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Director's Report to the National Advisory Council on Drug Abuse - September, 2002

Research Findings - Basic Research

Endocannabinoids Facilitate the Induction of LTP in the Hippocampus

The hippocampus is an important site in the memory process. Long-term potentiation (LTP) of neural activity, which is one type of neuronal plasticity, is commonly used in the study of the neuronal basis of memory and to assess effects of pharmacological agents. The interaction of glutamate and GABA systems plays an important role in the memory process and LTP. Alger and his colleagues characterized a cannabinoid-mediated generation of LTP, induced by depolarization-suppression of inhibition (DSI type of disinhibition). They found that following burst depolarization of the principal neurons in the hippocampus, the GABAergic IPSCs are suppressed for a short period of time. As a consequence of the disinhibition, they showed that a weak stimulus, which would not usually induce LTP, was capable of inducing NMDA-dependent LTP if it was applied soon after a preceding depolarizing pulse (conditioning stimulus), or under conditions in which the GABA receptor was blocked. The cannabinoid receptor activation, apparently through presynaptic interneurons, was necessary and sufficient for the DSI type of disinhibition/LTP in hippocampus. When it was blocked, the weak testing stimulus train failed to induce the DSI-mediated LTP, even though the hippocampus is still capable of generating LTP in response to standard (stronger) pulses. It is known that hippocampus neurons In Vivo often fire in bursts independently, and a burst of action potentials in a single cell can induce DSI. The DSI-mediated LTP is likely to be restricted in space for the short travel distance of released endocannabinoid. Such targeted LTP could underlie behavioral learning. Dr. Alger's work reveals two distinct types of LTP that are correlated with GABA and glutamate systems respectively. Exogenous cannabinoids will globally activate cannabinoid receptors, including those on the excitatory synapse and disrupt the exquisite temporal and special pattern of coding and recall mediated by endocannabinoids, and may contribute to the learning and memory deficiencies associated with cannabinoid drug abuse. Nature Neurosci., 5(8), pp. 723-724, 2002.

Cannabinoids Reduce Tolerance to Opioids

NIDA-grantee Dr. Sandra P. Welch of Virginia Commonwealth University and her colleagues have examined the interactions of cannabinioids and opioids in producing analgesia. Chronic exposure to opioids, such as morphine, results in a tolerance to the analgesic properties of the opioid. There is a concomitant decrease in opioid receptors (where morphine primarily acts) in the brain and spinal cord. Dr. Welch has found that tetrahydrocannabinol (THC) administration in mice reduced morphine-induced analgesic tolerance. Further, THC eliminated the down regulation of opioid receptors seen after chronic morphine administration. These data help to identify a mechanism of opioid tolerance, as well as demonstrate the interaction of cannabinioids and opioids at both the behavioral and cellular levels. Cichewicz, D.L., Haller, V.L., and Welch, S.P. Changes in Opioid and Cannabinoid Receptor Protein following Short-Term Combination Treatment with Tetrahydrocannabinol and Morphine, The Journal of Pharmacology and Experimental Therapeutics, 297, pp. 121-127, 2001.

