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Director's Report to the National Advisory Council on Drug Abuse
September, 1995

Research Findings

Basic Research

Cloning and Functional Characterization of a k3-Related Opioid Receptor:

Dr. Gavril W. Pasternak of Memorial Sloan-Kettering Cancer Center and of Cornell University Medical College and his research team have identified a putative opioid receptor from mouse brain (KOR-3), belonging to the G protein-coupled receptor family, that is distinct from the previously cloned µ, d, and k receptors.

Assignment of the clone to the opioid receptor family derives from both structural and functional studies. Its predicted amino acid sequence is highly homologous to that of the other opioid receptors, particularly in many of the transmembrane regions, where long stretches are identical to µ, d, and k1 receptors. Both cyclazocine and nalorphine inhibit cAMP accumulation on COS-7 cells stably expressing the clone. Northern analysis shows that the mRNA is present in brain but not in a number of other organs. Southern analysis suggests a single gene encoding the receptor. A highly selective monoclonal antibody directed against the native k3 receptor recognizes, in Western analysis, the clone expressed in COS-7 cells. The in vitro translation product is also labeled by the antibody. Additional clones reveal the presence of several introns, including one in the second extracellular loop and another in the first transmembrane region.

Antisense studies with an oligodeoxynucleotide directed against a region of the second extracellular loop reveal a selective blockade of k3 analgesia in vivo that is not observed with a mismatch oligodeoxynucleotide based upon the antisense sequence. The µ, d, and k1 analgesia is unaffected by this antisense treatment. Antisense mapping of the clone downstream from the splice site in the first transmembrane region reveals that six different antisense oligodeoxynucleotides all block k3 analgesia. In contrast, only one of an additional six different antisense oligodeoxynucleotides directed at regions upstream from this splice site is effective. This strong demarcation between the two regions raises the possibility of splice variants of the receptor. An additional clone reveals an insert in the 3' untranslated region. It is concluded that the antibody and antisense studies strongly associate KOR-3 with the k3-opioid receptor, although it is not clear whether it is the k3 receptor itself or a splice variant. (Yin-Xian Pan, Jie Cheng, Jin Xu, Grace Rossi, Elissa Jacobson, Jennifer Ryan-Moro, Andrew I. Brooks, Gary E. Dean, Kelly M. Standifer, and Gavril W. Pasternak. Cloning and Functional Characterization Through Antisense Mapping of a k3-Related Opioid Receptor. Mol. Pharmacol. 47:1180-1188 (1995))

PCP Induced Permanent Brain Damage:

Dr. John Olney (Washington University) is examining the neurotoxicity in the rat brain produced by antagonists of the NMDA (N-methyl-d-aspartate) subtype of glutamate receptor, such as phencyclidine (PCP), ketamine, or MK-801 (dizocilpine). The vacuolar injury (low dose) or death (high dose) of neurons in several corticolimbic regions of the adult rat brain is hypothesized by Dr. Olney to model schizophrenia, and NMDA receptor hypofunction has been proposed (by Dr. Olney and others) as a possible mechanism in schizophrenia.

Two studies focused on factors that influence vulnerability of rats to the NMDA antagonist neurotoxicity. In the first study (Farber, Wozniak, Price, Labruyere, Huss, St. Peter & Olney, Biological Psychiatry, in press), they found that this neurotoxic syndrome (now called the "Olney lesion" by most other workers in this field) has an age dependency resembling schizophrenia, with age of onset of full susceptibility being early adulthood. In the second study (Farber & Olney, 1994, Society for Neuroscience Abstracts), they confirmed their previous data showing that adult female rats are more sensitive than males to NMDA antagonist neurotoxicity, but extended this finding by demonstrating that during pregnancy they become hyposensitive.

The ability of a high dose of PCP to kill neurons in rat brain was described for the first time (Corso, Wozniak, Sesma & Olney, 1994, Society for Neuroscience Abstracts). A single 50 mg/kg IP dose in adult female rats killed many neurons in the posterior cingulate and retrosplenial cortices; occasionally it killed neurons in other neocortical and limbic regions. They also found that a high dose of MK-801 in adult male mice causes a permanent memory acquisition deficit (Brosnan-Watters, Wozniak, Nardi, Olney & Fix, 1994, Society for Neuroscience Abstracts). Scopolamine provides partial protection, and pentobarbital complete protection, against MK-801 induced neuronal necrosis (Wozniak, Nardi, Corso & Olney, 1995, Society for Neuroscience Abstracts). The necrotic action of NMDA antagonists does not appear to involve apoptotic cell death processes (Ishimaru, Der, Tenkova, Sesma, Thurston & Olney, 1995, Society for Neuroscience Abstracts).

