug-seeking remains intact over a prolonged time period. Data also implicate the medial prefrontal cortex and amygdala for mediating the cue-induced relapse and suggest that the D1 receptor may be an important substrate for the motivating effects of cocaine-related stimuli. Ciccocioppo, R., Paolo, P. and Weiss, F. Cocaine-Predictive Stimulus Induces Drug-Seeking Behavior and Neural Activation in Limbic Brain Regions After Multiple Months of Abstinence: Reversal by D1 Antagonists. PNAS, 98(4), pp. 1976-1981, 2001.
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