Slower Metabolism and Reduced Intake of Nicotine: Ethnic Differences

In the United States, about 90% of lung cancer cases are attributable to cigarette smoking. The incidence of lung cancer can, therefore, be taken as a population marker of cigarette-related disease. Considerable ethnic differences are seen in the prevalence of lung cancer. The lowest rates of lung cancer are seen in Asians and Latinos, with higher rates in whites and the highest rates in African-Americans. Ethnic differences in nicotine metabolism might, in part, explain ethnic differences in cigarette consumption and/or nicotine intake per cigarette and resultant tobacco-related cancer risk. Dr. Benowitz and colleagues compared the rate of nicotine metabolism and intake of nicotine per cigarette smoked among smokers of different ethnicities. Healthy volunteers, including Chinese-Americans, Latinos, and whites, received simultaneous infusions of deuterium-labeled nicotine and cotinine, a metabolite of nicotine. From blood and urine measurements, the disposition kinetics and the daily intake of nicotine from smoking were determined. Total and non-renal clearance of nicotine and cotinine and metabolic clearance of nicotine via the cotinine pathway were similar in Latinos and whites and statistically significantly lower in Chinese-Americans. Intake of nicotine per cigarette by Chinese-Americans was statistically significantly lower than that of Latinos. Among all the participants, there was a statistically significant positive correlation between nicotine clearance and daily intake of nicotine from cigarettes. The lower nicotine (and, therefore, tobacco smoke) intake per cigarette and the fewer cigarettes smoked per day, which may result, in part, from slower clearance of nicotine, may explain lower lung cancer rates in Chinese-Americans. Lower lung cancer rates among Latinos compared with whites, given their similar nicotine intake per cigarette, is probably due to smoking fewer cigarettes. The results with Chinese-Americans may have implications for dosing with nicotine medications to aid smoking cessation in Chinese-American smokers and perhaps in other Asian smokers. Benowitz, N.L., Perez-Stable, E.J., Herrera, B., and Jacob III, P. Slower Metabolism and Reduced Intake of Nicotine From Cigarette Smoking in Chinese-Americans. Journal of the National Cancer Institute, 94(2), pp. 108-115, 2002.

Biphalin Update

Biphalin is an opioid peptide first reported in 1991, which has a dimeric or "bivalent" enkephalin structure. It was designed as one of a class of compounds containing two pharmacophoric regions within one molecule, in this case separated by a two-atom hydrazide bridge. Biphalin possesses high affinity (nanomolar) for both the mu and delta receptors, and a lower affinity for the kappa receptor. It has been found to be more potent than morphine as an antinociceptive agent when given intracerebroventricularly to rats, and equipotent with morphine when given intraperitoneally. Interest in biphalin has persisted in part because of its diminished symptoms of dependence produced in the rat. Biphalin has limited ability to cross the blood brain barrier. However, this is somewhat enhanced by halogenation with chlorine, fluorine, or iodine on the phenyl rings of the phenylalanines present in the molecule. I-125-labeled biphalin has been shown to accumulate in the nucleus accumbens, the choroid plexus, and the pituitary, following intravenous injection. In recent work, Dr. Victor Hruby and his associates have reported new findings. The first is the crystal structure of biphalin sulfate, indicating a folded structure existing for both halves of the molecule. Specifically, amino acids 1-4 exhibited a random coil structure in the solid phase, and could be overlapped with the same four amino acid residues of the delta ligand DADLE. There was also overlap seen between some of the backbone atoms and the tyrosine group of TIPP-NH2 (a mu ligand) and corresponding atoms in residues 5-8 of biphalin. Secondly, because previous studies have shown that the minimum structural requirement for mu and delta activity in biphalin is the presence of one lipophilic amino acid beyond the hydrazide bridge, replacement of phenylalanine in position five with a dansyl group has been carried out, in order to produce a ligand with multiple receptor activity and fluorescence properties. The dansyl derivative of biphalin showed comparable mu/delta binding, antinociceptive properties in the rat model, and strong fluorescence emission at 600 nm, which may render this compound useful for pharmacokinetic studies. Misicka, A., Lipkowski, A., Kosson, D., Kosson, P., Lachwa-From, M., Brodzik-Bienkowska, A., and Hruby, V. Life Sciences. 70, pp. 893-897, 2002; Flippen-Anderson, J., Deschamps, J., George, C., Hruby, V., Misicka, A., and Lipkowski, A. J. Peptide Research, 59, pp. 123-133, 2002.

