Reduction in dopamine transporter mRNA after cessation of repeated cocaine administration

Molecular Brain Research, 22 (1994) 132 - 138 © 1994 Elsevier Science B.V. All rights reserved O169-328X/t)4/$07.00

132

BRESM 70717

Reduction in dopamine transporter mRNA after cessation of repeated cocaine administration Catherine Cerruti

a,

Nancy S. Pilotte

a

George Uhl

b

Michael J. Kuhar ~'*

a NIH, National Institute on Drug Abuse, Addiction Research Center, Neuroscience Branch, Baltimore, MD 21224, USA, b NIH, National Institute on Drug Abuse, Addiction Research Center, Molecular Neurobiology Branch, Baltimore, MD 21224, USA (Accepted 17 August 1993)

Key words: Dopamine transporter; Dopamine uptake; Gene expression; Transporter regulation; Cocaine

Male, Lewis rats were treated intravenously for 2 weeks with saline or cocaine using a dose and injection schedule that is similar to the doses and patterns of cocaine intake in self-administration studies. Ten days after cessation of treatment, dopamine transporter binding levels were decreased in the nucleus accumhens but not in the striatum. In situ hybridization studies revealed decreases in dopamine transporter mRNA that were restricted to cells of the interfascicular and caudal linear nuclei; these dopaminergic cell groups, found in the ventral tegmentum, project to the nucleus accumbens and other limbic areas. Other dopaminergic cell groups in midbrain which project mainly to other areas did not show a decrease in mRNA. These results indicate that gene expression can be altered many days after withdrawal from cocaine, and provide an example of transporter regulation by a change in gene expression.

INTRODUCTION Reuptake of extracellular dopamine is a primary means of terminating dopaminergic synaptic transmission. Because of the importance of the synaptic membrane dopamine transporter in this process, and because of its role in the action of various drugs such as psychostimulants, the dopamine transporter (DAT) has been the subject of many investigations. Recent molecular studies have shown that the DAT is an apparent 80,000 Da MW glycoprotein with heterogeneous forms defined by different carbohydrate side chains in different brain regions and cell types 7'19'29-31'36'37'43. cDNAs for DAT have been cloned from rat 16'25'46, cow 49 and human 17'5° brains. Antibodies prepared against peptides corresponding to segments of the deduced amino acid sequence from the cloned cDNA react with native DAT extracted from rat striatum 51. In situ hybridization studies with probes corresponding to segments of the cloned cDNA reveal striking and selective localiza-

* Corresponding author. Fax: (1) (410) 550-1672.

SSDI 0 1 6 9 - 3 2 8 X ( 9 3 ) E 0 1 5 7 - U

tion of related mRNA to cell groups well known to contain dopaminergic neurons 11A6'47'49'54. Regulatory influences on the DATs are not fully elucidated. However, chronic administration of psychostimulants such as cocaine can alter dopamine transport and ligand binding. Some of these changes are found after drug administration is stopped, i.e. during 'withdrawal' (see Discussion for references). For example, a preliminary report 45 found a significant loss of DAT binding in nucleus accumbens at 10 days after cessation of repeated cocaine administration compared to animals sacrificed on either the last day of repeated cocaine administration or the last day of repeated saline administration. This change was not found in the striatum, suggesting a relatively selective effect of drug on mesolimbic pathways. This association with limbic neurons suggested an association with cocaine's reinforcing and addicting properties. The delayed nature of this effect suggested that it might be associated with withdrawal or other long-term effects

133

MATERIALS AND METHODS

cells and is located medially above the caudal portion of the interpeduncular nucleus. The PN contains fusiform cells oriented parallel to the lateral borders of the interpeduncular nucleus. The PBP occupies the area bordered by the red nucleus, superior cerebellar peduncle and the medial lemniscus. Data for each nucleus was obtained at about the same rostro-caudal level. Differences in grain densities between cocaine-treated animals and the saline-treated control group were assessed with a non-parametric KolmogorovSmirnov 2-tailed test 32. This test is useful for comparing different distributions of grains.

