ibogaine signals addiction genes and methamphetamine alteration of long-term...

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Ann. N.Y. Acad. Sci. 965: 28–46 (2002). © 2002 New York Academy of Sciences.

Ibogaine Signals Addiction Genes and Methamphetamine Alteration of Long-Term Potentiation

EMMANUEL S. ONAIVI,a,b SYED F. ALI,c SANIKA S. CHIRWA,d JEAN ZWILLER,e NATHALIE THIRIET,f B. EMMANUEL AKINSHOLA,g AND HIROKI ISHIGUROb

aDepartment of Biology, William Paterson University, Wayne, New Jersey 07470, USAbMolecular Neurobiology Branch, IRP, NIDA-NIH, Baltimore, Maryland 21224, USAcNeurochemistry Laboratory, Division of Neurotoxicology Research, National Center for Toxicological Research/FDA, Jefferson, Arizona 72079, USAdDepartment of Anatomy and Physiology, Meharry Medical College, Nashville, Tennessee 37308,USAeINSERM U338, Center de Neurochemimie, Strasbourg, FrancefMolecular Neuropsychiatry, NIDA-NIH, Baltimore, Maryland 21224, USAgDepartment of Pharmacology, Howard University College of Medicine, Washington D.C. 20059, USA

ABSTRACT: The mapping of the human genetic code will enable us to identifypotential gene products involved in human addictions and diseases that havehereditary components. Thus, large-scale, parallel gene-expression studies,made possible by advances in microarray technologies, have shown insightsinto the connection between specific genes, or sets of genes, and human diseas-es. The compulsive use of addictive substances despite adverse consequencescontinues to affect society, and the science underlying these addictions in gen-eral is intensively studied. Pharmacological treatment of drug and alcohol ad-diction has largely been disappointing, and new therapeutic targets andhypotheses are needed. As the usefulness of the pharmacotherapy of addictionhas been limited, an emerging potential, yet controversial, therapeutic agent isthe natural alkaloid ibogaine. We have continued to investigate programs ofgene expression and the putative signaling molecules used by psychostimulantssuch as amphetamine in in vivo and in vitro models. Our work and that of oth-ers reveal that complex but defined signal transduction pathways are associat-ed with psychostimulant administration and that there is broad-spectrumregulation of these signals by ibogaine. We report that the actions of metham-phetamine were similar to those of cocaine, including the propensity to alterlong-term potentiation (LTP) in the hippocampus of the rat brain. This actionsuggests that there may be a “threshold” beyond which the excessive brainstimulation that probably occurs with compulsive psychostimulant use resultsin the occlusion of LTP. The influence of ibogaine on immediate early genes

Address for correspondence: Emmanuel S. Onaivi, Department of Biology, William PatersonUniversity, 300 Pompton Road, Wayne, NJ 07470. Voice: 973-720-3453; fax: 973-720-3730.

[email protected]@intra.nida.nih.gov

29ONAIVI et al.: IBOGAINE PHARMACOGENETICS

(IEGs) and other candidate genes possibly regulated by psychostimulants andother abused substances requires further evaluation in compulsive use, reward,relapse, tolerance, craving and withdrawal reactions. It is therefore temptingto suggest that ibogaine signals addiction gene products.

KEYWORDS: ibogaine; pharmacogenomics; pharmacotherapy: psychostimu-lant; gene chip; addiction; methamphetamine; haplotypes; SNPs; signal trans-duction; animal model

INTRODUCTION

The compulsive use of addictive substances despite adverse consequences con-tinues to affect society, and the science underlying these addictions in general re-mains poorly understood. This is because the pharmacological treatment of drug andalcohol addiction has largely been disappointing. The good news is that the mappingof the human genetic code will enable us to identify potential gene products involvedin human addictions and other diseases that have hereditary components. Indeed thecompulsive use of addictive substances leading to neuroadaptation activates signaltransduction pathways that regulate changes in gene expression.1 Thus, genetic vul-nerability and environmental factors are important determinants in transitions fromcasual drug use to compulsive use of addictive substances.2 Addiction is therefore apolygenic disorder that affects the brain and peripheral tissues and does not followsimple Mendelian monogenic inheritance. While our knowledge of the pharmacoge-nomics and pharmacogenetics of addiction has yet to produce therapeutic targets totreat drug addicts, few findings of positive allelic association rarely withstand repli-cation.3 A genome-wide, parallel search to determine at-risk genes and programs ofgene expression patterns using quantitative trait loci (QTLs) mapping of rodentstrains and DNA microarray analysis reveals at best genetic heterogeneity and com-plexity of addictions.3,4 Much effort recently has been focused on pharmaco-genomics and addiction to opiates,3 alcoholism and substance abuse,5 geneticinfluences on smoking, and candidate genes.6,7 Others have focused on the applica-tion of DNA microarrays to study human alcoholism,8 or changes in non-human pri-mate nucleus accumbens gene expression after chronic cocaine treatment,9 andlarge-scale analysis of gene expression changes during acute and chronic exposureto ∆9-THC in rats.10

Addiction is therefore a biological process11 and a brain disease12 that is notcaused by one single gene, but rather involves multiple vulnerable genes,3 with sig-nificant contribution from environmental factors, including the trigger by the avail-ability of abused substance(s). Thus, as the usefulness of pharmacotherapy ofaddiction has been limited, an emerging potential, yet controversial therapeuticagen, is the natural alkaloid, ibogaine. We have continued to investigate programs ofgene expression and the putative signaling molecules used by ibogaine and psycho-stimulants such as amphetamines in in vivo and in vitro models. Our work and thatof others reveal that complex but defined signal transduction pathways are associat-ed with the compulsive use of addictive substances and that the putative regulationof these signals by ibogaine may be linked to its broad spectrum of action on numer-ous biological systems.