Control of Synaptic Strength by Glial TNFalpha

The role of glia in synaptic plasticity has largely been ignored despite the fact that glia comprises 90 percent of all the cells in the brain. The Laboratories of Mark Von Zastrow of UCSF, Michael Beattie and Robert Malenka in the March 22, 2002 issue of Science show that glia control synaptic strength by releasing tumor necrosis factor alpha (TNFalpha). They found that application of media from glia cells increased the number of AMPA glutamate receptors and the frequency of spontaneous transmitter release of hippocampal neurons in culture. The increase in the number of AMPA receptors and the increase in the spontaneous transmitter release could be blocked by adding a TNFalpha antibody or the TNF receptor to the culture medium, which absorb TNF from the media and prevents TNF from activating receptors on neurons. In addition, an inhibitor of TNFalpha release from glia also blocked the increase in the number of AMPA receptors and the increase in spontaneous transmitter release. Similar results were found in hippocampal slice preparations where the number of AMPA receptors and the frequency of spontaneous transmitter release was decreased by blockers of TNFalpha. These results suggest that TNFalpha may play a role in forms of synaptic plasticity such as long-term potentiation which involves increased insertion of AMPA receptors into the post-synaptic membrane. It may also explain how TNFalpha may contribute to neural injury by increasing the number of AMPA receptors. Beattie, E.C., Stellwagen, D., Morishita, W., Bresnahan, J.C., Ha, B.K., Von Zastrow, M., Beattie, M.S., and Malenka, R.C. Control of Synaptic Strength by Glial TNFalpha. Science, 295(5563), pp. 2282-2285, 2002.

Brain-Derived Neurotrophic Factor is Essential for Opiate-Induced Plasticity of Noradrenergic Neurons

Withdrawal from chronic opiates is associated with increased firing of noradrenergic neurons in the locus coereleus. Withdrawal symptoms can be blocked by treating animals with clonidine, an alpha 2-adreneric agonist that decreases the firing of noradrenergic neurons in the locus coereleus. Biochemically, the increase in electrical impulses of locus coerelus neurons is associated with increased activity of the cAMP pathway and increased synthesis of tyrosine hydroxylase, the rate-limiting enzyme in norepinephrine synthesis. Exactly what regulates the processes of adaptation of noradrenergic neurons to chronic opiate treatment is not well understood. Brain Derived Neurotrophic Factor (BDNF) supports the survival of noradrenergic Locus Coereleus neurons and plays a significant role in synaptic plasticity. To test the role of BDNF in regulating adaptation of Locus Coereleus neuron to opiates, Dr. Akbarian generated a conditional knockout of the BDNF gene in which the gene was deleted in the adult mouse but not during development. Dr. Akbarian reports in the May 15, 2002 issue of the Journal of Neuroscience that deletion of the BDNF gene significantly attenuated opiate withdrawal without affecting tolerance. Deletion of BDNF blocked the upregulation of adenylyl cyclase, the enzymes responsible for cAMP synthesis, and induction of tyrosine hydroxylase. This report is consistent with work of Marc Caron's laboratory on beta-arrestin and work by Rafael Maldonado, Julie Blendy, and Gunter Schutz that found that the biochemical pathways regulating withdrawal are different from those regulating tolerance to opiates. It remains to be determined whether BDNF contributes to compulsive drug seeking behavior and relapse in the absence of physical dependence. Akbarian, S., Rios, M., Liu, R.J., Gold, S.J., Fong, H.F., Zeiler, S., Coppola, V., Tessarollo, L., Jones, K.R., Nestler, E.J., Aghajanian, G.K., Jaenisch, R. Brain-derived Neurotrophic Factor is Essential for Opiate-induced Plasticity of Noradrenergic Neurons. J. Neurosci., 22(10), pp. 4153-4162, May 15, 2002.

