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Effects of chronic cocaine administration on dopamine transporter mRNA and protein in the rat
Brain Research 750 Ž1997. 214–222
Research report
Effects of chronic cocaine administration on dopamine transporter mRNA and protein in the rat Sharon R. Letchworth ) , James B. Daunais, Ashlee A. Hedgecock, Linda J. Porrino Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Medical Center BouleÕard, Winston-Salem, NC 27157, USA Accepted 5 November 1996
Abstract Male Sprague–Dawley rats were administered cocaine Ž10, 15 or 25 mgrkg. or vehicle, i.p., once daily for 8 consecutive days and killed 1 h after the last injection. Acute cocaine administration produced dose-dependent increases in spontaneous locomotor activity. These levels of activity were further enhanced by 8 days of chronic treatment, indicating the emergence of behavioral sensitization. Chronic cocaine administration resulted in dose-dependent decreases in the density of dopamine transporter ŽDAT. mRNA in both the substantia nigra pars compacta and ventral tegmental area as shown by in situ hybridization histochemistry. Changes in DAT binding sites were assessed using w 3 Hxmazindol quantitative autoradiography. In contrast to the levels of mRNA, there were few changes in the number of w 3 Hxmazindol binding sites. Although the density of binding sites was unaltered in most regions, w 3 Hxmazindol binding was increased in the anterior nucleus accumbens. This study extends previous findings by demonstrating the dose-dependent nature of the changes in DAT mRNA that accompanies chronic cocaine administration. The levels of DAT binding sites within the dorsal and ventral striatum, however, were largely unchanged. This mismatch suggests that cocaine may differentially influence the gene expression of DAT in the ventral midbrain as compared to the density of DAT binding sites in the basal forebrain. q 1997 Elsevier Science B.V. All rights reserved. Keywords: Cocaine; Dopamine transporter; Sensitization; mRNA; In situ hybridization histochemistry; Autoradiography ; Rat
1. Introduction Cocaine binds to pre-synaptic dopamine, serotonin and norepinephrine transporters w18,32,33x, inhibiting the re-uptake of dopamine w13,21,35x, norepinephrine w14,21,35x and serotonin w4,21,36x from the synaptic cleft. Although cocaine acts at all monoaminergic transporters, many of the behavioral effects, particularly the reinforcing effects, have been attributed to its action at the dopamine transporter ŽDAT. w34,46x. The reinforcing and stimulant properties of cocaine and related drugs have been shown to correlate with their binding properties at the DAT w34,3x. For example, Ritz et al. reported that the potencies of these drugs in self-administration studies correlate with their potencies to inhibit the binding of the ligand, w 3 Hxmazindol, to rat striatal DAT protein w34x. In contrast, the same study showed little
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correlation between self-administration and ligand binding at serotonin and norepinephrine transporters. Moreover, mice lacking the DAT are indifferent to the stimulant effects of cocaine w11x. Since cocaine acts directly at the DAT, chronic cocaine exposure might be expected to produce changes in DAT regulation in the dopamine neurons of the mesolimbic and nigrostriatal pathways. Previous findings regarding alterations in the DAT binding sites that result from repeated administration of cocaine, however, have been inconsistent. Increases w1,44x, decreases w16,30,31,38,44x and no change w2,6,30,38,45x in the levels of DAT binding sites in both dorsal and ventral striatal regions have been reported. A clear understanding of the effects of chronic cocaine administration on the DAT, therefore, does not emerge from these reports. The discrepancies among these studies may result from paradigm differences in dose, route of administration, treatment schedule, length of administration, length of withdrawal, brain area examined or the ligand used. Furthermore, many studies that have examined changes in DAT protein report changes in binding density without addressing whether such alterations are the
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 3 8 4 - 4
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result of changes in the maximal number of binding sites Ž Bmax . or changes in the affinity of the protein for the ligand Ž K d .. The cloning and sequencing of the DAT w12,19,39,43x has enabled visualization of the DAT mRNA with in situ hybridization histochemistry within the cells that synthesize the transporter. Indeed, DAT mRNA has been localized to the dopaminergic cell bodies of the SNc and VTA w8,12,19,40x as well as in the olfactory bulb and in the arcuate nucleus of the hypothalamus w18,19,40,47x. Decreases in DAT mRNA in the ventral midbrain have been shown following repeated exposure to a single dose of cocaine w47x, during withdrawal w7x and in some human cocaine fatalities w42x. Previous studies have examined changes in either DAT binding sites or DAT mRNA as a result of chronic cocaine administration. Differences in the paradigms used in these studies make conclusions about the changes in the relationship of DAT mRNA to DAT binding sites, as a result of repeated cocaine treatment, difficult to draw. At present, no studies have fully described the relationship between levels of mRNA for the DAT expressed in the ventral midbrain and levels of DAT binding sites in the terminal fields of the striatum that result from chronic cocaine administration. The purpose of the present study, then, was to examine the dose-dependent effects of chronic exposure to cocaine on the regulation of the DAT binding sites and mRNA within the same animals.
2. Materials and methods 2.1. Animals and behaÕioral assessment Male Sprague–Dawley rats weighing 300–350 g ŽHarlan Laboratories, Indianapolis, IN, USA., housed tworcage in a temperature- and humidity-controlled room with a 12-h light–dark cycle Žlights on at 07:00 h., were used in these studies. Food and water were available ad libitum. All procedures were carried out in accordance with established practices as described in the NIH Guide for Care and Use of Laboratory Animals. In addition, all procedures were reviewed and approved by the Animal Care and Use Committee of the Bowman Gray School of Medicine of Wake Forest University. Animals were randomly selected to receive cocaine hydrochloride w10 Ž n s 9., 15 Ž n s 8. or 25 mgrkg Ž n s 8.x or saline vehicle w0 mgrkg Ž n s 8.x i.p. for 8 consecutive days. Cocaine was obtained from the National Institute on Drug Abuse and was dissolved in physiologic saline. Doses are expressed as the salt. Locomotor activity was measured in open-field Plexiglas test chambers Ž42 = 42 = 30 cm. by electronic counters that detected interruptions of eight independent IR photocell beams ŽOmnitech, Columbus, OH, USA.. Photocell counts were recorded and stored at 5-min intervals.