30 ANNALS NEW YORK ACADEMY OF SCIENCES

MOLECULAR SIGNALS OF ADDICTION GENES

After over four decades of intensive research on the brain reward pathways in thedevelopment and the compulsive use of addictive substances, the dopamine circuitsand hypothesis remain a difficult area and perhaps a major problem and hindranceto progress in unraveling the biology of addiction. For example, benzodiazepines,barbiturates, and inhalants do not increase dopamine levels, as do psychostimulantsand opiates and most importantly other systems like gamma amino butyric acid(GABA), cholinergic, glutamatergic, adrenergic systems, cannabinomimetic andcAMP transduction pathways, which are involved in reward and relapse circuits inthe brain. Additional evidence from knockout mice demonstrates that mutant micewithout dopamine receptors and transporters continue to self-administer psycho-stimulants, whereas mutant mice without metabotrophic glutamate receptor(mGluR5) are completely unresponsive to cocaine even though their dopaminergicsystem remains intact. The link between learning and memory processes in addictionfurther indicates the complexity and polygenic nature of substance abuse vulnerabil-ity. Transition into compulsive drug use induces the expression of a number of genesfrom several biochemical pathways resulting in neuroadaptive changes in the brain.1

Abused substances, as shown in FIGURE 1, are known to stimulate transcription ofspecific genes by activating the transcriptional regulators through appropriate tran-scriptional factors. Our working hypothesis is that ibogaine induces a synchronizedstate of programmed signal transduction and gene expression resulting in its broad-spectrum anti-addictive potential. As we reviewed recently,13 a number of complexgenetic markers and signaling molecules are stimulated or inhibited by transcription-al regulators and factors associated with specific programs of candidate genes. In-deed, a number of studies indicate that most drugs of abuse indirectly stimulate/inhibit transcription regulators and factors by a tangled, but precise web of signaltransducers (FIG. 1).1,13 Therefore, addictive substances are known to activate signaltransduction pathways that regulate gene expression in the brain and peripheral or-gan systems in human and perhaps animal models. Addiction from drug abuse is nowviewed as a brain disease, but we have to add that peripheral mechanisms, which arelargely ignored, also contribute to the neurobiological disturbances and behavioralpathologies, such as compulsive drug use and craving.1,14 Increasing evidence dem-onstrates that most drugs of abuse indirectly stimulate transcription of specific genesby increasing intracellular cAMP, which inevitably results in activation of multi-functional protein kinases and phosphorylation of several cellular proteins1 (FIG. 1).

The current understanding of how transcription factors regulate gene expressionis poor; however, the best characterized in the brain are the Fos/Jun family of imme-diate early gene (IEG) transcription factors and the CREB family of transcriptionfactors as possible mediators of the effects of drug abuse on the regulation of geneexpression.1,15 Another example is the claim that ∆FosB might be a relatively sus-tained molecular “switch” that contributes to a state of addiction.20 Some of the stud-ies reported here indicate that acute ibogaine injection induces expression of theIEGs, egr-1 and c-fos, in the mouse brain16 (TABLE 1). This may be one way in whichibogaine is able to block the action of abused substances that cause addiction. Thelist of brain circuits involved in drug abuse continues to grow. Significant amountsof data implicate some role for dopaminergic pathways in drug addiction, but other


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circuits like opioid peptides and GABA are also involved (FIG.1). Furthermore, it hasbeen demonstrated that the cannabinoid system may be involved in the neuronal pro-cesses underlying relapse to cocaine-seeking behavior in the rat model17. It is inter-esting that cannabinoid receptors, which are encoded by the CB1 gene, are one ofthe most abundant neuroreceptors in the mammalian brain. There is increasing evi-dence that cannabinoids inhibit neurotransmitter release via specific presynapticCB1 receptors.18 One of the recent advances in cannabinoid research is the identifi-cation of a series of endogenous modulators called endocannabinoids (e.g., ananda-mide, 2-arachidonyl glycerol and noladin ether).19 It appears that this previouslyunknown cannabinoid physiological control system might be a key player in the neu-ral mechanism of addiction. Cannabinoid receptor gene may be a piece of the geneticpuzzle in addiction and perhaps may be part of the solution. The cannabinoid recep-tor antagonist (SR 141716) prevents acquisition of drinking behavior in alcohol-pre-ferring rats, and SR 141716 has been found to block the acquisition, but not theexpression, of cocaine- and morphine-induced conditioned place preference inrats.21 These observations support the hypothesis that neurobiological events under-lie the acquisition to a greater extent than those events mediating the expression ofthe positive-reinforcing properties of abusive drugs, including alcohol. Taken to-gether, it appears that drugs of abuse alter intracellular messenger pathways intoshort- and long-term changes in target gene expression by the appropriate signal-reg-ulated transcription factors (FIG. 1). Unfortunately, the relationship between theseintracellular signaling and transcriptional regulations of the candidate genes in-volved with compulsive use of drugs is unclear at the moment.