Modulation of Postendocytic Sorting of G Protein-Coupled Receptors

Ligand-induced endocytosis contributes to the physiological regulation of a wide variety of signaling receptors. Many G protein-coupled receptors (GPCRs) are endocytosed by a mechanism involving receptor phosphorylation, interaction with beta-arrestins, and concentration in clathrin-coated pits. However, the functional consequences of GPCR endocytosis through this conserved cellular mechanism are diverse. Trafficking of internalized GPCRs by a rapid recycling pathway restores the complement of functional receptors in the plasma membrane and promotes resensitization of receptor-mediated signal transduction. In contrast, the sorting of internalized GPCRs to lysosomes promotes proteolytic down-regulation of receptors, leading to a prolonged attenuation of cellular signal transduction. Furthermore, the postendocytic sorting of certain GPCRs can itself be regulated under physiological condition.

Mu Opioid Receptors

(MORs) and delta opioid receptors (DORs) are structurally homologous GPCRs that mediate the actions of endogenously produced opioid neuropeptides and exogenously administered opiate drugs. Both receptors are endocytosed via clathrin-coated pits after agonist-induced activation, phosphorylation, and association with cytoplasmic beta-arrestin. However, it has been known that DOR, but not MOR, exhibits down-regulation and was rapidly proteolyzed after agonist-induced endocytosis. Moreover, DORs were found to concentrate in the perinuclear region of the cells and colocalized with the endosome and lysosome while MORs were localized in vesicles distributed throughout the cytoplasm that failed to colocalize with endosome and lysosome. Drs. Whistler and von Zastrow and their research team at the University of California and University of Lund, Sweden, using various cellular and molecular approaches, have identified a previously unknown protein with human, rat, and murine homologs that binds preferentially to the cytoplasmic tail of the DOR as a candidate heterotrimeric GTP binding protein (G protein)-coupled receptor-associated sorting protein (GASP). Disruption of the DOR-GASP interaction through receptor mutation or overexpression of a dominant negative fragment of GASP inhibited receptor trafficking to lysosomes and promoted recycling. The GASP family of proteins (noncovalent interactions with GPCRs), along with ubiquitinylation (covalent modification of GPCRs), may modulate lysosomal sorting and functional down-regulation of a variety of G protein-coupled receptors. Such complexity in postendocytic sorting machinery might be critical for generating the remarkable diversity and specificity with which signaling receptors are regulated in multicellular organisms.Whistler, J.L., Enquist, J., Marley, A., Fong, J., Gladher, F., Tsuruda, P., Murray, S.R. and von Zastrow, M. Modulation of Postendocytic Sorting of G Protein-Coupled Receptors. Science, 297(5581), pp. 615-620, July 26, 2002.

Potentiation of Opioid Analgesia in Dopamine2 Receptor Knock-out Mice: Evidence for a Tonically Active Antiopioid System

Dopamine (DA) systems are intimately involved with opioid actions. Pharmacological studies suggest an important modulatory effect of dopamine and its receptors on opioid analgesia. Dr. Gavril Pasternak and his research team at the Memorial Sloan-Kettering Cancer Center have now examined these interactions in a knock-out mouse model in which the D2 receptor has been disrupted. Loss of D2 receptors enhances, in a dose-dependent manner, the analgesic actions of the mu analgesic morphine, the kappa1 (k1) agonist U50,488H and the k3 analgesic naloxone benzoyl-hydrazone. The responses to the delta opioid analgesic [d-Pen2, d-Pen5]enkephalin were unaffected in the knock-out animals. Loss of D2 receptors also potentiated spinal orphanin FQ/nociceptin analgesia. Antisense studies using a probe targeting the D2 receptor revealed results similar to those observed in the mouse knock-out model. The modulatory actions of D2 receptors were independent of final sigma receptor systems because the final sigma agonist (+)-pentazocine lowered opioid analgesia in all mice, including the D2 knockout group. Thus, D2 receptors represent an additional, significant modulatory system that inhibits analgesic responses to mu and kappa opioids. King, M.A., Bradshaw, S., Chang, A.H., Pintar, J.E., and Pasternak, G.W. Potentiation of Opioid Analgesia in Dopamine2 Receptor Knock-Out Mice: Evidence for a Tonically Active Antiopioid System. J Neurosci., 21(19), pp. 7788-7792, October 1, 2001.