Stress and Sensitization in Animals:

Current research being conducted by Dr. Sydney Antelman at the University of Pittsburgh is directed to studying the hypothesis that sensitization is a time-dependent phenomenon. He and his collaborators have shown that cocaine administration to animals sensitized to the limits of their systems results in an "on-off" oscillatory pattern of physiological, neurochemical and behavioral responses. These results suggest that a host of systems - both neuronal and hormonal - when maximally sensitized, respond to further stimulation by "toggling" back and forth. This has implications for improving our understanding of cycling illnesses such as recurrent depression, manic disorders, and drug addictions.

Ibogaine: Preclinical Studies:

Dr. Stanley Glick (Albany Medical College) is studying the effects of ibogaine and ibogaine congeners on cocaine and morphine self-administration in the rat. He has shown that ibogaine blocks the rewarding effects of cocaine and morphine in the animals for several days after the motoric effects had dissipated, and that these effects last for up to 27 days, depending upon the dose. Marked decreases in dopamine levels were seen in brain levels using microdialysis methodology, indicating that the effects appear to be correlated. Dr. Glick has developed a sensitive (20 ng/ml) and highly specific GCMS analytical method for measuring ibogaine in biological samples. Using this technique, he has reported that ibogaine disappears rapidly from rat brain (40 mg/kg IP injection resulted in 3.3 ug/g in brain at 1 hour but only 190 ng/g at 19 hours). He is currently developing a method to measure noribogaine levels.

Finally, Dr. Glick reported that ibogaine (100 mg/kg/day IN x 3) resulted in bilaterally symmetrical, parasaggital strips of Purkinje cell degeneration in the rats. The degeneration was similar, but more severe, than Molliver and O'Hearn reported. A lower dose (40 mg/kg/day IP x 3) did not produce these effects.

Dopamine Receptors and Reinforcement:

Dr. James Belluzzi (University of California, Irvine) has been investigating the involvement of different dopamine receptor subtypes in the mediation of reinforcement (primary vs. conditioned vs. classical). Using a series of D1 (SKF 82958, SCH 23390) D2 (N-0923, [+]-PHNO, pergolide) and D3 compounds (7-OH-DPAT, PD-128,907), Dr. Belluzzi has concluded that cocaine-like self-administration is most closely mimicked by D1 agonists and that D1 receptors have a primary role in cocaine place preference and classical conditioning phenomena.

Prenatal Cocaine: Effects on the Offspring:

Dr. Larry Middaugh (Medical University of South Carolina) has been investigating the effects of prenatal cocaine in rats on the developing fetus and through the lifespan of the offspring. Dr. Middaugh has found that prenatal cocaine produces long-term alterations in the offspring's neural systems which utilize dopamine as the transmitter. This was seen by studies which showed a greater response to D2 agonists in the cocaine-treated offspring as compared to placebo-treated control offspring. Neither D1- nor D2-binding was altered in these cocaine-treated offspring, further suggesting an alteration in their neural systems rather than a change in their receptor binding.

Prenatal Cocaine: Effects on the Offspring:

Dr. Charles Mactutus (University of Kentucky) used a novel vascular port model to inject IV cocaine daily to pregnant rats and measure arterial sampled plasma levels of cocaine and metabolites in the dams. Studies in the offspring indicated an attentional disorder in the weanlings that apparently dissipated by 6 months of age. However, when these animals were given a challenge dose of cocaine at that time, they produced a markedly exaggerated behavioral response to the cocaine (sensitization).

Phospholipase A2 Modulation of the Dopamine Transporter:

In recent years arachidonic acid has been found to inhibit sodium dependent substrate transport including the dopamine transporter. Since dopamine stimulation has recently been reported to induce arachidonic acid release as a signal transduction pathway, it is conceivable that cocaine induced arachidonic acid release could indirectly inhibit dopamine uptake, in addition to the direct inhibition of dopamine uptake by cocaine. NIDA grantee Dr. Paul S. Berger of the University of California, San Francisco and his coworkers have observed that activation of phospholipase A2, the major enzyme which releases arachidonic acid, by local injections of the drug melittin into the VTA leads to a persistent sensitization of cocaine possibly by activating a dopaminergic signal transduction pathway.

Consistent with this hypothesis they have found that co-administration of quinacrine (a phospholipase A2 inhibitor) with cocaine prevents the development of sensitization to cocaine. These results are described in a manuscript in press in the journal Psychopharmacology and may be potentially useful in developing novel cocaine pharmacotherapies.