The application of DNA microarrays as discussed here to study candidate genesthat may be involved in human addiction has generated numerous genes whose rolein addiction may not be readily obvious. There are problems with pharmacogenom-ics and large variability in microarray data, as discussed below. The important ques-tions therefore are: (1) What is the significance of so many genes being transcribedafter compulsive substance use? and (2) How can we pin down what are the impor-tant genes that induce the drug-dependent state or a relapse state after cessation fromcompulsive substance use? The preliminary preclinical and clinical data obtained

TABLE 1. Effect of ibogaine on c-fos and egr-1 gene expression

egr-1 c-fos

Control Ibogaine Control Ibogaine

NAc 5.78 ± 0.61 7.50 ± 0.37* 1.45 ± 0.13 1.95 ± 0.41

CPU 7.23 ± 0.26 12.4 ± 0.72** 1.15 ± 0.06 2.40 ± 0.50*

FCx 9.65 ± 0.75 17.4 ± 1.3** 1.23 ± 0.13 3.30 ± 0.14*

DG 3.87 ± 0.31 11.5 ± 0.81** 1.53 ± 0.29 6.65 ± 0.76*

CA1 6.50 ± 0.52 7.70 ± 0.52 1.60 ± 0.26 2.75 ± 0.26*

CA3 4.87 ± 0.40 9.10 ± 1.10* 1.78 ± 0.26 2.40 ± 0.67

Septum 3.90 ± 0.71 8.10 ± 0.83** 1.47 ± 0.07 5,27 ± 0.77

*p < 0.01; ** p < 0.001, following Newmann-Keul’s t-test after ANOVA.


with ibogaine demonstrate a broad spectrum of action on multiple systems that mayaccount for its anti-addictive potential.

GENETIC APPROACHES FOR THE ANALYSIS OF ADDICTION

While significant advances have been made in demonstrating the role of specificgene products (or lack of) in animal models of addiction, Progress has been slow inidentifying genes that affect risk of addiction in humans. In addition the identifica-tion of the genes involved in complex polygenic traits may be difficult because theprinciples learned about single-gene single-disease inheritance may not be relevantto polygenic and multifactorial inheritance as in polysubstance abuse and addic-tions.24 It is even more difficult to pin down the cellular transcription cascades withthe behavioral manifestation of compulsive drug-seeking and drug-taking despitehorrendous consequences.20 Several studies have also linked genetic and environ-mental factors combining to influence the process by which repeated exposure leadsto addiction. For the genetic factors a large number of vulnerable candidate genesmay be involved in addictions, with no single gene sufficient or necessary to causeaddiction. For the environmental factors, the contribution of the environment is ob-vious in that genetic vulnerability can only lead to addiction when the substance ofabuse is readily available.3 Because drug addiction is a genetically complex andpolygenic disease involving different stages, a number of genetic approaches for theanalysis of addiction continue to evolve from pharmacogenetics to pharmacogenom-ic approaches in which the entire genome and its expression are evaluated to scanand map genetic variation across the entire human genome. Others have proposedthat micro- and minisatellite polymorphisms play a role in the expression of manygenes.24 The human genome is highly polymorphic and mutations in the human ge-nome lead to genetic polymorphisms in the population. The frequency of mutationin transgenes is now receiving considerable attention, since proteins synthesized inrecombinant DNA biological systems are subject to genetic alteration through mu-tation and selection.23 The complex polygenic trait in addiction may be multifacto-rial, since both genetic and environmental factors play a role in the cause.23,24 As aresult, these genes exists in the population with many functional alleleomorphicvariants. Thus, there is a reasonable chance that an individual will inherit a thresholdnumber of functional variants beyond which there is an appreciable effect on thephenotype.24 Therefore, disease markers in the human sequences can be targetedwith a complete genetic map of haplotypes and single nucleotide polymorphisms(SNPs). Haplotypes are sets of genetic markers that are close enough on a chromo-some to be inherited together. Similarly, SNPs act as markers in genome-wide scan-ning for disease-causing genes to be traced through generations to identify geneticdifferences between people affected and unaffected by addiction. So, SNPs and hap-lotypes can be used to unravel the genetic differences that make some people moreaddiction-prone than others. It can therefore be deduced that a SNP and haplotypicgenetic maps across the entire genome will be of great use in finding genes that areinvolved in addiction. However, using SNPs alone can be difficult and expensive,partly because it is currently hard to trace individual SNPs in the genome. But usinghaplotypes eases some of these difficulties, and makes it easier to identify variationin the genome because each haplotype contain a group of SNPs that tend to be in-


herited together. These haplotypes are found by analyzing genotype data to create aseries of markers that have linkage disequilibrium in a gene.