(1R)-2-[3R,4S)-3-Methyl-4-(N-phenyl-N-propionylamino) piperidin-1-yl]-1-phenylethyl p-bromobenzoate and N-{(3R,4S)-1-[(2S)-2-(4-bromo-phenyl)-2-hydroxyethyl] -3-methyl-piperidin-4-yl} -N-phenylacrylamide

Two brominated derivatives (both titled compounds) of the potent opioid, cis-b-hydroxy-3-methylfentanyl (ohmefentanyl) were studied and their absolute configuration were determined to assign the proper configuration of two of these stereoisomers and the compounds have the same stereochemistry at two of the three asymmetric C atoms. Ohmefentanyl is an extremely potent analgesic exhibiting high selectivity for the m-opioid receptor. It is one of the 'super potent' analogs of fentanyl that is more potent in producing antinociception than was predicted on the basis of its m-receptor affinity. With three asymmetric C atoms, the compound has eight possible stereoisomers. Four, two pairs of optical isomers, of the eight possible steroisomers would have cis arrangements of the substituents on C3 and C4. When the two pairs were separated, one pair was found to be 5.3 times more potent than the other and 6300 times more potent than morphine. The more active pair was referred to as ohmefentanyl. A second sample, designated as RTI-4614-4 was determined to be a mixture of all four cis isomers and was shown to be 25,000 times more potent than morphine. In view of the differing activities and isomeric compositions of ohmefentanyl and RTI-4614-4, it was clearly necessary to resolve ohmefentanyl into its four stereoisomers. Therefore two brominated derivatives of ohmefentanyl (titled compounds) were synthesized (Brine et al., J. Med. Chem., 38, 1547, 1995) to resolve the stereochemistry of ohmefentanyl. In this paper the absolute configuration of these two bromo-derivatives are reported. This study will help in the design of new opioid ligands for further development of better opioid therapies. Deschamps, J.R., George, C. and Flippen-Anderson, J.L. Acta Cryst., C58, pp. o362-o364, 2002.

Decreased Expression of the Transcription Factor NURR1 in Dopamine Neurons of Cocaine Abusers

NURR1 affects the transcriptional regulation of the dopamine transporter (DAT), a cocaine-sensitive gene. In this study, investigators looked at the postmortem brains of individuals who had died from cocaine overdoses and compared these to drug-free controls that were closely matched for age, sex, postmortem interval, and tissue pH. The investigators first confirmed that the NURR1 gene was expressed in the same regions of the brain as the phenotypic marker of dopamine neurons, the DAT gene. They observed NURR1 mRNA within almost every mid-brain dopamine cell. When they compared the NURR1 mRNA and protein levels in the ventral tier of the substantia nigra of cocaine abusers versus control tissue, they found very low levels of NURR1 in the cocaine abusers. Since other studies have shown that NURR1 regulates DAT expression In Vitro, they examined the level of DAT mRNA in the ventral tier dopamine cells and found that the level of DAT mRNA in cocaine abusers was 70-75% of the level observed in the control tissue. However, VMAT2, a distinct transporter protein expressed in high abundance in dopamine neurons, but not known to be regulated by NURR1, was unaltered in the tissue of cocaine abusers. This suggests that the decrease in NURR1 and DAT expression observed in cocaine abusers is gene-specific and not a general pathologic process within the dopamine neurons. Since DAT is a major site of action for cocaine in the brain, cocaine-induced decreases of DAT transcription could represent an important component of the compensatory mechanisms that occur as a result of chronic drug exposure. Bannon, M.J., Pruetz, B., Manning-Bog, A.B., Whitty, C.J., Michelhaugh, S.K., Sacchetti, P., Granneman, J.G., Mash, D.C., and Schmidt, C.J. Proc Natl Acad Sci, 99(9), pp. 6382-6385, April 30, 2002.

