Proc. Natl. Acad. Sci. USAVol. 91, pp. 385-389, January 1994Neurobiology

Cellular adaptation to opiates alters ion-channel mRNA levelsScoTT A. MACKLER* AND JAMES H. EBERWINEDepartment of Pharmacology, University of Pennsylvania School of Medicine, 36th and Hamilton Walk, Philadelphia, PA 19104

Communicated by George B. Koelle, June 10, 1993

ABSTRACT The chronic use of several drugs, includingopiates, results in the stereotypical behaviors characteristic ofaddiction. Alterations in gene expression have been associatedwith the use ofthese addictive drugs. Previous studies, however,have been limited to describing changes in amounts ofindividualmRNAs from single tissue samples. Cellular adaptation toopiates, reflected in the regulation of the expression of manydifferent mRNAs, seems likely to contribute to the complicatedbehaviors ofaddiction. The present studies examined coordinatealterations in the amounts ofmultiple mRNAs in the rat striatumand in NG108-15 cells after opioid stimulation or the precipi-tated withdrawal of opioid use. The experimental approachcombined amplification of the poly(A)+ RNA population withreverse Northerm blot analysis to simultaneously characterizethe relative changes in several mRNAs. Morphine treatment ofrats for 5 days was associated with a reduction in the amount ofstriatal RNA for the voltage-sensitive K+ channel without sig-nicant changes in other ion channels. In NG108-15 cellsstimulation with the 8-opiate receptor agonist [D-Ala2,D-Leujenkephalin (DADLE) alone and followed by naloxone(precipitated withdrawal) caused relative changes in the abun-dances ofseveral mRNAs. The composite effects of alterations inthe abundance of multiple mRNAs (and the proteins theyencode) in response to opioid use likely contribute to the devel-opment and maintenance of opiate-mediated behaviors.

Chronic use of opiates results in stereotypical behaviorsassociated with drug addiction, including tolerance and phys-ical dependence. The requirement for chronic drug exposurein the development ofthese behaviors and their characteristicappearances, by analogy to studies of learning in inverte-brates (1), suggest that changes in gene expression contributeto the development of drug addiction (2-4). Additionally,after opioid stimulation or withdrawal, the abundances ofseveral mRNAs are changed in discrete brain regions. Theseinclude mRNAs for c-fos in the striatum (5), tyrosine hy-droxylase in the locus coeruleus (6), and vasopressin andother neuropeptides in the hypothalamus (7) and the striatum(8). In cell culture, inhibition of RNA synthesis in the6-opiate-receptor-expressing NG108-15 cell line (9) has sug-gested a role for gene transcription in the up-regulation of6-opiate receptors (10). In NG108-15 cells, the mRNA for thea subunit of the stimulatory guanine nucleotide bindingprotein (Gs) also increased after exposure to morphine (11).

Previous studies have relied upon quantitation of individ-ual RNAs within a population of RNAs isolated from heter-ogeneous cell populations of the central nervous systemwhile cellular resolution was provided by in situ hybridiza-tion. Unfortunately, in situ hybridization can be used toidentify no more than two or three mRNAs in a single tissuesection. Limited amounts of neural tissue coupled with thepossibility that opiate-regulated mRNAs exist in low abun-dances in neurons complicate the identification of mRNAscritical to the development of tolerance and dependence. It is

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

likely that multiple gene products, rather than a singleprotein, are involved in the development of drug addiction.Relative changes in the expression of these genes may,therefore, affect the abundance of many proteins. Conse-quently, these proteins may act to alter cellular physiologyand result in tolerance and dependence.The present study utilized in vitro amplification of the

poly(A)+ RNA population (12) from the rat striatum andNG108-15 cells to show coordinate changes in the relativeamounts of mRNAs for several genes in response to opioiduse (Fig. 1). The synthesized RNA is amplified in a linearfashion and in amounts proportional to the original levels ofpoly(A)+ RNA [amplified antisense RNA (aRNA); refs. 12and 13]. The aRNA was used as a probe to simultaneouslyscreen cDNA clones encoding known mRNAs, a processcalled reverse Northern blot analysis (14) that generates anexpression profile of mRNA abundances.