Genotyping uses genomic DNA, PCR-RFLP, sequencing, and the like to examinethe association between genes and addiction. For example, a genetic association forcigarette smoking behavior was reported between allele 9 of a dopamine transportergene polymorphism (SLC6A3-9) and lack of smoking, late initiation of smoking,and length of quitting.25,26 After extracting DNA from peripheral blood, SLC6A33’, a variable number of tandem repeat (VNTR) genotypes were determined by PCRamplification and agarose gel electrophoresis by these investigators. In anotherstudy, alcohol and aldehyde dehydrogenase genotyping was undertaken to examinethe allele frequencies at the ADH1, ADH2, and ADH3 loci among Alaska natives.27

This is because alcohol and aldehyde dehydrogenase involved in alcohol metabolismare polymorhic and account for the ethnic differences in alcohol metabolism. Thestudy’s findings suggest that the Alaska natives are not protected from the risk of al-coholism in the way that Asians who possess the ALDH2*2 genotype are consideredto have a negative risk factor. Nor do there appear to be any generalized differencesbetween Alaska native alcoholics and members of the general population with re-spect to alcohol and aldehyde dehydrogenase.27 The role of ibogaine (if any) in thegenetics of alcohol and smoking addiction is provocative at best in the absence ofmicroarray data before and after treatment of any these addictions with ibogaine.

The use of microarrays to study gene expression in alcoholism and drug addictionhas yielded reams of data, but there is currently little information on the effect ofibogaine, if any, on “addiction genes.” But cell and animal studies have consistentlyindicated that changes in gene expression in the brain appear to be responsible fortolerance, dependence, craving, and relapse to substance abuse.28,29 Thus, DNA hy-bridization arrays for gene expression analysis30 has been applied to addiction re-search to simultaneously examine changes in the expression of thousands of genesboth in animal models of addiction and human addict samples. This is accomplishedby microarray hybridization of immobilized gene-specific sequences on a solid-statematrix (e.g., nylon membranes, glass microscope slides, or silicon/ceramic chips)with labeled nucleic acids from human addicts and controls who are not addicts. Thesamples could also be from animal models of various stages of addiction and theirrespective controls. A number of studies have applied DNA microarrays to study hu-man drug and alcohol addiction with a significant number of genes and gene prod-ucts identified. The application of DNA microarray to study human alcoholism usedpostmortem human brain tissue from the frontal cortex that had been exposed to thechronic effects of alcohol31 and nerve cells32 that have been exposed to a few daysof ethanol. The results demonstrated that 163 genes from the 4000 genes in the ar-rays differed by 40% or more from the frontal cortex tissue between alcoholics andnonalcoholics.31 These investigators found that addiction to alcohol alters gene ex-pression, which may change the programming and circuitry of the superior frontalcortex.31 Chronic cocaine-mediated changes in non-human primate nucleus accum-bens gene expression have been demonstrated by cDNA hybridization array analy-sis.33 In another study, large-scale cDNA microarrays were employed to assess geneexpression changes during acute and chronic exposure to ∆9-THC in rats in compar-ison to vehicle-treated animals.34 These studies and others further support and con-firm that changes in gene expression, particularly in the brain and perhaps in theperipheral organ systems, contribute to drug and alcohol and addiction.


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FIGURE 1 shows a number of the target genes whose expression is altered byabused substances. FIGURE 2 shows a prototypical example of a differential gene ex-pression pattern using an array probed with RNA from different treatment, and thescattergram example of the arrays is shown in FIGURE 3. The important question iswhether ibogaine has an effect in reversing the genetic effects of alcohol and drugabuse that contribute to addiction. Once a gene has been identified as playing somerole in addiction, gene targeting by homologous recombination makes it possible tocreate knockout animals without specific gene(s) of interest. Gene-targeting strate-gies therefore provide an avenue for studying the function of a gene after its deletion.Thus, transgenic mice or knockout mice, with null mutation of specific genes ob-tained by homologous recombination have been used to study the relevance of somegenes in different aspects of drug dependence and addiction.35 A number of exam-ples of genes inactivated using gene knockout techniques or mice transgenicallyoverexpressing specific genes, antisense oligodeoxynucleotide (ODN); and othergenetic manipulation strategies have been applied to study various aspects of addic-tion with a view to finding pharmacogenetic treatment for substance addiction. Micelacking genes for the 5HT1B, PKCγ, GABAA receptor subunits, dopamine receptorsubtypes, transporter and the vesicular monoamine transporter, tyrosine hydroxylase(Th), hypoxanthine-guanine phosphoribosyltransferase (HPRT), P-glucoprotein-mdr1a, p53, opioid receptor systems, pre-proenkephalin, isoforms of CREB, TGFα,IGF-I, fyn, neuronal acetylcholine receptor subunits (nAChR),35 substance P, andcannabinoid CB1 receptor have all been studied for their potential influence on al-cohol and drug response traits (FIG. 1).22 Some problems have been identified withthe use of transgenic animals, as discussed below; but the development of tissue-spe-cific inducible knock-out mice and other conditionally regulated transgenics maybecome useful in validating the role(s) of these candidate genes in addictions.