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Rats were habituated to experimental procedures for 2 days prior to testing. On these days, rats were injected with saline and placed in photocell chambers for 1 h. On each experimental day, animals were injected with cocaine or saline, then placed immediately in photocell chambers. Behavior was monitored continuously for 1 h on the first and final days of drug treatment Ždays 1 and 8.. On the final day of the drug treatment regimen Žday 8., animals were killed 1 h after the last injection. The brains were removed and quick-frozen in isopentane Žy408C., then stored at y708C until further use. Coronal sections Ž20 m m. were cut on a cryostat, thaw-mounted onto chrome-alumrgelatin-subbed slides, desiccated and frozen until processed for in situ hybridization histochemistry or quantitative autoradiography. 2.2. In situ hybridization histochemistry An antisense cDNA probe was designed complementary to bases 106–146 Žbased on the DAT6 probe w8x. of the rat DAT nucleotide sequence w12x. This is a sequence from a non-membrane spanning region that has low homology to other monoamine transporters w15,28x. The DAT probe was synthesized and gel-purified by the DNA Synthesis Core Laboratory of the Comprehensive Cancer Center, Bowman Gray School of Medicine. The probe was hybridized to tissue as previously described w9x. Briefly, tissue sections were post-fixed for 10 min in 4% paraformaldehyde in phosphate-buffered saline, pretreated for 10 min in 0.25% acetic anhydrider0.1 M triethanolamine–0.9% NaCl, defatted in chloroform and dried in ethanol. The DAT probe was 3X-labeled with a-w 35 Sxdeoxyadenosine triphosphate Ž) 1000 Cirmmol. ŽNew England Nuclear, Boston, MA, USA. using deoxynucleotidyl transferase ŽBoehringer Mannheim Biochemicals, Indianapolis, IN, USA.. The probe was extracted with phenolrchloroformrisoamyl alcohol and 4 M NaClr100% EtOH, then added to a hybridization buffer Ž50% formamide, 10% dextran sulfate, 4 = sterile sodium chloridersodium citrate buffer ŽSSC., 250 m grml yeast tRNA, 500 m grml sheared single-stranded DNA and 1 = Denhart’s solution.. Pre-treated slides were incubated with 0.5 = 10 6 cpm labeled prober25 m l hybridization bufferrtissue section for 20 h at 378C in a humid environment. Sections were then washed 5 = in 1 = SSC for 10 min each at room temperature followed by four 15-min 2 = SSCr50% formamide washes at 408C, then two final 1 = SSC washes for 30 min at room temperature and a final brief rinse in sterile water. Slides were then dried in alcohol and apposed to Hyperfilm-b max ŽAmersham, Arlington Heights, IL, USA. for 1 week in the presence of w 35 Sx-calibrated w 14 Cx standards ŽAmersham.. Following exposure to film, slides were emulsion-dipped ŽNTB-2 emulsion; Eastman Kodak, New Haven, CT, USA. and exposed for 2 weeks. Once developed, dipped slides were lightly Nissl-stained with thionin to determine correspond-
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ing cellular structure. Probe specificity was determined by hybridization of sections under the same conditions with labeled sense mRNA probe or treatment with 5 m grml RNase prior to antisense hybridization. 2.3. QuantitatiÕe autoradiography [ 3 H ] Mazindol binding. Desipramine-insensitive Hxmazindol autoradiography was carried out based on procedures described by Javitch et al. w17x as adapted by Marshall et al. w25x and Sharpe et al. w38x. Briefly, tissue sections were pre-incubated at 48C in buffer Ž50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.9. for 5 min, then incubated for 40 min at 48C in buffer containing 4 nM w 3 Hxmazindol Ž19 Cirmmol. ŽNew England Nuclear. in the presence of 0.3 m M desipramine. Sections were then rinsed in two consecutive 3-min washes in buffer at 48C, with a final 10-s rinse in cold water. They were immediately dried under a stream of cold air and placed on Ultrofilm ŽLeica, Cambridge, MA, USA. for 4 weeks along with tritium standards ŽAmersham.. Non-specific binding was determined in the presence of 1 m M unlabeled mazindol or 30 m M benztropine ŽResearch Biochemicals, International, Natick, MA, USA.. No difference was detectable between w 3 Hxmazindol densities as defined by either mazindol or benztropine. Saturation analyses were determined autoradiographically using 1-, 2-, 4-, 8-, 15and 30-nM concentrations of w 3 Hxmazindol. Non-specific binding was determined as described above. w3
2.4. Densitometry and data analysis After appropriate exposure times, films were developed with Kodak GBX developer, fixed and rinsed. Analysis of autoradiography for both in situ hybridization histochemistry and ligand-binding experiments were conducted by quantitative densitometry with a computerized imageprocessing system ŽMCID, Imaging Research, St. Catharines, Ontario, Canada.. Specific mRNA hybridization was quantified from the autoradiograms. With the
background standardized, measurements were made in the SNc and the VTA in four adjacent sectionsranimal. Values of these regions for each animal were determined from optical densities Ž 14 C nCirmg tissue. and a calibration curve obtained by densiometric analysis of the autoradiograms of the standards. The values were then converted to 35 S equivalents, generated from the known 14 C nCirmg tissue plotted against 35 S dpmrmg tissue as determined from brain paste standards. In addition, the area of specific mRNA hybridization was measured in m m2 w 3 HxMazindol binding sites were measured in eight precommisural regions of the striatum and in the SNc and VTA as illustrated in Fig. 1. Optical density measurements are made in a minimum of four sectionsrbrain area examined. Tissue equivalent values Žfmolrmg of wet weight tissue. were determined from the optical densities and from a calibration curve obtained by densiometric analysis of the autoradiograms of 3 H standards. Specific binding was determined by digitally subtracting non-specific binding from the total binding as measured in adjacent sections. K d and Bmax values were determined from Scatchard analysis of saturation binding data, using the curve-fitting program Ligand w27x. 2.5. Statistical analysis Spontaneous locomotor activity was analyzed using a two-way analysis of variance ŽANOVA. for repeated measures, with dose as the independent variable and time as the repeated measure. In situ hybridization histochemistry data, as well as quantitative autoradiography data, for each brain area were analyzed by means of a one-way ANOVA followed by Dunnett’s t-test for multiple comparisons. 3. Results 3.1. BehaÕior Rates of locomotor activity following cocaine administration were measured by the total number of interruptions
Fig. 1. Schematic diagrams of coronal sections through the rat brain illustrate areas in which w 3 Hxmazindol binding was measured autoradiographically, as adapted from the rat brain atlas of Paxinos and Watson w29x. A: Žbregma 2.2 mm. anterior striatum 1, anterior accumbens 2, olfactory tubercle 3. B: Žbregma 1.6 mm. olfactory tubercle 3, accumbens shell 4, accumbens core 5, dorsomedial caudate 6, dorsolateral caudate 7. C: Žbregma 0.2 mm. dorsomedial caudate 6, dorsolateral caudate 7, ventral caudate 8. D: Žbregma y4.8 mm. substantia nigra pars compacta 9, ventral tegmental area 10.
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Fig. 2. Autoradiograms of in situ hybridization to DAT mRNA reveal intense labeling in the SNcrVTA ŽA. and an absence of labeling in the caudate area ŽB.. Pre-treatment with RNase also resulted in an absence of labeling in the SNcrVTA regions ŽC..
Fig. 3. High magnification photomicrographs of in situ hybridization to DAT mRNA in large dopamine-containing cells of the SNc ŽA. but not in smaller noradrenergic locus coeruleus cells ŽB. as visualized in light-field on emulsion-dipped slides. Scale bar, 10 m m.
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Table 1 Effects of repeated cocaine administration on spontaneous activity in rats Treatment
Vehicle Cocaine Ž10 mgrkg. Cocaine Ž15 mgrkg. Cocaine Ž25 mgrkg.