Cocaine treatment

RESULTS

of c o c a i n e an d o t h e r p s y c h o s t i m u l a n t drugs. In this study, we f u r t h e r c h a r a c t e r i z e t h e s e c h a n g e s by comparing D A T

binding and m R N A

levels in animals

sacrificed 10 days after r e p e a t e d a d m i n i s t r a t i o n o f coc a i n e or saline.

Male, Lewis (Charles River, Wilmington, MA) rats were treated with cocaine or saline as previously described 45. Lewis rats were utilized because they acquire self-administration behavior more rapidly than several other strains 15. Briefly, the treatment consisted of infusing 1 mg/kg of cocaine hydrochloride through a jugular catheter every 12 rain over a 2 h period. Control groups received saline (0.1 ml/kg). This schedule of administration mimics the total dose and pattern of cocaine intake in animals self-administering the drug 4°. Rats were treated Monday through Friday in the morning over a 2 week period and were killed 10 days after the last treatment.

In vitro binding assay o f dopamine transporters A n i m a l s w e r e t r e a t e d with c o c a i n e or saline, a nd 10 days af t er s t o p p i n g t r e a t m e n t , tissues w e r e assayed for D A T b i n d i n g as d e s c r i b e d in M e t h o d s . T h e S T R exhibited m o r e [ 3 H ] W I N 35,428 b i n d i n g t h a n t h e N A C in b o t h groups of animals. F u r t h e r , t h e r e was no differe n c e in b i n d i n g b e t w e e n saline- and c o c a i n e - t r e a t e d

Binding of DA T Binding to DAT was measured in the terminal fields of the mesolimbic and nigrostriatal dopamine neurons. For this experiment, animals were sacrificed and the brain was removed quickly and placed ventral side up within an ice-cold metal brain matrix (Harvard Apparatus). Brain slices (2 mm wide) containing the striatum (STR) and nucleus accumbens (NAC) were obtained. A dorsal portion of the caudate nucleus and the NAC were removed, frozen and stored at - 20°C until assayed. To measure dopamine transporters, [3H]WIN 35,428 (spec. act. 81.7 Ci/mmol) binding assays were carried out in homogenates as previously described 9 with 0.4 nM [3H]WIN 35,428 and with 30/zM cocaine used to estimate non-specific binding.

an i m al s in t h e S T R . H o w e v e r , in animals w i t h d r a w n f r o m cocaine, a significant d e c r e a s e o f 2 3 % in b i n d i n g was d e t e c t e d in the N A C ( T a b l e I).

In situ hybridization studies A s previously f o u n d 11'47, highest grain densities w e r e m e a s u r e d o v e r cells in t h e substantia nigra pars c o m p a c t a (SNpc) and t h e lowest grain densities w e r e f o u n d in the I F an d C L nuclei. I n t e r m e d i a t e levels w e r e f o u n d over the o t h e r cell g r o u p s including t h e PBP,

In situ hybridization procedure and analysis Five cocaine-treated animals and four saline-treated control animals were prepared for in situ hybridization as previously described 11'47'48. Rats were anesthetized with sodium pentobarbital and sacrificed by intracardiac perfusion with a paraformaldehyde (0.5%) - glutaraldehyde (1%) buffer. After freezing, 10/xm sections from midbrain regions known to contain dopaminergic cell bodies were thaw-mounted onto Denhardt's pretreated microscope slides. Sections were treated with 0.2 N HCI and proteinase K, dehydrated, dried, and hybridized with an 35S-labeled 45 base oligonucleotide at 37°C overnight in a complex buffer tl'a7,4s. Aliquots of the same preparation of radiolabeled probe were used with all sections. Following washes at room temperature and at 50°C, emulsion was applied, autoradiograms were developed, and tissue was stained 47'48. The time of exposures for both treatment and control groups were 2 weeks except for more caudal sections containing the CL nucleus where the exposure was 3 weeks. At 4 weeks of exposure, grain densities in some areas approached saturation. Neurons were identified on the basis of size, shape and presence of nucleoli. Cells with the highest densities of autoradiographic grains were selected and overlying grain densities were measured using a 100× oil objective and eyepiece reticule.. Background levels were assessed by measuring the density of autoradiographic grains in random locations (excluding ventral midbrain) over the tissue. Cells with overlying autoradiographic Brains were assigned to ventral tegmental subnuclei and substantia nigra pars compacta (SNpc) according to published criteria 1°,2°,24,34. The subnuclei of the ventral tegmental area included the interfascicular nucleus (IF), the rostral linear nucleus (RL), the caudal linear nucleus (CL), the paranigral nucleus (PN), and the parabrachiat pigmented nucleus (PBP). The IF was located medial, rostral and dorsal to the interpeduncular nucleus and is composed of small, round cells most of which are densely packed. The RL is another midline nucleus that contains loosely grouped cells, some large, located above the IF and between the oculomotor nerve fibers. The CL contains ventrodorsally oriented