Opiates and Neonatal Respiratory Function:

Morphine-6-beta-D-glucuronide (M6G), an active metabolite of morphine, causes respiratory depression similar to the parent compound. However, in neonatal animals, the potency of M6G with respect to respiratory depression increases with aging. As there are no developmental changes in the distribution of M6G in the brain, an alteration in opioid receptors may explain these differences. Drs. Laine Murphey and George D. Olsen, in a recent short communication, report developmental changes of mu receptors in the brain stem of neonatal guinea pigs and the significance of increased mu receptors in the augmented potency of M6G respiratory effects during the first week after birth. (L.J. Murphey & G.D. Olsen, Developmental Brain Research 85:146-148,1995.)

Marijuana and Pregnancy:

NIDA supported researchers, Dr. S.K. Dey and his associates from the University of Kansas Medical Center, Kansas City, recently reported that both brain- and spleen-type cannabinoid receptor mRNAs are expressed in the preimplantation mouse embryo; whereas in the mouse uterus, only the brain-type cannabinoid receptor mRNA is expressed. They also observed that both oviduct and uterus had the capacity to synthesize the putative endogenous cannabinoid ligand, anandamide, suggesting the preimplantation mouse embryo and the uterus as targets for cannabinoid ligand-receptor signaling. Although the significance of these findings especially in the preimplantation mouse embryo with the cannabinoid binding sites several fold higher than those in the brain needs to be determined, these findings are still important from the public health view/concern. From these results, the P.I. suggests that the reported adverse effects of cannabinoid use or abuse seen during pregnancy and in uterine disorders could be due to an aberrant expression of the endogenous ligand in the reproductive tract and of the receptors in the embryo and uterus. (S.K. Das, B.C. Paria, I. Chakraborty and S.K. Dey, Proc. Natl. Acad. Sci. 92: 4332-4336, 1995; B.C. Paria, S.K. Das and S.K. Dey, Proc. Natl. Acad. Sci., in press.)

Excitotoxic Insult Due to Ibogaine Leads to Delayed Induction of NADPH-diaphorase and Neuronal NOS in Purkinje Cells.

The cover of NeuroReport, vol. 6, #12, August, 1995, featured plates of immunohistochemical data from an accompanying journal article by Elizabeth O'Hearn, M.D., Peisu Zhang, and Mark Molliver, M.D. of Johns Hopkins University School of Medicine. Mature cerebellar Purkinje cells do not normally express neuronal nitric oxide synthase (nNOS) or NADPH-diaphorase activity, but this study demonstrates the induction of these enzymes in response to ibogaine treatment. Ibogaine treatment leads to the rapid, dose-dependent degeneration of Purkinje cells due to excitotoxic injury; however, the induction of nNOS in surviving Purkinje cells follows a delayed time course. The functional significance of nNOS induction following neuronal injury is unknown. It also remains to be determined whether nNOS expressing Purkinje cells go on to degenerate or whether nNOS is associated with recovery from sub-lethal neuronal injury.

Morphine Stimulates Growth of HIV in Brain Cell Cultures:

In early studies, Peterson and colleagues observed an increase in the production of HIV in co-cultures of humanymphocytes with the addition of either cocaine or morphine. More recently, they have also shown this relationship extends to co-cultures of human microglia with a HIV-growth-culture-clone U1. That is, morphine stimulates HIV growth in this co-culture in the presence of a cytokine-stimulant LPS; this upregulation is naloxone sensitive. In a second study, two new antiviral agents were tested as inhibitors of HIV growth in these cultures and were observed to be more potent than AZT. Morphine amplifies HIV-1 expression in chronically infected promonocytes co-cultured with human brain cells. (J Neuroimmunology 50:167-75, 1994, Peterson, P; Gekker, G; Hu, S.; Anderson, W.R.; Kravitz, F.; Portughese, P.S.; Balfour, Jr., H.H.; Chao, C.C.)

Previous studies have shown that morphine promotes the replication of human immunodeficiency virus (HIV)-1 in peripheral blood mononuclear cell co-cultures. In one recent study the hypothesis that morphine would amplify HIV-1 expression in the chronically infected promonocytic clone U1 when co-cultured with lipopolysaccharide-stimulated human fetal brain cells was tested. Marked upregulation of HIV-1 expression was observed in these co-cultures (quantified by measurement of HIV-1 p24 antigen levels in supernatants) and treatment of brain cells with morphine resulted in a bell-shaped dose-dependent enhancement of viral expression. The mechanism of morphine's amplifying effect appears to be opioid receptor-mediated and to involve enhanced production of tumor necrosis factor by microglial cells.