MATERIALS AND METHODSSupplies and Reagents. All enzymes were purchased from

Boehringer Mannheim except for avian myeloblastosis virusreverse transcriptase (Seikagaku America, Rockville, MD) andT7 RNA polymerase (Epicentre Technologies, Madison, WI).Radionucleotides were purchased from New England Nuclear.

In Situ Transcription (IST) of Rat Striatal Poly(A)+ RNA.Five adult male Sprague-Dawley rats were made opiate-tolerant by daily subcutaneous implantation of delayed-release pellets ofmorphine for 4 days (75 mg on day 1, 150 mgon day 2, 225 mg on day 3, and 300 mg on day 4; see ref. 3).An identical number of rats were implanted in the samemanner with placebo pellets. On the fifth day, the rats werekilled and the brains were frozen in liquid nitrogen. Coronalsections (11 gm thick) through the striatum were cut with acryostat at -14°C, fixed in4% (wt/vol) paraformaldehyde for5 min, and frozen at -80°C. An oligonucleotide primer[consisting of the T7 RNA polymerase promoter sequencepositioned 5' to an oligo(dT) segment of 18-24 thymidines]was annealed (2 ng/,l) to the RNA in each section at roomtemperature for 16 h (12-15). Equal numbers ofcontrol slideswent through the hybridization procedure without the addi-tion of an oligonucleotide primer (to determine the back-ground level of endogenous priming). The tissue sectionswere washed in 5 x standard saline citrate (SSC) for 6 h andthen in 0.5 x SSC for 1 h. In situ transcription proceeded for60 min at 370C in 150 .l {120 mM KCI/5 mM MgCl2/50 mMTris-HCl, pH 8.3/250 t,M dATP/250 ,uM dGTP/250 ,MTTP/50 uM dCTP/25 ,.Ci[a-32P]dCTP (1 Ci = 37 GBq)/RNAsin (20 units/jd)/avian myeloblastosis virus reversetranscriptase (0.5 unit/A.)}. The synthesis of cDNA wasverified by autoradiography to detect the incorporation of[a-32P]dCTP into cDNA (Fig. 2A). First-strand cDNA tran-scripts were removed from the tissue sections by alkaline

Abbreviations: DADLE, [D-Ala2,D-Leu5]enkephalin; G., stimula-tory guanine nucleotide binding protein; aRNA, amplified antisenseRNA; IEG, immediate early gene; IST, in situ transcription; GFAP,glial fibrillary acidic protein.*Current address: Department of Medicine, Philadelphia VeteransAffairs Medical Center, Philadelphia, PA 19104.

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386 Neurobiology: Mackler and Eberwine

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FIG. 1. Outline of the experimental approach. Poly(A)+ RNA ineither tissue sections or isolated from cell culture is transcribed intocDNA with the T7-polymerase-promoter-containing oligonucleo-tide. The aRNA is hybridized to cDNA blots, and the blots arewashed and exposed to autoradiographic film.

denaturation. The conversion of single-stranded cDNA intotemplate for aRNA amplification is described elsewhere (14).

Pharmacological Treatment of NG108-15 Cells. Similarnumbers ofNG108-15 cells were treated with normal medium(control group), 10 nM [D-Ala2,D-Leu5]enkephalin (DADLE;stimulated group), orDADLE treatment followed by additionof 100 uM naloxone (precipitated-withdrawal group). Opiatetreatment periods were 8, 24, or 48 h (naloxone was added tothe withdrawal groups 8 h prior to the end of the treatmentperiod). The three treatments did not significantly alter thenumber of cells. The cells were harvested and total RNA wasisolated by the GTC/LiCl method (16). Poly(A)+ RNA wasisolated by two passages through a oligo(dT)-cellulose col-umn. The oligo(dT) T7 primer was annealed to the poly(A)+RNA via three cycles of 80°C/ice, and cDNA was preparedas described for aRNA amplification (14).aRNA Amplification. The cDNA templates were suspended

in 10 ,ul and freed of unincorporated dNTPs by drop dialysisagainst 50 ml of double-distilled H20. Equal volumes ofcDNAtemplates, =100o of the total amount, were amplified (14).Transcription proceeded for 3-4 h at 37°C. The size distributionof the aRNA was determined by electrophoresis of =20,000cpm ofincorporated radioactivity from each reaction mixture ina 1.1% agarose/2.5 M formaldehyde denaturing gel (Fig. 2B)followed by drying and film autoradiography of the gel.