Synapses are Fundamental in Processing Information within the Central Nervous System

One of the hallmarks of synaptic transmission is its plasticity: neural activity channeled through synapses modulates the efficacy of subsequent transmission. Temporally correlated activity may also modulate neuronal networks by creating and eliminating synapses. Such use-dependent plasticity may provide a cellular mechanism for the encoding of persistent memories. Dr. Yukiko Goda and her colleagues at the University of California at San Diego have pioneered the technique of photoconductive excitation of individual neurons cultured on a silicon chip and demonstrated that activity-dependent morphological synaptic plasticity occurs by video-imaging GFP-actin at individual synapses. A single tetanus transiently moved presynaptic actin toward and postsynaptic actin away from the synaptic junction. Repetitive spaced tetani induced glutamate receptor-dependent stable restructuring of synapses. The presynaptic actin redistributed and new puncta indicative of active synapses were formed within 2 hours. Their results indicate that activity-dependent presynaptic structural plasticity facilitates the formation of new active presynaptic terminals. These data are among the first to be reported as a result of NIDA's CEBRA (Cutting Edge Basic Research Award) mechanism, which focuses on innovative science. Colicos, M.A., Collins, B.E., Sailor, M.J., and Goda, Y. Remodeling of Synaptic Actin Induced by Photoconductive Stimulation. Cell, 107, pp. 605-616, 2001.

Synaptic Mechanisms Explain Nicotine's Prolonged Effects

Scientists studying nicotine's effects on the brain have faced a puzzling question: Why do dopamine (DA) cells projecting from the ventral tegmental area (VTA) to the nucleus accumbens continue to release dopamine for more than an hour In Vivo, after even a single exposure to nicotine, when the nicotinic acetylcholine receptors (nAChRs) on those cells desensitize rapidly? Now, investigators at the University of Chicago report that this phenomena can be explained by examining the system of inhibitory and excitatory neurons providing inputs to the VTA dopamine neurons. Drs. Huibert D. Mansvelder, J. Russel Keath, and Daniel S. McGehee undertook a series of electrophysiological studies examining the GABAergic and glutamatergic inputs to the VTA dopamine neurons. They report that each cell type is modulated by a different type of nicotinic receptor. "7 type receptors on the excitatory glutamatergic inputs mediate enhancement of glutamatergic transmission during exposure to nicotine, and desensitize less than the nicotinic receptors on the GABAergic neurons. The nicotinic receptors on the GABAergic neurons appear similar to those on the dopamine neurons themselves, and likely contain "4 and $2 subunits, according to the investigators. Initially, nicotine activates these receptors, but then they rapidly desensitize. As a result, the inhibitory input to the dopamine neurons becomes suppressed, leading to disinhibtion of the dopamine neurons. The receptors on the GABAergic neurons remain desensitized for a longer period of time than those on the glutamatergic neurons. The net effect of these different receptor behaviors on various synaptic inputs in the system is thus a shift toward excitation of the dopamine neurons which persists for some time. Indeed, as the Chicago research team points out: "If the DA neuron is depolarized sufficiently, the enhancement of glutamatergic transmission can induce a long-term potentiation of these inputs, as we reported previously." The investigators also looked at the role of endogenous acetylcholine (ACh) inputs. Their findings supported the idea that desensitization of nAChRs by nicotine or chronic ACh can reduce the excitatory drive on the GABAergic neurons. Again, this action effectively disinhibits the dopamine neurons and increases their excitability. Thus, different nAChR's with different spatial and temporal responses located on different cell types constitute components of a system whose behavior leads to the release of dopamine, which, in turn, appears correlated with the reinforcing properties of nicotine. This work was supported by the Netherlands Organization for Scientific Research, the National Institute on Drug Abuse, the National Institute of Neurological Disorders and Stroke, and the Brain Research Foundation. Mansvelder, H.D., Keath, J.R. and McGehee, D.S. Synaptic Mechanisms Underlie Nicotine-Induced Excitability of Brain Reward Areas, Neuron, 33, pp. 905-919, March 14, 2002.