Locomotor activity Day 1
Day 8
8683"1090 17336"1666 a 28101"2750 a 37857"2530 a
5673"1081 22378"1392 a,b 32163"1472 a,b 46697"4543 a,b
Data shown are mean"S.E.M. cumulative photocell counts during 60-min test period for ns8. a P - 0.05 significantly different from saline. Bonferroni’s multiple comparison following two-way ANOVA for repeated measures. b P - 0.05 different from day 1. Bonferroni’s multiple comparison following two-way ANOVA for repeated measures.
of photocell beams and are shown in Table 1. As expected, cocaine administration elicited dose-dependent increases in spontaneous activity. In addition, administration of each dose of cocaine resulted in an enhancement of locomotor activity on day 8 of treatment as compared to day 1. The augmented levels of spontaneous behavior on day 8 demonstrate the emergence of behavioral sensitization to the effects of chronic cocaine administration in these rats. This emergence of sensitization was used as an indication that the animals had properly received the drug. Rats that did not show such enhancements, therefore, were excluded from assessments of DAT mRNA and DAT binding sites. 3.2. In situ hybridization histochemistry 3.2.1. Specificity of the DAT probe A 40-mer antisense oligonucleotide probe was synthesized complementary to a region of the DAT mRNA that had minimal similarity to other known sequences. Intense labeling was evident within the SNc and VTA ŽFig. 2A. following hybridization with the DAT probe. Labeling was also seen in the arcuate nucleus of the hypothalamus, another area known to contain dopaminergic cell bodies
Žnot shown.. Dense labeling occurred specifically within cell bodies as illustrated with emulsion-dipped slides ŽFig. 3A.. To ensure the specificity of the chosen probe sequence for DAT mRNA as compared to other monoamine transporter mRNAs, serotonin- and norepinephrine-containing cell groups were examined. The fact that no labeling was seen in cells of the locus coeruleus ŽFig. 3B., an area of norepinephrine cell bodies, or in the serotonin-containing cells of the raphe nucleus Žnot shown. confirms that the probe chosen was specific for the DAT and did not label either the closely related norepinephrine or serotonin transporter mRNA. Additional evidence of probe specificity is demonstrated in Fig. 2B. This figure illustrates that the striatum, which contains dopaminergic projections but no dopaminergic cell bodies, contained only background levels of hybridization signal. Finally, hybridization of sections containing the SNcrVTA with a sense probe Žnot shown. or pre-treatment with RNase ŽFig. 2C. failed to produce any specific binding. These findings indicate that the probe utilized in these experiments binds to RNA rather than other cellular structures. 3.2.2. Effects of cocaine Quantitative analysis of the in situ hybridization histochemistry autoradiograms revealed that chronic cocaine dose-dependently decreased DAT mRNA density in both the SNc and the VTA ŽFig. 4, left.. The mean density of DAT mRNA in the VTA was significantly reduced by 19 and 26% following the administration of 15 and 25 mgrkg cocaine, respectively, as compared to saline controls. In addition, DAT mRNA density was similarly reduced significantly in the SNc following treatment with 15 and 25 mgrkg cocaine Žy23 and y25%, respectively.. In contrast, chronic cocaine exposure did not significantly alter the area of DAT mRNA hybridization in either the SNc or the VTA ŽFig. 4, right., suggesting that the total number of cells expressing DAT mRNA did not decrease.
Fig. 4. Effects of chronic cocaine administration on the density Žleft. and area Žright. of DAT mRNA hybridization in the SN and VTA. Data presented as mean " S.E.M. Chronic cocaine administration resulted Žleft. in dose-dependent decreases in DAT mRNA density Ž ) P - 0.05.. There were no significant changes Žright. in the area of DAT mRNA hybridization.
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Table 2 w 3 HxMazindol binding expressed as fmolrmg of wet weight tissue Žmean " S.E.M.. Cocaine
1 Anterior caudate 2 Anterior accumbens 3 Olfactory tubercle 4 Accumbens shell 5 Accumbens core 6 Dorsomedial caudate 7 Dorsolateral caudate 8 Ventral caudate 9 Substantia nigra 10 Ventral tegmental area
0 mgrkg
10 mgrkg
15 mgrkg
25 mgrkg
206.5 " 13 Ž7. 115.8 " 8 Ž6. 119.5 " 4 Ž7. 79.1 " 3 Ž8. 150.1 " 5 Ž8. 194.8 " 7 Ž8. 215.3 " 9 Ž8. 118.5 " 6 Ž8. 69.4 " 4 Ž3. 97.1 " 16 Ž3.
228.7 " 10 Ž9. 143.0 " 10 a Ž9. 126.9 " 8 Ž8. 81.7 " 5 Ž9. 153.7 " 8 Ž9. 202.3 " 7 Ž9. 224.9 " 9 Ž9. 137.4 " 7 Ž9. 82.4 " 9 Ž5. 98.3 " 8 Ž5.
215.2 " 18 Ž6. 116.5 " 3 Ž6. 111.1 " 7 Ž8. 76.2 " 3 Ž8. 136.7 " 3 Ž8. 196.4 " 4 Ž8. 212.5 " 5 Ž8. 138.6 " 7 Ž6. 75.1 " 5 Ž5. 97.2 " 5 Ž4.
226.9 " 14 Ž6. 133.3 " 5 Ž6. 133.9 " 6 Ž8. 74.0 " 2 Ž8. 139.9 " 3 Ž8. 186.8 " 8 Ž8. 204.9 " 10 Ž8. 126.0 " 7 Ž8. 76.5 " 3 Ž4. 96.4 " 3 Ž4.
Number of animals in which measurements were made are indicated in parentheses. a P - 0.05 different from saline. Dunnett’s t-test for multiple comparisons following a one-way ANOVA.
3.3. QuantitatiÕe autoradiography The density of w 3 Hxmazindol binding to DAT was measured in eight precommissural areas containing dopaminergic terminal regions as well as in the SNc and VTA as shown in Fig. 1. These data are displayed in Table 2. In contrast to the decreases in DAT mRNA levels, chronic cocaine treatment generally did not produce significant changes in w 3 Hxmazindol binding. Furthermore, the changes in DAT binding sites were not dose-dependent. In only one area, the anterior accumbens, was an increase Žq35%. in w 3 Hxmazindol binding measured after repeated administration of cocaine but only at the 10-mgrkg dose. Scatchard analysis conducted in the anterior accumbens revealed that the data from the saturation analysis fit a one-site model. Chronic cocaine administration resulted in increases in Bmax of the dopamine transporter in the anterior accumbens Ž Bmax s 1063 fmolrmg tissue in the saline group; Bmax s 1194 fmolrmg tissue in the 10mgrkg group., with no change in the K d in this area Ž K d s 21.9 nM for both saline and 10-mgrkg groups.. This indicates that the increase in binding density in the anterior accumbens as measured with quantitative autoradiography is a result of an increase in the number of w 3 Hxmazindol binding sites rather than a change in the affinity of the dopamine transporter for w 3 Hxmazindol. Furthermore, cocaine-induced changes in mRNA hybridization levels in the ventral midbrain region Ždescribed above. did not correlate with w 3 Hxmazindol-labeled DAT binding sites in any measured brain area.