FIGURE 3. Scattergram of the example of the array results from FIGURE 2.


Mapping of quantitative trait loci (QTLs) is growing in importance in contributingto the identification of chromosomal “hot spots” that contain specific genes that in-fluence all aspects of substance addiction. The specific regions of the chromosomesthat contain specific genes that have been implicated in alcohol and drug addiction isshown in FIGURE 4. These chromosomal “hot spots” are by no means exhaustive be-cause other abused substances influence genes on chromosome 1, 2, 7, and Y thathave not been listed in FIGURE 4. These unbiased searches of the whole genome usingvarious markers indicate the complexity of alcohol and drug addiction.

METHAMPHETAMINE ALTERS LONG-TERM POTENTIATION

Ibogaine in a number of studies has been shown to influence multiple central ner-vous system pathways, which has led us to postulate that the broad spectrum of ac-

FIGURE 4. Addiction “hot spots” in human chromosomes. These “hot spots” are asso-ciations between genetic variants and substance abuse. ABBREVIATIONS: CCK, cholecystoki-nin; DRD1-5, dopamine receptor D1-5; CCKAR, cholecystokinin receptor type A; DAT1,dopamine transporter; HTR1B, serotonin receptor 1B; CNR1, cannabinoid receptor type 1;OPRM1, opioid receptor mu 1; DBH, dopamine beta-hydroxylase; VMAT2, vesicularmonamine transporter 2; CCKBR, cholecystokinin receptor B; TH, tyrosine hydroxylase;TPH, trypophan hydroxylase; EAAT2, glutamate transporter; ALDH2, aldehyde dehydroge-nase 2; HTR2A, serotonin receptor 2A; AHH, aryl hydrocarbon hydroxylase; 5HTT, serotonintransporter; CYP2A6, cytochrome P450 subfamily 2A6; COMT, catechol-O-methyltrans-ferase; CYP2D6, cytochrome P450 subfamily 2D6; MAOA, monoamine oxidase A.


tion of ibogaine-like compounds may be important in their anti-addictionpotential13,36 The previous study in the mouse model of withdrawal indicated thatibogaine reversed the withdrawal aversions from chronic administration of psycho-stimulants like cocaine.36 The follow-up studies described below examined the invivo and in vitro action of another psychostimulant, methamphetamine. Metham-phetamine is an indirect-acting catecholaminergic agent used in the treatment of hy-perkinetic states in children or in the management of obesity.37 It is a simplechemical derivative of ephedrine, an active ingredient of several cold remedies. Syn-thesis of methamphetamine is simple and straightforward, a factor that probably ac-counts for its widespread availability. This is of particular concern sincemethamphetamine produces prominent euphoric effects, a property that renders itsubject to abuse38. Inappropriate doses or prolonged use of methamphetamine causepsychosis, disturbances in perception, and memory dysfunction.39 The exact neuralmechanisms that underlie these behavioral responses are not completely known.However, animal studies have shown that amphetamines induce phosphorylation ofseveral proteins, including cAMP-responsive element binding protein (CREB). Theyalso induce immediate early gene expressions, particularly those involving theCREB pathway.40 One intriguing feature is that CREB phosphorylation and targetgene activations require both DA1 dopaminergic and NMDA glutamatergic receptoractivations.41 Both NMDA receptors, and CREB pathways have been implicated incellular processes involved in cognition. For example, memory consolidation isthought to require the stabilization of changes in synaptic efficacy through new syn-aptic growth. A cascade of biochemical events involving excitatory amino acid re-ceptors, CREB-mediated gene expressions, and new protein synthesis triggers thesestructural changes.42, 43

Thirty Long-Evans hooded rats were used in the study and randomly divided intogroups of 10 animals. Two groups were given methamphetamine (1 or 10 mg/kg i.p.)every day for 90 days, whereas the third group was treated with saline solution dur-ing the same period. All animals were subjected to behavioral assessments prior toand at weekly intervals during treatments. In brief: spontaneous locomotor activityand stereotype behavior were measured in computer-controlled-activity cagesequipped with infrared emitters and detectors. After 90 days of treatment, animalswere prepared for the removal of both hippocampal lobes. One set of hippocampallobes was collected in vials according to treatment groups and stored in liquid nitro-gen for the molecular analysis. The other hippocampuses were sliced to preserve agreater portion of the Schaffer collateral-commissural projections terminating in thesame plane in CA1 regions and were randomly selected and transferred onto a re-cording slice chamber for the electrophysiological studies (a total of 8–10 sliceswere examined per animal group). Glass microelectrodes filled with saline solutionwere used to record population spikes (PS) in the CA1 region. Baseline responseswere activated at 0.2 Hz, whereas 100 Hz for 1 sec was used for LTP. The populationspikes were amplified via a 2-channel AC differential pre-amplifier (band pass: 100Hz to 1 KHz). Rectangular pulses (60–100 µA, 0.1 msec) were delivered throughphotoelectric current isolation units regulated by a Grass stimulator. The amplifiedvoltage potentials were viewed on an oscilloscope and taped for off-line analysis.The changes in DA1 and DA2A receptor gene expression in the hippocampus wasperformed by semi-quantitative competitive reverse transcriptase-polymerase chain


reaction (RT-PCR), using constructed PCR mimics as normalized internalstandards.44