and the R L an d P N nuclei (Figs. 1 an d 2). B a c k g r o u n d levels o f a u t o r a d i o g r a p h i c grains, m e a s u r e d r a n d o m l y over ar eas excluding t h e v e n t r a l m e s e n c e p h a l o n , w e r e still l o w er t h a n t h e a v e r a g e grain densities associated with t h e s e cells ( b e t w e e n 1.75 an d 2.5%). C o m p a r i s o n o f t h e grain densities b e t w e e n t h e saline an d t h e c o c a i n e - t r e a t e d animals r e v e a l e d significant d i f f e r e n c e s in two cell groups, t h e I F ( P < 0.02) and C L ( P < 0.002) nuclei (Figs. 1, 2 an d 3). T h e distribution o f grain densities shown in t h e h i s t o g r a m s was shifted to significantly l o w er levels in t h e tissues f r o m the c o c a i n e - t r e a t e d animals. T h e r e d u c t i o n in a v e r a g e grain density p e r cell was 2 2 % in t h e I F an d 36% in

TABLE I

[ 3H]WIN 35,428 binding in nucleus accumbens (NAC) and striatum (STR) 10 days after cessation of repeated infusions of saline or cocaine Data are presented as mean ± S.E.M. (n = number of animals) dpm [3H]WlN 35,428 specifically bound per mg tissue wet weight. See text for details.

Region

Saline

Cocaine

% Change

NAC STR

510+ 22.1 (9) 1 112± 105.5 (11)

394± 26.5(10) 1070± 150.6 (11)

-23% * n.s.d.

* P < 0.004, two-tailed t test, 17 df. n.s.d. = no significant difference.

134

the CL. The distributions were not significantly different for the other cell groups of dopaminergic neurons. The total number of cells measured in the salinetreated group and cocaine-treated group, in the SNpc, were 134-171 (saline-cocaine); in PBP, 101-111; in CL, 116-133; in IF, 148-152; in PN, 129-157; and in RL, 41-60 respectively. The data were derived from sections exposed for 2 weeks (3 weeks for caudal sections containing CL). When some sections were exposed for 4 weeks, average grain densities were 2-fold higher (data not shown) indicating that our data were gathered under conditions where autoradiograms were not saturated. In a separate series of measurements, the total number of cells with overlying autoradiographic grains per coronal section was not changed by the cocaine treatment in IF (8.28 + 1.83, n = 18 sections from saline-treated vs. 7.23 + 2.01, n = 22 from cocaine-treated) or CL (14.88 + 3.64, n = 8 from

COCAINE

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9-9

10-19

20-29

30-39

40-49

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70-70

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90-99

)100

80-80

90-00

>I00

oI p<0.02

30

20

10

0

5~9

10~19

20"29

30-09

40-49

50-59

90-69

70-79

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'

'

'

p
COCAINE

8o~ SNpc

..