4. Discussion The present data demonstrate that repeated exposure to cocaine results in dose-dependent decreases in DAT mRNA within both the substantia nigra pars compacta and the
ventral tegmental area of rats. In contrast, the density of DAT binding sites expressed in the dorsal and ventral striatum, as measured by w 3 Hxmazindol binding in the same animals, was generally unaltered, with the exception of the anterior portions of the nucleus accumbens. In this area, increases in DAT binding sites were seen after chronic cocaine administration. There is, therefore, a lack of concordance between the effects of chronic cocaine exposure on the levels of DAT mRNA and the density of DAT binding sites as examined in the same animals. The results of the present experiment are largely consistent with, but also extend, previous reports on the effects of chronic cocaine exposure on DAT mRNA. Decreases in DAT mRNA were found in rat SNc after repeated injections of a single dose of cocaine, with no change after acute cocaine exposure w47x. More limited decreases in DAT mRNA, however, were found in rats during withdrawal from a chronic cocaine administration regimen designed to mimic cocaine self-administration and were restricted to the interfascicular nucleus and caudal linear nuclei, both subnuclei of the ventral tegmental area w7x. Message for the DAT in the SNc has also been shown to decrease in human cocaine fatalities as compared to agematched controls w42x. The present data extend these previous findings in two ways. First, they demonstrate that the mRNA decreases that occur as a result of chronic cocaine administration are clearly a dose-dependent phenomenon, with higher doses producing greater reduction in DAT mRNA density as measured by in situ hybridization histochemistry. Second, the present data also show that the decreases in DAT mRNA that accompany repeated cocaine administration are not associated with parallel changes in w 3 Hxmazindol binding to DAT in the same animals as assessed by quantitative autoradiography. The general lack of change in DAT binding sites by chronic cocaine treatment, as observed in the present study with w 3 Hxmazindol, is also consistent with the findings of
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previous studies. Several studies have shown that chronic cocaine administration did not alter the levels of DAT binding sites in rats, for example, no change in the density of w 3 Hxmazindol-labeled DAT sites occurred immediately after 10 days of i.v. infusions of cocaine w38x. In addition, a challenge dose of cocaine 7 days after 1 week of cocaine administration produced no change in w 3 Hxmazindol binding w6x. The absence of changes in DAT binding sites was also seen in w 3 HxWIN 35,428 binding in rats immediately after chronic cocaine administration w30x. Similarly, Allard et al. found no changes in w 3 HxGBR 12,935 binding in the striatum shortly after chronic administration of cocaine w2x. Finally, w 3 HxWIN 35,428 and w 3 HxGBR 12,935 binding sites were unaltered after 4 weeks of exposure to a low dose of cocaine w45x. Increases in DAT protein as a result of chronic cocaine administration, however, have also been reported. Wilson et al. found that levels of both w 3 HxWIN 35,428 and w 3 HxGBR 12,935 binding were increased in the nucleus accumbens and striatum of rats on the final day of a chronic cocaine self-administration regimen w44x. Also, w 3 HxBTCP binding to DAT sites in striatal homogenates was increased after repeated cocaine administration in rats sacrificed shortly after their last exposure to cocaine w1x. Increases in the density of w 3 HxWIN 35,428 binding have been shown in human cocaine fatalities w41x whereas decreases in DAT binding sites, as measured with w 3 Hxmazindol binding, have been demonstrated in cocaine addicts who had cocaine on board at the time of death w16x. Although the effects of chronic cocaine exposure on the levels of DAT binding sites can vary considerably and appear to depend in part on the treatment regimen and ligand used, there remains a clear lack of concordance between the effects of cocaine on DAT binding sites and the regulation of the DAT mRNA from which the transporter protein is synthesized. Similar discrepancies between changes in mRNA levels and protein-binding sites as a result of repeated cocaine treatment have been previously reported for dopamine D 1 receptors w24x as well as dopamine D 2 receptors w20x. Taken together, these findings suggest that non-parellel changes in mRNA levels and protein-binding sites can occur at a given time point as a result of chronic cocaine administration. The present data were collected at a single time point and may not, therefore, represent a complete picture of the dynamic relationship between mRNA and protein. Since mRNA is the precursor to protein, changes in the mRNA system may precede changes in the protein system. It is possible that the decreases in DAT mRNA content predict a pending decrease in DAT protein as is the case in the leu-enkephalin system w23x where increases in protein are preceded by increases in mRNA. In addition, the half-lives of most proteins are much longer than their mRNA precursors. For example, the half-life of the closely related glutamate transporter ŽGlut-1. mRNA has been shown to be 0.5–1 h w26x while that of the Glut-1 protein appears to
be 19 h in an adipocyte cell line w37x. While the half-life of the DAT mRNA is not known, the half-life of the DAT protein appears to be 6 days w10x. The longer half-life of the protein suggests that there may be a time lag in the effects seen in the protein after changes in the mRNA are evident. In the present study, no correlations were found between the levels of DAT ligand binding and the enhancement of locomotor activity that accompanies chronic cocaine administration. This is consistent with a number of studies that have attempted to relate behavioral sensitization to changes in the levels of ligand binding to DAT protein w6,22x. Koff et al. w22x demonstrated sensitization to repeated cocaine administration in five strains of mice but only two strains exhibited alterations in dopamine transporter binding. Moreover, no changes in w 3 Hxmazindol binding was observed in rats that exhibited behavioral sensitization to chronic cocaine administration w6x. In contrast, Boulay et al. w5x noted the concurrence of behavioral sensitization and a decrease in w 3 Hxmazindol binding in the accumbens shell. This decrease, however, may have been the result of the 14-day withdrawal period between the repeated cocaine administration and the cocaine challenge, rather than to behavioral sensitization itself, similar to previous studies using a withdrawal paradigm w30,31,38,44x. While decreases in DAT mRNA were observed in the present study in rats that exhibited sensitization to the behavioral activating effects of chronic cocaine administration, the mRNA is not the site of dopamine uptake inhibition by cocaine. Furthermore, there is a clear lack of correlation between mRNA for DAT and ligand binding to DAT. Thus, it appears that chronic cocaine administration is associated with both alterations in the DAT and behavioral sensitization, but it is unlikely that behavioral sensitization is a direct consequence of these changes. In summary, chronic cocaine administration dose-dependently decreased DAT mRNA within the cells of the SNc and VTA as visualized by in situ hybridization histochemistry but generally did not alter levels of DAT binding sites as assessed by w 3 Hxmazindol binding. Whereas chronic exposure to cocaine appears to alter the regulation of DAT mRNA in the ventral midbrain, the present data indicate that such changes can be dissociated from the levels of expression of DAT binding sites in striatal regions.
Acknowledgements The authors thank Dr. Jacqueline McGinty for expert training and extensive consultation on the in situ hybridization histochemistry technique. The authors also thank Dr. David Friedman and Dr. David Lyons for their helpful comments on this manuscript. This research was supported by NIDA Grants DA07522 and DA07246.