FIGURE 5 shows the behavioral effects of methamphetamine during the chronictreatment. The spontaneous locomotor activity and sterotype behaviors were signif-icantly increased, demonstrating the well-established pyschostimulant sensitization,probably attributable to increased release of dopamine. Overall, therefore, the chron-ic treatment with methamphetamine produced significant increases in DA1 andDA2A gene expression in rat hippocampus, as shown in FIGURE 6. In terms of bio-electrical recordings, the basic criterion for detection of LTP was that there be a post-tetanic response enhancement of at least 40% above baseline population spike, sus-

FIGURE 5. Effects of chronic methamphetamine on locomotor activity and stereotypebehavior. Methamphetamine was administered 10 min prior to behavioral testing, and eachtrial was run for 20 min, respectively. Data represents the mean + SEM (n = 10) in each case.Both locomotor (A) and stereotype (B) behaviors were found to be significantly different,from 2–3 weeks (p <0.05, ANOVA with Dunnett’s multiple comparisons tests).


tained beyond 15 min. In this regard, high-frequency titanic stimulation readily pro-duced LTP in 5 of 5 hippocampal slices obtained from the control animals (PSamplitude: pre-tetanus, 0.95 + 0.02 mV; 30 min post-tetanus, 1.45 + 0.04 mV; N =5). In contrast, 2 of 6 hippocampal slices obtained from the low-dose group exhibitedLTP (PS amplitude: pre-tetanus, 1.2 + 0.2 mV; 30 min post-tetanus 1.42 + 0.11 mV;N = 6), and none of the slices from the high-dose group developed LTP (PS ampli-tude: pre-tetanus, 0.8 + 0.03 mV; 30 min post-tetanus, 0.68 0.04 mV, N = 4 slices).We report that similar to the actions of cocaine, methamphetamine increased bothstereotypic and spontaneous locomotor activity and increased the expression of DA1and DA2A receptor in the hippocampus of these animals. Furthermore, while synap-tic transmission remained unaltered, the propensity to induce long-term potentiation(LTP) in the hippocampus was altered by methamphetamine. We deduced that if theeffects of methamphetamine were mediated via increased release of dopamine, thenthe data would suggest that there might be a “threshold” beyond which excessive ac-tivation of the dopaminergic system, as probably occurs with chronic methamphet-amine administration, results in antagonism of LTP expression. This is consistentwith reports that methamphetamine is internalized into nerve terminals via thedopamine transporter and that the process results in a competitive blockade ofdopamine re-uptake.45 Within the terminal, methamphetamine presumably displacesdopamine in vesicles, and this contributes towards the enhanced release of dopamineinto the synaptic cleft. Methamphetamine itself activates dopamine receptors indi-rectly, albeit with a lower efficacy. The foregoing actions invariably heighten behav-ioral responses mediated by dopamine receptor activations, including enhancedlocomotion and stereotypy.

The significant increases in DA1 and DA2A transcripts is suggestive of an in-creased synthesis of DA1 and DA2A receptors transduced by coupling to guaninenucleotide–binding G-protein that regulate the activities of adenylate cyclase and

FIGURE 6. The amount of specific mRNA for DA1 and DA2A receptors. Data showthat DA1 and DA2A gene expression increased in the hippocampal cells in rats chronicallytreated with methamphetamine, relative to control aimals given saline. Asterisk denotes sig-nificant differences (p <0.05, ANOVA with Dunnett’s multiple comparison tests).