Reverse Northern Blot Analysis. This analysis was performed(14) with minor modification. The candidate cDNAs chosen forthis study were those that might be expected to play criticalroles in cellular functioning, that contained the 3' end of thecorresponding mRNA (important because the synthesized

FIG. 2. (A) Appearance of tissue sections containing the striatumfrom a morphine-tolerant rat. IST with the oligonucleotide primerresults in incorporation of [32P]dCTP in cellular-rich regions (Upper).Addition of all components for IST except the oligonucleotide primerresults in much lower levels of synthesis (Lower). (B) The sizedistribution of the aRNA probes is demonstrated by separation in adenaturing 1.1% agarose gel. Approximately equal counts of aRNAobtained from two brain sections and one NG108-15 cell cultureexperiment are shown. Total NG108-15 RNA served as a marker forrRNA. (C) An expression profile employing aRNA from a morphine-tolerant striatal section displays relative differences in hybridizationsignals for several cDNA clones.

aRNA will always contain more of the 3' region ofthe mRNA,see Fig. 1), and that did not contain long poly(A)+ tail regions[to which the poly(U) region of the aRNA would hybridize].

Statistical Analysis. Each autoradiogram was analyzed byscanning laser densitometry. The densitometer readings forglial fibrillary acidic protein (GFAP) (for the IST-derivedaRNA probes) and a retinoic acid receptor (for the aRNAsamples obtained from the NG108-15 cells) were selected asreferences for normalization because of their high relativehybridization signals (14). The quantitated signals were cal-culated as a percentage ofthe internal reference value in eachautoradiogram. Values for each cDNA in a single blot werenormalized using the internal standard, thus permitting directcomparison ofrelative values among several blots. The meanof each group was used in a two-tailed Student's t test witha P value of

Proc. Natl. Acad. Sci. USA 91 (1994) 387

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FIG. 3. Results of scanning densitometry for expression profiles from rat striatal sections. Each bar (mean ± SEM) in this and Figs. 4 and5 presents data from at least three experiments. Individual values were calculated as the percentage of the signal for GFAP. (A) Ion channels.m, morphine; c, control; GABAA, ytamino-butyric acid type A receptor. (B) IEGs. (C) G-protein-coupled receptors. P1i and (32, (81- andP2-adrenergic receptors, respectively.

Upper Right). 32P-labeled aRNAs from these samples wereused to probe Southern blots containing multiple cDNA clones(Fig. 1 Lower). Fig. 2 shows one result from combining ISTwith aRNA synthesis using rat striatal poly(A)+ RNA. Acoronal section of the striatum from a rat made tolerant tomorphine shows incorporation of [a-32P]dCTP in all cellular-rich regions. This level of incorporation was higher than thatobtained in comparable sections without the addition of anoligonucleotide to initiate cDNA synthesis (Fig. 2A). The sizedistribution of the synthesized aRNAs ranged from a fewhundred bases to >2000 bases (Fig. 2B). It is important to notethat none ofthe synthesized aRNAs could result from the highlevel of endogenous background shown in Fig. 2A becauseonly the specifically primed material would contain the T7RNA polymerase promoter site. Hybridization ofthe aRNA toseveral candidate rat cDNAs revealed that the relative signalsdiffer between many of these clones. Fig. 2C contains anexample of such a result from the striatum of a morphine-tolerant rat. Differing intensities of the hybridization signalsare apparent in this autoradiogram, with more intense signalsoverlying the cDNAs for GFAP and the voltage-sensitive Na+and Ca2+ channels and with less intense signals overlying thecDNA for the voltage-sensitive K+ channel. The expressionprofiles represent specific hybridization ofindividual RNAs inthe aRNA population to their corresponding cDNA clones.Hybridization signals were not observed for blots containingvector plasmid DNA without cDNA inserts or prokaryoticDNA used as a molecular size marker.