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Research report
Effects of chronic cocaine administration on dopamine transporter mRNA and protein in the rat Sharon R. Letchworth ) , James B. Daunais, Ashlee A. Hedgecock, Linda J. Porrino Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Medical Center BouleÕard, Winston-Salem, NC 27157, USA Accepted 5 November 1996
Abstract Male Sprague–Dawley rats were administered cocaine Ž10, 15 or 25 mgrkg. or vehicle, i.p., once daily for 8 consecutive days and killed 1 h after the last injection. Acute cocaine administration produced dose-dependent increases in spontaneous locomotor activity. These levels of activity were further enhanced by 8 days of chronic treatment, indicating the emergence of behavioral sensitization. Chronic cocaine administration resulted in dose-dependent decreases in the density of dopamine transporter ŽDAT. mRNA in both the substantia nigra pars compacta and ventral tegmental area as shown by in situ hybridization histochemistry. Changes in DAT binding sites were assessed using w 3 Hxmazindol quantitative autoradiography. In contrast to the levels of mRNA, there were few changes in the number of w 3 Hxmazindol binding sites. Although the density of binding sites was unaltered in most regions, w 3 Hxmazindol binding was increased in the anterior nucleus accumbens. This study extends previous findings by demonstrating the dose-dependent nature of the changes in DAT mRNA that accompanies chronic cocaine administration. The levels of DAT binding sites within the dorsal and ventral striatum, however, were largely unchanged. This mismatch suggests that cocaine may differentially influence the gene expression of DAT in the ventral midbrain as compared to the density of DAT binding sites in the basal forebrain. q 1997 Elsevier Science B.V. All rights reserved. Keywords: Cocaine; Dopamine transporter; Sensitization; mRNA; In situ hybridization histochemistry; Autoradiography ; Rat
1. Introduction Cocaine binds to pre-synaptic dopamine, serotonin and norepinephrine transporters w18,32,33x, inhibiting the re-uptake of dopamine w13,21,35x, norepinephrine w14,21,35x and serotonin w4,21,36x from the synaptic cleft. Although cocaine acts at all monoaminergic transporters, many of the behavioral effects, particularly the reinforcing effects, have been attributed to its action at the dopamine transporter ŽDAT. w34,46x. The reinforcing and stimulant properties of cocaine and related drugs have been shown to correlate with their binding properties at the DAT w34,3x. For example, Ritz et al. reported that the potencies of these drugs in self-administration studies correlate with their potencies to inhibit the binding of the ligand, w 3 Hxmazindol, to rat striatal DAT protein w34x. In contrast, the same study showed little
) Corresponding author. Fax: q1 Ž910. 716-8501; E-mail: [email protected]
correlation between self-administration and ligand binding at serotonin and norepinephrine transporters. Moreover, mice lacking the DAT are indifferent to the stimulant effects of cocaine w11x. Since cocaine acts directly at the DAT, chronic cocaine exposure might be expected to produce changes in DAT regulation in the dopamine neurons of the mesolimbic and nigrostriatal pathways. Previous findings regarding alterations in the DAT binding sites that result from repeated administration of cocaine, however, have been inconsistent. Increases w1,44x, decreases w16,30,31,38,44x and no change w2,6,30,38,45x in the levels of DAT binding sites in both dorsal and ventral striatal regions have been reported. A clear understanding of the effects of chronic cocaine administration on the DAT, therefore, does not emerge from these reports. The discrepancies among these studies may result from paradigm differences in dose, route of administration, treatment schedule, length of administration, length of withdrawal, brain area examined or the ligand used. Furthermore, many studies that have examined changes in DAT protein report changes in binding density without addressing whether such alterations are the
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 3 8 4 - 4
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result of changes in the maximal number of binding sites Ž Bmax . or changes in the affinity of the protein for the ligand Ž K d .. The cloning and sequencing of the DAT w12,19,39,43x has enabled visualization of the DAT mRNA with in situ hybridization histochemistry within the cells that synthesize the transporter. Indeed, DAT mRNA has been localized to the dopaminergic cell bodies of the SNc and VTA w8,12,19,40x as well as in the olfactory bulb and in the arcuate nucleus of the hypothalamus w18,19,40,47x. Decreases in DAT mRNA in the ventral midbrain have been shown following repeated exposure to a single dose of cocaine w47x, during withdrawal w7x and in some human cocaine fatalities w42x. Previous studies have examined changes in either DAT binding sites or DAT mRNA as a result of chronic cocaine administration. Differences in the paradigms used in these studies make conclusions about the changes in the relationship of DAT mRNA to DAT binding sites, as a result of repeated cocaine treatment, difficult to draw. At present, no studies have fully described the relationship between levels of mRNA for the DAT expressed in the ventral midbrain and levels of DAT binding sites in the terminal fields of the striatum that result from chronic cocaine administration. The purpose of the present study, then, was to examine the dose-dependent effects of chronic exposure to cocaine on the regulation of the DAT binding sites and mRNA within the same animals.
2. Materials and methods 2.1. Animals and behaÕioral assessment Male Sprague–Dawley rats weighing 300–350 g ŽHarlan Laboratories, Indianapolis, IN, USA., housed tworcage in a temperature- and humidity-controlled room with a 12-h light–dark cycle Žlights on at 07:00 h., were used in these studies. Food and water were available ad libitum. All procedures were carried out in accordance with established practices as described in the NIH Guide for Care and Use of Laboratory Animals. In addition, all procedures were reviewed and approved by the Animal Care and Use Committee of the Bowman Gray School of Medicine of Wake Forest University. Animals were randomly selected to receive cocaine hydrochloride w10 Ž n s 9., 15 Ž n s 8. or 25 mgrkg Ž n s 8.x or saline vehicle w0 mgrkg Ž n s 8.x i.p. for 8 consecutive days. Cocaine was obtained from the National Institute on Drug Abuse and was dissolved in physiologic saline. Doses are expressed as the salt. Locomotor activity was measured in open-field Plexiglas test chambers Ž42 = 42 = 30 cm. by electronic counters that detected interruptions of eight independent IR photocell beams ŽOmnitech, Columbus, OH, USA.. Photocell counts were recorded and stored at 5-min intervals.