phospholipase C.45 Specifically, the stimulation of DA1 receptor increases cAMPand potentiates the activation of certain voltage-gated Ca2+ channels and multifunc-tional Ca2+/calmodulin-dependent kinase II (CaMKII). In addition it is known thatCREB is activated via phosphorylation by cAMP-dependent protein kinase A andCamKII, which in turn causes synergistic increases in CREB-mediated transcrip-tions and gene expression. While it was unexpected to discover a decrease in the pro-pensity to induce LTP in slices obtained from methamphetamine-treated rats in thehippocampus, some reports suggest that dopaminergic systems partly mediate LTP.Indeed the expression of long-term potentiation in the striatum of methamphet-amine-sensitized rats which was suppressed by NMDA receptor antagonist was re-ported by Nishioku et al.47 and that tetanic stimulation induced long-termdepression (LTD) of the field in the striatum of saline-treated rats. LTP in the hip-pocampus and LTD in the cerebellum are well-established models of synaptic plas-ticity that require the activation of certain classes of AMPA and NMDA glutamatereceptors, which has recently been linked to the recall of the memories of addic-tion.48 A single dose of cocaine exposure in vivo was shown to induce long-term po-tentiation in VTA dopamine neurons, which may be involved in the early stages ofthe development of drug addiction.49 There are extrinsic dopaminergic inputs to thehippocampus, such as the mesolimbic dopaminergic pathway, that interact with DA1and DA5 dopaminergic receptors, presumably on CA1 pyramidal cells.50 Also, theuse of retrograde fluorescent tracers showed that dopaminergic fibers projectingfrom the VTA and substantia nigra areas innervate the CA1 field. Thus the inductionand maintenance of LTP seems to be dependent on de novo RNA transcription andprotein synthesis.51 The signal transduction mechanisms that initiate these processesmay involve the dopamine/cAMP/CREB cascades.46 Moreover, dopamine receptorantagonists abolish the maintenance of LTP across CA3/CA1 synapses.52,53 In par-ticular, blockade of either DA1 or DA2 receptor decreased the magnitude of the late-phase of LTP, two or more hours after induction.52 In contrast, LTP-producing high-frequency stimulation of the CA1 Schaffer collateral synapses is facilitated by DA1/DA5 dopamine receptor activation.52,53 How then do we explain the observed lowLTP production in treated rats? Presuming that methamphetamine actions were me-diated via dopamine/cAMP/CREB and gene expression in the hippocampus, it isfeasible that some of these mechanisms are the same as those required for LTP.54 Ifthese mechanisms are already “maximally” activated by the drug, additional activa-tion as a consequence of tetanic stimulations to produce LTP may not occur, result-ing in occlusion of LTP development53. Interestingly, greater LTD was observed incocaine-treated animals.49 In summary, chronic methamphetamine administrationproduced behavioral sensitization in rats that may be associated with enhanced ex-pression of DA1 and DA2A gene expression and a modification to the characteristicsof synaptic transmission in the hippocampus. The data obtained add to the growingbody of evidence indicating that modification of the hippocampus by drugs of abusemay play a role in altering nervous system plasticity, which may partly underlie dys-functional mnemonic behaviors. It appears that the nature of recall of memories forthe compulsive drug use and relapses that sustain addiction may be associated withthe neural circuits involved with learning and memory. It is therefore tempting tospeculate that the effects of ibogaine on psychostimulant abuse may involve actionon the molecular processes associated with signals of addiction genes in learning andmemory.


IBOGAINE PHARMACOGENOMICS

Because pharmacogenomics may be defined as the science of the development ofdrug therapies based on knowledge of the human genome, pharmacogenetic data areneeded to determine a role (if any) of ibogaine pharmacogenomics. The pharmacoge-netic approach that accesses genetic variation between individual’s responses to drugsand in animal studies with both selected lines and inbred strains have provided clearevidence of genetic influence. Although, the genes identified by the pharmacogeneticapproach indicate the presence or influence of genes in addiction, identifying the spe-cific genes that make an animal sensitive or resistant to a given drug effect is diffi-cult.22 However, the advent of new powerful molecular techniques including genome-wide scanning and microarray approaches that lead to the identification of specificgenes or regions of the chromosomes will allow the function of candidate genes to beanalyzed. The promise of pharmacogenomics in addiction research will pay off whentherapies based on human genome using variation in DNA and gene expression areused to find new therapeutic targets. The determination of at-risk programs of geneexpression patterns involved in addiction may ultimately lead to the identification ofnew drug targets from the analysis of the genome and expression data.

IBOGAINE INDUCES IMMEDIATE EARLY GENES

There are excellent reviews on the immediate early genes (IEGs)54,55 and otherinducible transcription factors (ITFs).56 IEGs are a set of transcription factors thatare expressed rapidly after stimulation of a variety of cell surface receptors. It haspreviously been reported that ibogaine injection induces expression of IEGs, egr-1and c-fos, in mouse brain.16 Whether the IEGs could be a common biochemicalmechanism(s) that underlie addiction is not established, but all drugs of abuse acti-vate IEGs in the brain with differences in brain regions activated. Thus, differentclasses of abused substances activate IEGs in different brain regions and in somecases overlapping areas of IEG activation have been reported.55 While common neu-ropharmacological mechanisms responsible for the activation of IEG expression inforebrain involve dopaminergic and glutamatergic systems, this may not be limitedto addictive substances. There are no reports of addiction to ibogaine, which also ac-tivates IEGs, as shown in TABLE 1. Furthermore, not all addictive substances activatethe release of dopamine in the brain, and therefore the expression of IEGs may beviewed as one of the markers for neuronal activation in the CNS that may not be thecommon biochemical mechanisms in addiction.