Reverse Northern Blot Analysis of Striatal aRNA in Morphine-Treated Rats. Comparison ofthe expression profiles after 5 daysof morphine administration with placebo-treated rats revealeda significant decrease in the hybridization signal for the Kv2voltage-sensitive K+ channel (from 83 ± 24% to 2 ± 2%; P <0.05; Fig. 3). There was also a reduction in hybridization ofaRNA to the Kv1 voltage-sensitive K+ channel (from 156 +57% to 50 ± 25%), without clear changes in other ion channelsstudied. During the same period of drug exposure, a trend

toward an increase in c-fos mRNA levels (55 ± 43% comparedto 156 ± 7%) without alterations in c-jun mRNA or selectedmembers of the G-protein-receptor group occurred.

Analysis of NG108-15 Cells After DADLE Treatment. Ex-periments were next performed in vitro to study a "homoge-neous" population of opiate-receptor-expressing cells and tofurther describe the time course ofchanges inmRNA amountsafter opiate use and precipitated withdrawal. Treatment ofNG108-15 cells with DADLE for 24 h or more results inmaximal 8-opiate receptor down-regulation (17). Precipitatedwithdrawal using naloxone produces maximal receptor up-regulation at 8 h (10). In the present experiments, equalnumbers of cells from these treatment groups were detachedfrom the plates and processed foraRNA amplification (Fig. 1).

Stimulation with DADLE resulted in a reduction in thesignals for the Kvl channels (Fig. 4A; from 9.13 ± 2.82% incontrol cells to 0.36 ± 0.36% at 48 h of treatment; P < 0.05)and Kv2 channels (from 5.36 ± 3.46% to 0.08 ± 0.08% at 48h of treatment). The decline was largest after 48 h ofDADLEapplication. In comparison, the signal for a voltage-sensitiveCa2+ channel remained unchanged while that for a voltage-sensitive Na+ channel increased slightly [statistically signif-icant at 8 h (23.55 ± 7.62% vs. 11.04 ± 2.19%; P < 0.05)]. Inaddition, increases in the hybridization signals were associ-ated with DADLE treatment at all three periods for the asubunit of G, (Fig. 4B) and for the two immediate early genes(IEGs) c-fos and c-jun [Fig. 4C; for c-jun, 58.25 ± 18.34% (P

388 Neurobiology: Mackler and Eberwine

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FIG. 5. Results of scanning densitometry for naloxone-treated NG018-15 cells. Time points at 24 and 48 h represent precipitated withdrawalafter 8 h of DADLE stimulation. (A) Ion channels. (B) G-protein subunit. (C) IEGs.

to 14.87 ± 2.77%; P < 0.05). Naloxone (precipitated with-drawal) also led to an increase in the signal for the Na+ channel[significant at 48 h (35.23 ± 9.99% vs. 11.04 ± 2.19%; P <0.05)], the G, a subunit (not statistically significant), c-fos(15.73 ± 7.32% vs. 1.99 ± 0.98% at 48 h; P < 0.05), and c-jun[29.41 ± 13.63% at 8 h (P < 0.05), 43.86 ± 13.61% at 24 h (P< 0.01), and 48.61 ± 19.47% at 48 h (P < 0.05) compared to1.39 ± 1.28%]. Naloxone treatment alone for 8 h had a greatereffect on c-jun mRNA levels than c-fos mRNA levels.To test whether the above results correlate with the more

standard Northern blot technique, radiolabeled cDNA probesfor G, and c-jun mRNAs were hybridized to a set of blotscontaining equal amounts of total RNA from NG108-15 cells inthe three treatment groups. Both DADLE treatment alone andfollowed by naloxone (precipitated withdrawal) for 24 h in-creased the absolute amount ofG. mRNA compared to control(respectively, 2.27-fold and 1.28-fold). DADLE treatment alone(a 4.56-fold increase) and followed by naloxone (precipitatedwithdrawal) (3.19-fold increase) were also associated with in-creases in mRNA for c-jun after 24 h of treatment.