Antiviral activities of the reverse transcriptase inhibitors U-90152 and 3'-azido-2',3' dideoxythymidine and the protease inhibitor U-75875 were compared in two culture models of human immunodeficiency virus type 1 brain infection. In a model involving acutely infected microglial cells, U-90152 was the most active, whereas in a model using chronically infected promonocytes, U-75875 was the most active. (Anti-Human Immunodeficiency Virus Type 1 Activities of U-90152 and U-75875 in Human Brain Cell Cultures, P.K. Peterson, G. Gekker, S. Hu, and C.C. Chao. Antimicrobial Agents Chemotherapy 38:2465-8;1994.)

Gender Differences in Responses to Acute Cocaine:

Gender differences after acute cocaine administration have received little attention despite the fact that males and females respond differently to many other drugs. Dr. Scott Lukas and colleagues of McLean Hospital and Harvard Medical School have recently reported (in press) the results of a study examining seven male and seven female occasional cocaine users who received either an intranasal dose of cocaine hydrochloride (0.9 mg/kg) or placebo powder. Women studied during the follicular and luteal phase of their menstrual cycle had peak plasma cocaine levels of 77.5±13.6 ng/ml and 61.3±17.5 ng/ml, respectively. Male subjects achieved the highest peak plasma cocaine levels (144.4± 17.5 ng/ml), detected cocaine effects significantly faster than females and also experienced a greater number of episodes of intense good effects or "euphoria." Cocaine-induced tachycardia paralleled the subjective effects and plasma levels. Peak increases in heart rate occurred more rapidly in males. These data suggest that there are significant gender differences in the responses to acute intranasal cocaine.

Alcohol Pretreatment Increases Preference for Cocaine over Monetary Reinforcement:

Dr. Steven Higgins and colleagues at the University of Vermont previously have reported a laboratory procedure in which subjects can choose between either cocaine or a monetary reinforcer. They find that choice of cocaine over the monetary alternative reinforcer decreases as an orderly function of increases in the amount of the monetary reinforcer. This laboratory arrangement was developed as an analog of a current behavioral treatment for cocaine dependence that increases cocaine abstinence by systematically increasing the availability of alternative nondrug reinforcers. Dr. Higgins and his colleagues (in press) have now reported that when subjects were given alcohol prior to experimental sessions, choice for cocaine over the monetary reinforcer was significantly increased. The researchers note that these data are consistent with prior reports that alcohol abuse is associated with poorer treatment outcomes in cocaine abusers. Given that the blood alcohol levels were equivalent to those obtained under conditions of social drinking, Dr. Higgins and colleagues recommend alcohol abstinence for individuals seeking to reduce or eliminate their cocaine use.

Effects of Prenatal Exposure to Cocaine on Infant Cognition and Emotional Regulation:

Dr. Linda Mayes and her colleagues at the Yale Child Center assessed the effects of prenatal cocaine exposure on cognitive and psychomotor performance beyond the newborn period. In a recent study, at 3-months of age, 61 cocaine-exposed infants and 47 non-cocaine-exposed infants participated in an infant-control habituation and novelty responsiveness procedure. The task assessed the organization of looking behavior and attention to determine infants early stages of cognitive processing. The Bayley Scales of Infant Development (BSID) was also administered to assess cognitive and psychomotor development. Based on their results, the authors concluded that a substantial proportion of cocaine-exposed infants were more reactive, or aroused by novel stimuli, than non-cocaine-exposed infants. A marked increase in reactivity, or negative affective state can prevent an infant from attending to stimuli. Increased reactivity may reflect differences in temperament, which may have an impact on parental responsiveness. Starting at 5 months of age, parental responsiveness to an infant can influence later cognitive development. Cocaine-using mothers may already be less responsive to their infants which may further place highly reactive cocaine-exposed infants at risk. The potential of reduced parental responsiveness of cocaine-using mothers and increased reactivity in cocaine-exposed infants can have a profound influence on an infant's cognitive performance. Importantly, these conditions could negatively affect later development of attention regulation and learning ability. (Mayes, et al., Pediatrics, 95(4):539-545, 1995).

Motivational Effects of Marijuana:

At the July 19-20, National Conference on Marijuana Use: Prevention, Treatment, and Research, Dr. Don R. Cherek reported on studies conducted at the University of Texas-Houston which demonstrated that marijuana smoking can produce diminished motivation. This controlled laboratory study measured the acute effects of marijuana smoking among 18-22 year old male subjects who had histories of occasional marijuana use and had 10-12 years of education. Marijuana smoking produced diminished motivation to perform a work task which required increasing effort over one hour sessions. This reduced motivation could only be partially reversed by large increases in the monetary value of the incentives. These effects of marijuana on motivation were directly related to the potency of the marijuana smoked. Future research will examine characteristics of the smoker which may contribute to these effects.

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