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Rats were habituated to experimental procedures for 2 days prior to testing. On these days, rats were injected with saline and placed in photocell chambers for 1 h. On each experimental day, animals were injected with cocaine or saline, then placed immediately in photocell chambers. Behavior was monitored continuously for 1 h on the first and final days of drug treatment Ždays 1 and 8.. On the final day of the drug treatment regimen Žday 8., animals were killed 1 h after the last injection. The brains were removed and quick-frozen in isopentane Žy408C., then stored at y708C until further use. Coronal sections Ž20 m m. were cut on a cryostat, thaw-mounted onto chrome-alumrgelatin-subbed slides, desiccated and frozen until processed for in situ hybridization histochemistry or quantitative autoradiography. 2.2. In situ hybridization histochemistry An antisense cDNA probe was designed complementary to bases 106–146 Žbased on the DAT6 probe w8x. of the rat DAT nucleotide sequence w12x. This is a sequence from a non-membrane spanning region that has low homology to other monoamine transporters w15,28x. The DAT probe was synthesized and gel-purified by the DNA Synthesis Core Laboratory of the Comprehensive Cancer Center, Bowman Gray School of Medicine. The probe was hybridized to tissue as previously described w9x. Briefly, tissue sections were post-fixed for 10 min in 4% paraformaldehyde in phosphate-buffered saline, pretreated for 10 min in 0.25% acetic anhydrider0.1 M triethanolamine–0.9% NaCl, defatted in chloroform and dried in ethanol. The DAT probe was 3X-labeled with a-w 35 Sxdeoxyadenosine triphosphate Ž) 1000 Cirmmol. ŽNew England Nuclear, Boston, MA, USA. using deoxynucleotidyl transferase ŽBoehringer Mannheim Biochemicals, Indianapolis, IN, USA.. The probe was extracted with phenolrchloroformrisoamyl alcohol and 4 M NaClr100% EtOH, then added to a hybridization buffer Ž50% formamide, 10% dextran sulfate, 4 = sterile sodium chloridersodium citrate buffer ŽSSC., 250 m grml yeast tRNA, 500 m grml sheared single-stranded DNA and 1 = Denhart’s solution.. Pre-treated slides were incubated with 0.5 = 10 6 cpm labeled prober25 m l hybridization bufferrtissue section for 20 h at 378C in a humid environment. Sections were then washed 5 = in 1 = SSC for 10 min each at room temperature followed by four 15-min 2 = SSCr50% formamide washes at 408C, then two final 1 = SSC washes for 30 min at room temperature and a final brief rinse in sterile water. Slides were then dried in alcohol and apposed to Hyperfilm-b max ŽAmersham, Arlington Heights, IL, USA. for 1 week in the presence of w 35 Sx-calibrated w 14 Cx standards ŽAmersham.. Following exposure to film, slides were emulsion-dipped ŽNTB-2 emulsion; Eastman Kodak, New Haven, CT, USA. and exposed for 2 weeks. Once developed, dipped slides were lightly Nissl-stained with thionin to determine correspond-
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ing cellular structure. Probe specificity was determined by hybridization of sections under the same conditions with labeled sense mRNA probe or treatment with 5 m grml RNase prior to antisense hybridization. 2.3. QuantitatiÕe autoradiography [ 3 H ] Mazindol binding. Desipramine-insensitive Hxmazindol autoradiography was carried out based on procedures described by Javitch et al. w17x as adapted by Marshall et al. w25x and Sharpe et al. w38x. Briefly, tissue sections were pre-incubated at 48C in buffer Ž50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.9. for 5 min, then incubated for 40 min at 48C in buffer containing 4 nM w 3 Hxmazindol Ž19 Cirmmol. ŽNew England Nuclear. in the presence of 0.3 m M desipramine. Sections were then rinsed in two consecutive 3-min washes in buffer at 48C, with a final 10-s rinse in cold water. They were immediately dried under a stream of cold air and placed on Ultrofilm ŽLeica, Cambridge, MA, USA. for 4 weeks along with tritium standards ŽAmersham.. Non-specific binding was determined in the presence of 1 m M unlabeled mazindol or 30 m M benztropine ŽResearch Biochemicals, International, Natick, MA, USA.. No difference was detectable between w 3 Hxmazindol densities as defined by either mazindol or benztropine. Saturation analyses were determined autoradiographically using 1-, 2-, 4-, 8-, 15and 30-nM concentrations of w 3 Hxmazindol. Non-specific binding was determined as described above. w3
2.4. Densitometry and data analysis After appropriate exposure times, films were developed with Kodak GBX developer, fixed and rinsed. Analysis of autoradiography for both in situ hybridization histochemistry and ligand-binding experiments were conducted by quantitative densitometry with a computerized imageprocessing system ŽMCID, Imaging Research, St. Catharines, Ontario, Canada.. Specific mRNA hybridization was quantified from the autoradiograms. With the
background standardized, measurements were made in the SNc and the VTA in four adjacent sectionsranimal. Values of these regions for each animal were determined from optical densities Ž 14 C nCirmg tissue. and a calibration curve obtained by densiometric analysis of the autoradiograms of the standards. The values were then converted to 35 S equivalents, generated from the known 14 C nCirmg tissue plotted against 35 S dpmrmg tissue as determined from brain paste standards. In addition, the area of specific mRNA hybridization was measured in m m2 w 3 HxMazindol binding sites were measured in eight precommisural regions of the striatum and in the SNc and VTA as illustrated in Fig. 1. Optical density measurements are made in a minimum of four sectionsrbrain area examined. Tissue equivalent values Žfmolrmg of wet weight tissue. were determined from the optical densities and from a calibration curve obtained by densiometric analysis of the autoradiograms of 3 H standards. Specific binding was determined by digitally subtracting non-specific binding from the total binding as measured in adjacent sections. K d and Bmax values were determined from Scatchard analysis of saturation binding data, using the curve-fitting program Ligand w27x. 2.5. Statistical analysis Spontaneous locomotor activity was analyzed using a two-way analysis of variance ŽANOVA. for repeated measures, with dose as the independent variable and time as the repeated measure. In situ hybridization histochemistry data, as well as quantitative autoradiography data, for each brain area were analyzed by means of a one-way ANOVA followed by Dunnett’s t-test for multiple comparisons. 3. Results 3.1. BehaÕior Rates of locomotor activity following cocaine administration were measured by the total number of interruptions
Fig. 1. Schematic diagrams of coronal sections through the rat brain illustrate areas in which w 3 Hxmazindol binding was measured autoradiographically, as adapted from the rat brain atlas of Paxinos and Watson w29x. A: Žbregma 2.2 mm. anterior striatum 1, anterior accumbens 2, olfactory tubercle 3. B: Žbregma 1.6 mm. olfactory tubercle 3, accumbens shell 4, accumbens core 5, dorsomedial caudate 6, dorsolateral caudate 7. C: Žbregma 0.2 mm. dorsomedial caudate 6, dorsolateral caudate 7, ventral caudate 8. D: Žbregma y4.8 mm. substantia nigra pars compacta 9, ventral tegmental area 10.
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Fig. 2. Autoradiograms of in situ hybridization to DAT mRNA reveal intense labeling in the SNcrVTA ŽA. and an absence of labeling in the caudate area ŽB.. Pre-treatment with RNase also resulted in an absence of labeling in the SNcrVTA regions ŽC..
Fig. 3. High magnification photomicrographs of in situ hybridization to DAT mRNA in large dopamine-containing cells of the SNc ŽA. but not in smaller noradrenergic locus coeruleus cells ŽB. as visualized in light-field on emulsion-dipped slides. Scale bar, 10 m m.
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Table 1 Effects of repeated cocaine administration on spontaneous activity in rats Treatment
Vehicle Cocaine Ž10 mgrkg. Cocaine Ž15 mgrkg. Cocaine Ž25 mgrkg.