DISCUSSIONTolerance and withdrawal are the physiological responses tochronic opiate use. Identification of the genes whose expres-sion are regulated by the use of opioid drugs is necessary tounderstand the molecular events underlying tolerance andwithdrawal. Regulation of neuronal mRNA levels by opioiduse almost certainly results from interactions among heter-ogeneous central nervous system cells. The present studyidentifies mRNA molecules that change in abundance inresponse to opioid use (Fig. 6). The tissues examined in-cluded the striatum of the adult rat (an opiate-receptor-richregion with normal synaptic connections maintained amongheterogeneous neuronal populations) and NG108-15 cells (ahomogeneous cell population in which the cellular environ-ment is easily regulated).

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Stimulation of & and ,-opiate receptor subtypes leads tochanges in K+ and Ca2+ conductances in postsynaptic neuronsexamined in vitro (18-20). In the present study, chronic opioidadministration, which is thought to cause opiate-receptordown-regulation in the central nervous system (21), wasassociated with decreases in the relative levels ofmRNAs fortwo K+ channels (Fig. 3A). This result suggests a decrease inthe amount of K+ channel proteins leading to a down-regulation of K+ channel function. Chronic treatment of ratswith morphine resulted in tolerance to opioid-induced in-creases in K+ flux in vitro of locus coeruleus neurons (20).Studies with single opiate-responsive neurons in the locuscoeruleus (18) and individual neurons in the spinal cord (22)have provided electrophysiological evidence for K+ channelactivation and membrane hyperpolarization after acute opioidadministration. The observed decrease in striatal K+ channelmRNA and a potential reduction in K+ channel functioningmay reflect differences between acute or chronic opiate treat-ment, striatal-specific differences in responsiveness, oropiate-receptor-subtype-specific effects. A nonspecific general re-duction in ion-channel gene expression induced by chronicopioid use is not supported by the present data (Fig. 3A).No significant increases in relative levels of c-fos or c-jun

mRNAs occurred after 5 days of morphine treatment (Fig.3B). In accordance with these findings, IEG expressionusually declines within hours of activation in response toseveral types of stimuli (22). Previous experiments investi-gating opioid effects (5) and opioid withdrawal (23) haveshown increases in levels of many IEGs. However, theabsence of significant changes in either c-fos or c-jun mRNAlevels in the present study does not preclude a possibleselective and prolonged activation of c-fos, c-jun, or otherIEG mRNAs that could occur within distinct classes ofneurons in the central nervous system (2).The results of morphine treatment in adult rats are distinct

from DADLE treatment of NG108-15 cells over a period of

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FIG. 6. Overview of results in a hypothetical cell. Opiate administration (M, morphine in the striatum; D, DADLE in NG108-15 cells) andnaloxone-precipitated withdrawal in NG108-15 cells (W) are shown. NM, nuclear membrane; CM, cell membrane. See text for other details.

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8-48 h. This treatment was associated with changes in theabundances of several mRNAs (Fig. 4). Experiments usingthis homogeneous population of opiate-receptor-expressingcells avoids the under representation of opiate-regulatedmRNAs in tissue samples containing diverse types ofneuronsand glia (such as in the striatum). A relative reduction occurredin Kvl and Kv2 mRNA levels, similar to that observed in therat striatum. This early and pronounced decrease in signals forthese K+ channels also seems likely to represent a down-regulation of K+ channels in response to continued opiate-receptor stimulation. In contrast to these fmdings, a significantincrease in relative levels of mRNA for a brain voltage-sensitive Na+ channel occurred after 8 h of DADLE treat-ment. The relative increase in Na+ channelmRNA remains forthe entire 48-h period of study. NG108-15 cells contain only5-opiate receptors (9), whereas the rat striatum contains u-, &-,and K-opiate receptors (24). It is possible that the differenteffects on the relative amounts of Na+ channel mRNA ob-served in this study are due to activation of distinct opiatereceptor subtypes. Alternatively, the increased expressionmay result from the interaction of heterogenous cells withindifferent neuronal and glial environments.The increase in G. mRNA in NG108-15 cells afterDADLE