Coordinated Release of ATP and GABA May Offer Novel Synaptic Flexibility to Hypothalamic Circuits

Adenosine triphosphate (ATP) is recognized as the major energy source within cells; however it also can serve as a neurotransmitter, in a process called "purinergic" transmission. (Adenine is a purine.) As a neurotransmitter, ATP interacts with a family of receptors generally known as P2X receptors. Although these receptors are expressed throughout the central nervous system, little is known about them, because of difficulties in detecting small amplitude synaptic currents, and because of a lack of P2X receptor antagonists. One brain region which expresses P2X receptors prominently is the hypothalamus. NIDA grantee Dr. Lorna Role undertook the task of determining if P2X receptor expression underlies a significant contribution of ATP to synaptic transmission in the hypothalamus. Through electrophysiological In Vitro studies on neurons from the lateral hypothalamus, she found evidence for robust and reliable action potential-dependent ATP P2X receptor-mediated purinergic transmission in preparations of embryonic chick neurons and in preparations of neurons from postnatal mice; moreover, based on recordings from pairs of pre- and postsynaptic neurons, she found that postsynaptic currents mediated by ATP and GABAA receptors originate from coordinated release of ATP and GABA from individual neurons. From her findings, Dr. Role proposes that the GABA and ATP may be stored within the same synaptic vesicle. Dr. Role notes that GABA is the primary inhibiting neurotransmitter in the hypothalamus, and that inhibitory circuits in the lateral hypothalamus may play an important role in feeding. During development, however, GABA can exert depolarizing effects on neurons. Dr. Role suggests that during development, the coordinate release of ATP and GABA may provide important synergy for excitatory influences on the developing synapses. At mature synapses, according to Dr. Role, activation of GABAA receptors may enhance calcium influx through P2X receptors. Thus, the coordinated release of the two transmitters may serve as a means for synaptic tuning of hypothalamic circuits in developing versus mature animals. Understanding lateral hypothalamic circuits is crucial because they integrate autonomic and limbic information, and are important in behavioral arousal. This work demonstrates the importance of examining the effects of multiple systems simultaneously and the properties of the systems at different temporal stages. Young-Hwan, J. and Role, L.W. Coordinated Release of ATP and GABA at In Vitro Synapses of Lateral Hypothalamic Neurons, The Journal of Neuroscience, 22(12), pp. 4794-4804, June 12, 2002.

cAMP Mediated Transcription Mapping during Morphine Withdrawal

cAMP response element binding protein (CREB) is a genetic transcription factor which recognizes the cAMP-response element (CRE) promoter site. A variety of stimuli can lead to CREB activating the transcription of target genes. For example, chronic opiate exposure upregulates CREB in the locus ceruleus (LC) of the brain. This has long been of interest since CREB also has been implicated in long term synaptic processes associated with learning and memory. But does morphine dependence actually influence transcription mediated by the CRE response element? To answer this question, Dr. Eric Nestler of the University of Texas Southwestern Medical Center and a team of investigators studied morphine dependent transgenic animals containing a marker to tell if and where transcription occurred during naltrexone precipitated withdrawal. The transgenic animals contained a genetic construct in which a "reporter" is made in such a way that it is under the control of CRE-consensus elements. In other words, they used the "reporter" molecule, beta-galactosidase, as a visual indicator for CRE-mediated transcription. They also used cell markers to identify specific neuronal cell populations where changes in gene expression occurred. They observed changes in gene transcription in several brain regions consistent with expected physiological effects, such as those concerned with arousal, reward, mood, and affective responses. For example, they found morphine withdrawal affected expression of beta-galactosidase in a mixed population of cells in the nucleus accumbens. The authors suggest that CRE-mediated transcription during withdrawal may be a homeostatic mechanism responding to molecular adaptations which developed during chronic opiate exposure. Observing such transcription also may serve as a marker of neuronal plasticity during withdrawal, according to the research team. Shaw-Lutchman, T.Z., Barrot, M. Wallace, T., Gilden, L., Zachariou, V., Impey, S., Duman, R.S., Storm, D. and Nestler, E.J. Regional and Cellular Mapping of cAMP Response Element-mediated Transcription during Naltrexone-precipitated Morphine Withdrawal. Journal of Neuroscience, 22(9), pp. 3663-3672, May 1, 2002.