10

0

5-9

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5-9

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saline-treated vs. 13.6 ± 3.22, n = 10 from cocainetreated tissues). DISCUSSION

>100

z0 ! 0

Neuronal Hybridization Density [sliver grelns/(lOpm)

30-89

Fig. 2. Frequency distribution histograms of hybridization grain densities in rostral linear nucleus (RL), interfascicular nucleus (IF) and caudal linear (CL) nucleus. Significant differences were found using Kolmogorov-Smirnov test. See text for details.

n.s.d.

5-9

20-29

Neuronal Hybridization Densl ty [sllver 9 r a l n s l ( 1 0 / / m ) : ]

>100

°"

2o

0

90-99

PBP

l 0

30-39

10"19

2 ]

Fig. 1. Frequency distribution histograms of the hybridization grain densities over cells and background grain density. The most intensely labeled cells in substantia nigra pars compacta (SNpc), parabrachial pigmentosous (PBP) and paranigral (PN) nucleus were examined as described in methods. The animals and tissue treatments are described in Methods. There were no significant differences (n.s.d.) between the saline groups and cocaine-treated groups.

The finding of a decrease in DAT binding in the NAC 10 days after withdrawal of cocaine as reported by Sharpe et al. 45 has been confirmed and extended in this study. We show that the cocaine-induced change in binding is significant when compared to an identically prepared, saline injected control group, whereas Sharpe et al. 45 restricted their comparison to non-withdrawn groups of animals. Pilotte and coworkers (in preparation) have recently extended these experiments to show that the decrease in DAT is not found at 1, 3 or 6 days, although it occurs at 10 days in a highly reproducible fashion. The reduction in DAT binding is also dose-dependent and long lasting. Thus, altered DAT could be a significant factor underlying behavioral and biochemical changes during cocaine withdrawal. The occurrence of change in the NAC but not in the STR emphasizes the importance of limbic regions in

cocaine

saline

PBP

Fig. 3. High magnification photomicrographs after in situ hybridization in different subnuclei including the interfascicular (IF), caudal linear (CL) and parabrachial pigmented (PBP) nuclei after saline or cocaine treatment. Arrows mark cells with overlying elevated grain densities (X 960).

136 psychostimulant action. We observed a 23% decrease in [3H]WlN 35428 binding in NAC with no change in STR. Other investigators also observed no change in rat striatum after abstinence from cocaine 3'6, and Izenwasser and Cox z3, found a decrease in uptake in the nucleus accumbens. However, some inconsistencies in the literature are likely to be due to differences in routes and patterns of drug administration26 (i.v. vs. subcutaneous or i.p., intermittent vs. bolus), or to differences in strains of rats (Lewis vs. Sprague-Dawley). Our experimental conditions were selected to mimic, as closely as possible, drug taking in animal models of human addiction. The intermittant pattern of cocaine administration used here mimics the pattern used by self-administering animalsa°; also, the Lewis strain of rats acquires self-administration behavior more rapidly than some other strains 15. The significant decreases in hybridization density occurred only in the IF and CL nuclei which contain dopaminergic cells that project to the medial NAC, the septum, and the orbitofrontal cortex 1°'27'34. No change was observed in the SNpc which projects to the striaturn. Also, no changes were observed in: the PBP which projects mainly to the amygdala but also to the suprarhinal cortex, anteromedial striatum and lateral accumbens; the PN which projects mainly to the lateral habenula, anteromedial striatum and lateral accumbens and the RL which projects essentially to the septum and cortex 1°'27'3a. Thus, the changes associated with withdrawing from cocaine under these conditions were restricted to the mesolimbic system well known to be implicated in the rewarding effects of cocaine 28. Also, the cellular decrease in mRNA (22% in IF and 36% in CL) corresponds anatomically to the decrease in DAT binding (23%) in the nucleus accumbens and is presumably at least one of the causes of the decreased DAT binding. However, other kinds of regulatory changes that may contribute to the decrease, such as phosphorylation of the DAT protein 4 and reduction in axonal flow 5 cannot be ruled out. In any case, the fact that mRNA levels are changed many days after withdrawal of cocaine indicates that gene expression can be altered well into the withdrawal period. The changes in DAT gene expression in these cell groups are presumably indirect effects of inhibition of dopamine uptake by cocaine. Xia et al. 54 recently reported that DAT mRNA was decreased at 4 h after a last injection of a chronic regimen of cocaine administration but not at 72 h. We did not use conditions similar to those of Xia et al. 54, but their results support the general concept that dopamine uptake inhibition can regulate DAT gene expression. Potentiation of dopamine's action on presynaptic or postsynaptic re-