Locomotor activity Day 1
Day 8
8683"1090 17336"1666 a 28101"2750 a 37857"2530 a
5673"1081 22378"1392 a,b 32163"1472 a,b 46697"4543 a,b
Data shown are mean"S.E.M. cumulative photocell counts during 60-min test period for ns8. a P - 0.05 significantly different from saline. Bonferroni’s multiple comparison following two-way ANOVA for repeated measures. b P - 0.05 different from day 1. Bonferroni’s multiple comparison following two-way ANOVA for repeated measures.
of photocell beams and are shown in Table 1. As expected, cocaine administration elicited dose-dependent increases in spontaneous activity. In addition, administration of each dose of cocaine resulted in an enhancement of locomotor activity on day 8 of treatment as compared to day 1. The augmented levels of spontaneous behavior on day 8 demonstrate the emergence of behavioral sensitization to the effects of chronic cocaine administration in these rats. This emergence of sensitization was used as an indication that the animals had properly received the drug. Rats that did not show such enhancements, therefore, were excluded from assessments of DAT mRNA and DAT binding sites. 3.2. In situ hybridization histochemistry 3.2.1. Specificity of the DAT probe A 40-mer antisense oligonucleotide probe was synthesized complementary to a region of the DAT mRNA that had minimal similarity to other known sequences. Intense labeling was evident within the SNc and VTA ŽFig. 2A. following hybridization with the DAT probe. Labeling was also seen in the arcuate nucleus of the hypothalamus, another area known to contain dopaminergic cell bodies
Žnot shown.. Dense labeling occurred specifically within cell bodies as illustrated with emulsion-dipped slides ŽFig. 3A.. To ensure the specificity of the chosen probe sequence for DAT mRNA as compared to other monoamine transporter mRNAs, serotonin- and norepinephrine-containing cell groups were examined. The fact that no labeling was seen in cells of the locus coeruleus ŽFig. 3B., an area of norepinephrine cell bodies, or in the serotonin-containing cells of the raphe nucleus Žnot shown. confirms that the probe chosen was specific for the DAT and did not label either the closely related norepinephrine or serotonin transporter mRNA. Additional evidence of probe specificity is demonstrated in Fig. 2B. This figure illustrates that the striatum, which contains dopaminergic projections but no dopaminergic cell bodies, contained only background levels of hybridization signal. Finally, hybridization of sections containing the SNcrVTA with a sense probe Žnot shown. or pre-treatment with RNase ŽFig. 2C. failed to produce any specific binding. These findings indicate that the probe utilized in these experiments binds to RNA rather than other cellular structures. 3.2.2. Effects of cocaine Quantitative analysis of the in situ hybridization histochemistry autoradiograms revealed that chronic cocaine dose-dependently decreased DAT mRNA density in both the SNc and the VTA ŽFig. 4, left.. The mean density of DAT mRNA in the VTA was significantly reduced by 19 and 26% following the administration of 15 and 25 mgrkg cocaine, respectively, as compared to saline controls. In addition, DAT mRNA density was similarly reduced significantly in the SNc following treatment with 15 and 25 mgrkg cocaine Žy23 and y25%, respectively.. In contrast, chronic cocaine exposure did not significantly alter the area of DAT mRNA hybridization in either the SNc or the VTA ŽFig. 4, right., suggesting that the total number of cells expressing DAT mRNA did not decrease.
Fig. 4. Effects of chronic cocaine administration on the density Žleft. and area Žright. of DAT mRNA hybridization in the SN and VTA. Data presented as mean " S.E.M. Chronic cocaine administration resulted Žleft. in dose-dependent decreases in DAT mRNA density Ž ) P - 0.05.. There were no significant changes Žright. in the area of DAT mRNA hybridization.
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Table 2 w 3 HxMazindol binding expressed as fmolrmg of wet weight tissue Žmean " S.E.M.. Cocaine
1 Anterior caudate 2 Anterior accumbens 3 Olfactory tubercle 4 Accumbens shell 5 Accumbens core 6 Dorsomedial caudate 7 Dorsolateral caudate 8 Ventral caudate 9 Substantia nigra 10 Ventral tegmental area
0 mgrkg
10 mgrkg
15 mgrkg
25 mgrkg
206.5 " 13 Ž7. 115.8 " 8 Ž6. 119.5 " 4 Ž7. 79.1 " 3 Ž8. 150.1 " 5 Ž8. 194.8 " 7 Ž8. 215.3 " 9 Ž8. 118.5 " 6 Ž8. 69.4 " 4 Ž3. 97.1 " 16 Ž3.
228.7 " 10 Ž9. 143.0 " 10 a Ž9. 126.9 " 8 Ž8. 81.7 " 5 Ž9. 153.7 " 8 Ž9. 202.3 " 7 Ž9. 224.9 " 9 Ž9. 137.4 " 7 Ž9. 82.4 " 9 Ž5. 98.3 " 8 Ž5.
215.2 " 18 Ž6. 116.5 " 3 Ž6. 111.1 " 7 Ž8. 76.2 " 3 Ž8. 136.7 " 3 Ž8. 196.4 " 4 Ž8. 212.5 " 5 Ž8. 138.6 " 7 Ž6. 75.1 " 5 Ž5. 97.2 " 5 Ž4.
226.9 " 14 Ž6. 133.3 " 5 Ž6. 133.9 " 6 Ž8. 74.0 " 2 Ž8. 139.9 " 3 Ž8. 186.8 " 8 Ž8. 204.9 " 10 Ž8. 126.0 " 7 Ž8. 76.5 " 3 Ž4. 96.4 " 3 Ž4.
Number of animals in which measurements were made are indicated in parentheses. a P - 0.05 different from saline. Dunnett’s t-test for multiple comparisons following a one-way ANOVA.
3.3. QuantitatiÕe autoradiography The density of w 3 Hxmazindol binding to DAT was measured in eight precommissural areas containing dopaminergic terminal regions as well as in the SNc and VTA as shown in Fig. 1. These data are displayed in Table 2. In contrast to the decreases in DAT mRNA levels, chronic cocaine treatment generally did not produce significant changes in w 3 Hxmazindol binding. Furthermore, the changes in DAT binding sites were not dose-dependent. In only one area, the anterior accumbens, was an increase Žq35%. in w 3 Hxmazindol binding measured after repeated administration of cocaine but only at the 10-mgrkg dose. Scatchard analysis conducted in the anterior accumbens revealed that the data from the saturation analysis fit a one-site model. Chronic cocaine administration resulted in increases in Bmax of the dopamine transporter in the anterior accumbens Ž Bmax s 1063 fmolrmg tissue in the saline group; Bmax s 1194 fmolrmg tissue in the 10mgrkg group., with no change in the K d in this area Ž K d s 21.9 nM for both saline and 10-mgrkg groups.. This indicates that the increase in binding density in the anterior accumbens as measured with quantitative autoradiography is a result of an increase in the number of w 3 Hxmazindol binding sites rather than a change in the affinity of the dopamine transporter for w 3 Hxmazindol. Furthermore, cocaine-induced changes in mRNA hybridization levels in the ventral midbrain region Ždescribed above. did not correlate with w 3 Hxmazindol-labeled DAT binding sites in any measured brain area.