Chemokines as Modulators of Pain

Chemokines are recently studied peptides interacting with glia and neurons in the brain and also on blood leukocytes. It has been known for awhile that opioids inhibit the chemotactic activity of these chemokines, thus impacting on their neuroimmune functions. This group has focused on the chemotaxis of peripheral blood monocytes. Herein they find that opioids activate chemokine receptors in these cells leading to immunosuppression. They postulate this crosstalk between these two receptors may also lead to desensitization of the opioid receptors so that chemokines build up in inflammatory states. Further studies should elucidate the basic mechanism of how opioids and chemokines function together in pain or inflammatory processes. The chemokines use G protein-coupled receptors to regulate the migratory and proadhesive responses of leukocytes. Based on observations that G protein-coupled receptors undergo heterologous desensitization, they have examined the ability of chemokines to also influence the perception of pain by cross-desensitizing opioidGprotein-coupled receptors function In Vitro and In Vivo. The authors found that the chemotactic activities of both m- and d-opioid receptors are desensitized following activation of the chemokine receptors CCR5, CCR2, CCR7, and CXCR4 but not of the CXCR1 or CXCR2 receptors. Furthermore, they also found that pretreatment with RANTES/CCL5, the ligand for CCR1, and CCR5 or SDF-1a /CXCL12, the ligand for CXCR4, followed by opioid administration into the periaqueductal gray matter of the brain results in an increased rat tail flick response to a painful stimulus. Because chemokine administration into the periaqueductal gray matter inhibits opioid-induced analgesia, the authors propose that the activation of proinflammatory chemokine receptors down-regulates the analgesic functions of opioid receptors, and this enhances the perception of pain at inflammatory sites. Szabo, I., Chen, X.H., Xin, L., Adler, M.W., Howard, O.M.Z., Oppenheim, J.J., and Rogers, J. Heterologous Desensitization of Opioid Receptors by Chemokines Inhibits Chemotaxis and Enhances the Perception of Pain. PNAS, 99, pp. 10276-10281, 2002.

Inhibition of Morphine-potentiated HIV-1 Replication in Peripheral Blood Mononuclear Cells with the Nuclease-resistant 2-5A Agonist Analog, 2-5A(N6B)

The compound, 2-5AN6B is a nuclease-resistant 2-5A agonist analog. In these studies using morphine-treated peripheral blood mononuclear cell cultures researchers found that 2-5AN6B, but not AZT or saquinavir completely reversed morphine-induced potentiation of HIV-1 infection. Treatment of peripheral blood mononuclear cell cultures with 2-5AN6B increased RNase L activity in control peripheral blood mononuclear cell cultures, in morphine-treated peripheral blood mononuclear cell cultures and in morphine-treated, HIV-1-infected peripheral blood mononuclear cells. The researchers also found that 2-5AN6B enhanced expression of both IFN-" and IFN-(. The increased expression of IFN-( was associated with a significant increase in expression of RANTES and monocyte chemotactic protein-1, chemokines that may inhibit HIV-1 infection by blocking viral attachment to CCR2 and CCR5 co-receptors. By adding 2-5AN6B to the cultured morphine-treated peripheral blood mononuclear cells, the researchers were able to reverse the morphine-potentiated HIV-1 infection of the cells. Homan, J.W., Steele, A.D., Martinand-Mari, C., Rogers, T.J., Henderson, E.E., Charubala, R., Pfleiderer, W., Reichenbach, N.L., and Suhadolnik, R.J. J Acquir Immune Defic Syndr.,30(1), pp. 9-20, May 1, 2002.


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