use is supported by findings from the Northern blot analysisusing acDNA clone for G, to probe NG108-15 RNA and fromprevious experiments (11). The significance of this increasein a G-protein group whose function is not directly altered byopiate use may be due to a compensatory up-regulatorymechanism. These data suggest that increased activity of theinhibitory G protein Gi, the G protein coupled to the 6-opiatereceptor, may lead to an increase in G, mRNA and proteinlevels to counter the increase in Gi activity. The unchangedlevels of rIfRNAs encoding the a subunit of G, and theG-protein-coupled 13i- and P2-adrenergic receptors (Fig. 3C)suggest that heterologous regulation of receptor mRNA lev-els does not occur at the tissue level in the rat striatum bymorphine modulation.

Precipitated withdrawal by naloxone also produces changesin the relative amounts of several mRNA molecules. A trendtoward a decrease in K+ channel mRNAs again appears (Fig.SA) but does not result in as large a reduction in mRNA, asseen with DADLE use alone (Fig. 4A and SA). Naloxoneexposure for 8 h without priorDADLE administration resultedin a small decrease in K+ channel mRNA levels. Naloxonemay thus modulate endogenous activation of K+ channelmRNA in NG108-15 cells. However, opioid pretreatment maylimit this effect of naloxone. Also, an increase in the signal forthe Ca2+ channel occurs with precipitated withdrawal. Theeffects of increases in Ca2+ and Na+ channels (a significantincrease in Na+ channel signal is noted at 48 h) on neuronalactivity suggests a mechanism to explain the neural hyperex-citability, which is characteristic of withdrawal in animal andclinical studies. This may occur by lowering the threshold forinitiation of action potentials in excitable cells that containmore voltage-sensitive Na+ or Ca2+ channels.

Increases in c-fos and c-jun mRNAs occur with bothnaloxone alone and after DADLE treatment (precipitatedwithdrawal) (Fig. 5C). These increases persist for 48 h andmay play a role similar to that hypothesized for IEG activa-tion after opiate-receptor stimulation. In NG108-15 cells,activation of c-fos and c-jun mRNAs occurs with bothDADLE and naloxone application. Previous work supports arole for IEGs in the cellular response to agonist and antag-onist treatments (5, 23, 25).

Fig. 6 summarizes what has been learned from the presentstudies. After opiate-receptor challenge (Fig. 6, position A),as yet ill-defined second messengers (arrow B) elicit a ge-

nomic DNA response resulting in an increase in c-fos andc-jun mRNA levels (position C) as well as other mRNAs(unpublished results). It is also possible that ion-channelmodulation by opiates (arrow H) results in the observedincrease in c-fos and c-jun mRNA levels (arrow I). Theincreased mRNA levels likely result in increased Fos and Junprotein levels (4), which in turn will alter the transcriptionrates of downstream genes (arrow E). The changes that areseen in Ca2+, Na+, and K+ channel mRNA abundances(position F), if reflected in protein level changes (position G),may alter ion flow. This may directly alter mRNA stability ortranscription of selective genes (arrow I).The observed changes in mRNA abundances in expression

profiles are likely a consequence ofaltered transcription rates.However, mRNA degradation may also account for the ob-served results. Regardless of the mechanism, changes in theamounts of several mRNAs will result in altered proteinsynthesis and, consequently, cellular function. The presentstudy shows that cellular adaptation to opiate drug presenceand withdrawal includes changes in the amounts of multiplemRNAs. This correlation between opiate tolerance and al-tered gene expression is an initial step in the analysis ofadaptive processes induced by opiate use. The relative ratiosof these and other untested mRNAs whose abundance isreflective of altered protein levels or function that likelyunderlie the molecular basis of cellular adaptation to opioids.Future studies of the proteins encoded by opiate-regulatedmRNAs, in particular with respect to their altered abundanceratios compared to other molecules, will be required to deter-mine how they are involved in the behaviors ofdrug addiction.

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