4. Discussion The present data demonstrate that repeated exposure to cocaine results in dose-dependent decreases in DAT mRNA within both the substantia nigra pars compacta and the
ventral tegmental area of rats. In contrast, the density of DAT binding sites expressed in the dorsal and ventral striatum, as measured by w 3 Hxmazindol binding in the same animals, was generally unaltered, with the exception of the anterior portions of the nucleus accumbens. In this area, increases in DAT binding sites were seen after chronic cocaine administration. There is, therefore, a lack of concordance between the effects of chronic cocaine exposure on the levels of DAT mRNA and the density of DAT binding sites as examined in the same animals. The results of the present experiment are largely consistent with, but also extend, previous reports on the effects of chronic cocaine exposure on DAT mRNA. Decreases in DAT mRNA were found in rat SNc after repeated injections of a single dose of cocaine, with no change after acute cocaine exposure w47x. More limited decreases in DAT mRNA, however, were found in rats during withdrawal from a chronic cocaine administration regimen designed to mimic cocaine self-administration and were restricted to the interfascicular nucleus and caudal linear nuclei, both subnuclei of the ventral tegmental area w7x. Message for the DAT in the SNc has also been shown to decrease in human cocaine fatalities as compared to agematched controls w42x. The present data extend these previous findings in two ways. First, they demonstrate that the mRNA decreases that occur as a result of chronic cocaine administration are clearly a dose-dependent phenomenon, with higher doses producing greater reduction in DAT mRNA density as measured by in situ hybridization histochemistry. Second, the present data also show that the decreases in DAT mRNA that accompany repeated cocaine administration are not associated with parallel changes in w 3 Hxmazindol binding to DAT in the same animals as assessed by quantitative autoradiography. The general lack of change in DAT binding sites by chronic cocaine treatment, as observed in the present study with w 3 Hxmazindol, is also consistent with the findings of
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previous studies. Several studies have shown that chronic cocaine administration did not alter the levels of DAT binding sites in rats, for example, no change in the density of w 3 Hxmazindol-labeled DAT sites occurred immediately after 10 days of i.v. infusions of cocaine w38x. In addition, a challenge dose of cocaine 7 days after 1 week of cocaine administration produced no change in w 3 Hxmazindol binding w6x. The absence of changes in DAT binding sites was also seen in w 3 HxWIN 35,428 binding in rats immediately after chronic cocaine administration w30x. Similarly, Allard et al. found no changes in w 3 HxGBR 12,935 binding in the striatum shortly after chronic administration of cocaine w2x. Finally, w 3 HxWIN 35,428 and w 3 HxGBR 12,935 binding sites were unaltered after 4 weeks of exposure to a low dose of cocaine w45x. Increases in DAT protein as a result of chronic cocaine administration, however, have also been reported. Wilson et al. found that levels of both w 3 HxWIN 35,428 and w 3 HxGBR 12,935 binding were increased in the nucleus accumbens and striatum of rats on the final day of a chronic cocaine self-administration regimen w44x. Also, w 3 HxBTCP binding to DAT sites in striatal homogenates was increased after repeated cocaine administration in rats sacrificed shortly after their last exposure to cocaine w1x. Increases in the density of w 3 HxWIN 35,428 binding have been shown in human cocaine fatalities w41x whereas decreases in DAT binding sites, as measured with w 3 Hxmazindol binding, have been demonstrated in cocaine addicts who had cocaine on board at the time of death w16x. Although the effects of chronic cocaine exposure on the levels of DAT binding sites can vary considerably and appear to depend in part on the treatment regimen and ligand used, there remains a clear lack of concordance between the effects of cocaine on DAT binding sites and the regulation of the DAT mRNA from which the transporter protein is synthesized. Similar discrepancies between changes in mRNA levels and protein-binding sites as a result of repeated cocaine treatment have been previously reported for dopamine D 1 receptors w24x as well as dopamine D 2 receptors w20x. Taken together, these findings suggest that non-parellel changes in mRNA levels and protein-binding sites can occur at a given time point as a result of chronic cocaine administration. The present data were collected at a single time point and may not, therefore, represent a complete picture of the dynamic relationship between mRNA and protein. Since mRNA is the precursor to protein, changes in the mRNA system may precede changes in the protein system. It is possible that the decreases in DAT mRNA content predict a pending decrease in DAT protein as is the case in the leu-enkephalin system w23x where increases in protein are preceded by increases in mRNA. In addition, the half-lives of most proteins are much longer than their mRNA precursors. For example, the half-life of the closely related glutamate transporter ŽGlut-1. mRNA has been shown to be 0.5–1 h w26x while that of the Glut-1 protein appears to
be 19 h in an adipocyte cell line w37x. While the half-life of the DAT mRNA is not known, the half-life of the DAT protein appears to be 6 days w10x. The longer half-life of the protein suggests that there may be a time lag in the effects seen in the protein after changes in the mRNA are evident. In the present study, no correlations were found between the levels of DAT ligand binding and the enhancement of locomotor activity that accompanies chronic cocaine administration. This is consistent with a number of studies that have attempted to relate behavioral sensitization to changes in the levels of ligand binding to DAT protein w6,22x. Koff et al. w22x demonstrated sensitization to repeated cocaine administration in five strains of mice but only two strains exhibited alterations in dopamine transporter binding. Moreover, no changes in w 3 Hxmazindol binding was observed in rats that exhibited behavioral sensitization to chronic cocaine administration w6x. In contrast, Boulay et al. w5x noted the concurrence of behavioral sensitization and a decrease in w 3 Hxmazindol binding in the accumbens shell. This decrease, however, may have been the result of the 14-day withdrawal period between the repeated cocaine administration and the cocaine challenge, rather than to behavioral sensitization itself, similar to previous studies using a withdrawal paradigm w30,31,38,44x. While decreases in DAT mRNA were observed in the present study in rats that exhibited sensitization to the behavioral activating effects of chronic cocaine administration, the mRNA is not the site of dopamine uptake inhibition by cocaine. Furthermore, there is a clear lack of correlation between mRNA for DAT and ligand binding to DAT. Thus, it appears that chronic cocaine administration is associated with both alterations in the DAT and behavioral sensitization, but it is unlikely that behavioral sensitization is a direct consequence of these changes. In summary, chronic cocaine administration dose-dependently decreased DAT mRNA within the cells of the SNc and VTA as visualized by in situ hybridization histochemistry but generally did not alter levels of DAT binding sites as assessed by w 3 Hxmazindol binding. Whereas chronic exposure to cocaine appears to alter the regulation of DAT mRNA in the ventral midbrain, the present data indicate that such changes can be dissociated from the levels of expression of DAT binding sites in striatal regions.
Acknowledgements The authors thank Dr. Jacqueline McGinty for expert training and extensive consultation on the in situ hybridization histochemistry technique. The authors also thank Dr. David Friedman and Dr. David Lyons for their helpful comments on this manuscript. This research was supported by NIDA Grants DA07522 and DA07246.
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