ceptors by reuptake blockade is likely to be involved in the mediation of the observed changes on DAT gene expression. It is possible that the decrease in mRNA is mediated by input from feedback neurons from nucleus accumbens to A1013'21. Such anatomical connections have been found and may contain Gabaergic or glutamatergic neurons (see Oades and Halliday ~4 and Bjrrklund and Lindvall w for references). It is well known that neurotransmitter receptor activation can alter gene expression 33 and cocaine and amphetamine can indirectly induce immediate early gene expression through activation of dopamine receptors 8'12'18'39. Other influences may underlie the change in mRNA as well. For example, A10 has an input from serotonin (5-HT)-containing cells m and cocaine could cause activation of 5-HT receptors on these cells by blocking 5-HT uptake. Additional experiments will be necessary to elucidate the causes of the mRNA changes. Several other studies show various changes in dopaminergic neurons as a result of withdrawal of chronic cocaine. Electrophysiologic studies 1'2j have provided evidence for autoreceptor subsensitivity in A10 neuronal cell bodies up to four days of withdrawal, but not at later times. Also, the number of spontaneously active neurons in the ventral tegmental area is markedly reduced 10-14 days after withdrawal of cocaine 2. Similarly, Yi and Johnson 55 studied [3H]dopamine release and found evidence for autoreceptor subsensitivity at 7 days after cessation of cocaine. Studies of dopamine effiux with microdiatysis also identify neuronal changes in NAC during drug withdrawal. This work has revealed mainly decreases in dopamine efflux 22'35'41"42'44. Other reports indicate changes in dopamine uptake, amphetamine-stimulated dopamine release, and various other receptors and behaviors during cocaine withdrawal 14'23'38'56'57. Taken together, the above findings suggest that withdrawal from cocaine appears to be characterized by a period of decreased dopamine effiux, autoreceptor subsensitivity, a decrease in number of spontaneously active neurons, and decreased DAT binding. Beitner-Johnson et al. 5, found that chronic cocaine treatment also resulted in decreases in neurofilament protein which is involved in axonal transport in mesolimbic dopaminergic cells. These several findings suggest that the withdrawal period may be characterized by a severely dysfunctional mesolimbic dopaminergic neuron. If similar changes are shown to occur in humans, treatment strategies could be attempted with the goal of reversing such changes. Although the reports mentioned above have utilized

137

animals, studies with human subjects and PET imaging also reveal brain changes during withdrawal. PET studies of cocaine abusers show changes in regional brain glucose metabolism at various times up to 4 months of cocaine withdrawal. In the first week of withdrawal, glucose metabolism increased in basal ganglia and orbitofrontal cortex 53. However, at later times, up to 4 months, glucose metabolism was decreased in frontal regions and correlated with cocaine dose and years of abuse 52. Also, changes in cerebral blood flow were found in anterior brain areas of cocaine users (see Volkow et al. s2 for additional references). While changes in nucleus accumbens were not reported in humans as they have been in animals, the frontal cortical changes reported in human brain imaging studies presumably reflect changes in mesolimbic dopaminergic function, which is compatible with findings in animal studies. Acknowledgements. The authors acknowledge the technical support of Donna Walther and the clerical support of Ms. B. Cepl, and also the help and advice of Dr. Stevens Smith regarding statistics. ABBREVIATIONS

CL DAT 5-HT IF NAC PBP PN RL SNpc STR

,

caudal linear nucleus dopamine transporter serotonin interfascicular nucleus nucleus accumbens parabrachial pigmented nucleus paranigral nucleus rostral linear nucleus substantia nigra pars compacta striatum

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43

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