A number of studies have shown that different groups of addictive substances in-creases the expression of certain IEGs or ITFs in the brain, although each drug ap-pears to induce a particular neuroanatomical pattern of expression, suggestingactivation of distinct sets of neurons.16,55 For example, patterns of c-fos expressionhave been studied after administration of alcohol and surprisingly, these patterns aresignificantly different from the ones produced by cocaine.55 Since these genes encodetranscription factors, their activation is likely to play a key role in the transduction ofshort-lived environmental signals into long-lasting changes in the cell function. Acti-vation of c-fos in the brain can be induced by a diverse group of stimuli.58 The induc-ibility of c-fos can now be regarded as a tool to study neuronal activation in different


systems in the brain, while egr-1 has been shown to be involved in neuronal plasticityof hippocampus, especially in LTP.59 The ibogaine study reported here used adultC57 mice from the NCTR breeding colony. The mice were injected with saline oribogaine, 50 mg/kg, i.p. (n = 6–8), and 30 minutes later animals were given an over-dose of pentobarbital (50 mg/kg, i.p) and perfused transcardially with 0.9% saline (10ml), followed by 1% paraformaldehyde in phosphate-buffered saline (pH 7.2, 50 ml).The brains were frozen in isopentane at –40oC, and stored at –80oC. Coronal tissuesections (10 µm thick) were thaw-mounted onto gelatin-coated glass slides and storedat –80oC. In situ hybridization with riboprobe for egr-1 and c-fos was performed aspreviously described by Thiriet et al.60 with [35S]uridine triphosphate (UTP)-labeledRNA probes. In brief: sections were delipidated, acetylated, and prehybridized for 10min at 60oC in 50% formamide, 1 × SSC (150 mM NaCl and 15 mM sodium citrate,pH 7.0), dehydrated and air-dried. Thirty microliters of the labeled probe, diluted to60,000 dpm/µL with hybridization buffer (50% formamide, 4 × SSC, 10% dextransulfate and 10 mM dithiothreitol), were placed on tissue sections and covered withcover slips. Hybridization was carried out overnight at 52oC. Hybridization mediumwas then washed off and the sections were washed twice in 50% formamide, 1 × SSCat 55oC for 1 hr, followed by two washes in 2 × SSC (5 min, room temperature). Sec-tions were incubated in a 10-mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl,1 mM EDTA, and 6.10−3 U/ml RNase for 30 min at 37oC. The slides were then rinsed,dehydrated, and exposed to X-ray film for 2–4 days. To obtain quantitative results,densitometry was determined with a Biocom 2000 image analyzer and the resultswere expressed in kilobecquerel per gram tissue, using [14C]microscales for calibra-tion as previously described.60

Acute injection of ibogaine produced the induction of IEGs, erg-1 and c-fos, inselected regions of mouse brain (TABLE 1). In situ hybridization for egr-1 showed aprominent high signal in caudate-putamen (Cpu), frontal cortex (FCx), septal area,as well as in the CA3 and dentate gyrus (DG) subfields of hippocampus. The egr-1mRNA was less induced in nucleus accumbens (NAc) and hippocampal CA1. Sim-ilar treatment of ibogaine increased c-fos mRNA in Cpu, FCx, and septum, as wellas DG and CA1 region of hippocampus. No prominent induction of egr-1 and c-foswas found in other regions of the brain. Quantitative analysis revealed that ibogaineinduced statistically significant expression of egr-1 and c-fos in those areas of thebrain when compared to controls (TABLE 1). It is noteworthy that induction of bothgenes was highest in DG (197% and 334% increase for egr-1 and c-fos, respective-ly). IEG expression was only induced by about 30% in the Nac, an increase that didnot reach statistical significance in the case of c-fos induction. The data show thatacute ibogaine injection induced IEGs, egr-1 and c-fos, in different regions of themouse brain. It has been reported that stimulants like cocaine or amphetamines in-duce IEGs, egr-1 and c-fos, as well as the transcription factors, AP-1 and NFκB, indifferent brain regions.16 Because of the complex pharmacology and broad spectrumof action of ibogaine, this activation of IEGs may be linked to increase in dopamin-ergic, serotonergic, and glutamatergic neurotransmission.16 Evidence that glutamatemay be more essential and central than dopamine in addiction was obtained fromknockout mice.57 This is because in dopamine receptor subtypes and transporterknockout mice, cocaine remains addictive, whereas in the knockout mice, withoutmetabotrophic glutamate receptor (mGluR5), the mutant mice are completely unre-sponsive to cocaine, even though their dopaminergic system remains intact.57


CURRENT PROBLEMS IN PHARMACOGENOMICS

Some of the current problems in pharmacogenomics are summarized below.

• lack of complete genetic maps of SNPs, SSLPs, and haplotypes that can pre-dict variation across the entire human genome and be capable of identifyingdisease genes (e.g., addiction genes);

• the requirement of accurate and cheap genotyping;

• problems associated with compensation in gene knockout animals;

• site-specific action in the brain;

• changes in levels or processing of proteins or peptides (these are thought to bemore relevant to function than changes in gene expression, or the productionof new mRNAs; it is known that changes in some mRNA levels do not alwaysproduce changes in proteins);

• lack of unifying biochemical mechanism for addiction;

• ethical considerations (e.g., the use individual versus pooled samples); and

• large variability in array data.

This list is by no means exhaustive as analysis of the human genome continues withthe promise that specific therapies may be tailored to individual problems in the fu-ture, particular in the field of substance abuse.

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