Brain Research, 578 (1992) 235-243 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

235

BRES 17646

Effects of repeated injections of cocaine on catecholamine receptor binding sites, dopamine transporter binding sites and behavior in rhesus monkey Gail M. Farfel a, Mark S. Kleven a, William L. Woolverton a'b, Lewis S. Seiden a'b and Bruce D. Perry b'c aDepartment of Pharmacological and Physiological Sciences, bDepartment of Psychiatry, and CDepartmem of Pediatrics, Drug Abuse Research Center, The University of Chicago, Chicago, IL 60637 (USA) (Accepted 10 December 1991) Key words: Cocaine; Binding site; Receptor; Dopamine; Caudate; Rhesus monkey

In order to determine if repeated injections of cocaine produced long-lasting alterations in catecholaminergic binding sites, rhesus monkeys were treated with saline (1.0 ml/15 kg) or cocaine (3.0-4.0 mg/kg) four times daily for 14 consecutive days and sacrificed two weeks after the last injection. The densities of dopamine D 1 receptor binding sites, dopamine transporter binding sites and fl adrenergic receptor binding sites were significantly decreased in caudate nucleus to 51%, 17% and 61% of control, respectively, two weeks after repeated cocaine injections. There were no differences in D 2 receptor binding site densities in the caudate, nor were there differences in binding sites between groups in the other brain regions examined: prefrontal cortex (D 1, D2, dopamine transporter, fl), nucleus accumbens (D 1, D2, dopamine transporter) and substantia nigra (D2). Behavioral observation showed that the cocaine-treated monkeys became sensitized to the repeated injections. Early in the regimen, these animals displayed stereotypic grooming, buccal movements and visual checking after each injection that differed significantly from the saline-treated animals. As the regimen progressed, the frequency of grooming decreased while the frequencies of visual tracking and splayed legs increased in a manner consistent with the development of behavioral sensitization. Together, these findings suggest that the caudate nucleus may be more sensitive than other dopamine-containing brain regions to long-lasting pre- and post-synaptic effects of repeated cocaine administration, and that the changes seen in dopaminergic neurons may be related to behavioral sensitization.

INTRODUCTION Cocaine is a psychomotor stimulant which shares physiological and behavioral effects with D-amphetamine and its congeners 9'24'51, although they differ in their chemical structures. Repeated exposure to either D-amphetamine, D-methamphetamine or cocaine has been demonstrated to cause sensitization to stereotypic behaviors, convulsions and pharmacological kindling 4'19'22'24' 37,45. Furthermore, repeated exposure to several amphetamine analogs has been shown to induce long-lasting changes in dopamine (DA) neurons, including decreases in levels of D A and its metabolites, decreases in tyrosine hydroxylase activity, decreases in the Vmax for [3H]DA uptake, morphological degeneration of nerve terminals and changes in densities of D A receptor binding sites 4' 29,42 Due to the similarity of the behavioral effects of cocaine and D-amphetamine, it is important to ascertain whether repeated exposure to cocaine causes long-last-

ing alterations in D A neurons analogous to those of the amphetamines. In contrast to D-amphetamine or its congeners, chronic administration of cocaine generally has not been found to cause long-lasting reductions in concentrations of D A or its metabolites ~8"26'52, although there has been a report of changes in metabolite concentrations for up to 12 weeks after the last injection 2~. Alterations in D A neurons which have been associated with chronic cocaine administration include long-lasting decreases in D A metabolism and either increases or decreases in stimulated D A release ~7'1s'21'35. Changes in D2 receptor binding site densities have also been reported; Goeders and Kuhar 1° reported decreases in D2 binding sites in the striatum and increases in the nucleus accumbens immediately following 15 days of cocaine injections, and Peris and coworkers 35 have reported increases in nucleus a'ccumbens D2 binding sites at 24 hours, but not at one week, following eight days of cocaine injections. Recently, we re-

Correspondence: L.S. Seiden, Dept. of Pharmacological and Physiological Sciences, The University of Chicago, 947 E. 58th Street, Chicago, IL 60637, USA.

236 p o r t e d that rats g i v e n single daily injections o f c o c a i n e for 15 c o n s e c u t i v e days h a d d e c r e a s e d densities o f D 1 b i n d i n g sites in frontal c o r t e x and c o r p u s s t r i a t u m b u t n o t in nucleus a c c u m b e n s two w e e k s after the last injection 25. N o d e c r e a s e in D : binding site densities was obs e r v e d at two w e e k s . T h e p r e s e n t study was d e s i g n e d to e x a m i n e the effects of r e p e a t e d c o c a i n e injections on rec e p t o r b i n d i n g sites in rhesus m o n k e y brain. MATERIALS AND METHODS

Animals The subjects were nine male rhesus monkeys (Macaca mulatta) weighing 10-16 kg at the start of the experiment, with no history of previous participation in drug studies. Monkeys were housed in individual cages in a room maintained at 22-26°C on a 12 h darklight cycle (lights on from 07.00-19.00 h). Subjects were maintained on ad libitum food (Purina Monkey Chow, Ralston-Purina Co., St. Louis, MO) before and after the drug regimen. Due to the anorexic effect of cocaine, drug-treated animals were fed preferred foods such as fruit, chocolate candy and crackers in addition to ad libitum chow during the injection regimen. Saline-treated animals were restricted to 65 g of chow plus one quarter of an apple or orange per day during the injection regimen to control for decreased food intake in the drug-treated group. Water was available ad libitum throughout the experiment, and each animal received one children's chewable vitamin daily (Vi-Daylin, Ross Laboratories, Columbus,OH). Qualitative notations regarding food intake ('Refused apple', 'Ate all chow') were recorded for each monkey each day during the injection regimen although quantitative records of intake were not kept daily. Quantitative measurements were recorded when needed to aid in evaluating the health of the monkeys.

Methods At the start of the experiment, the monkeys were assigned to one of two groups, balanced for weight and approximate age. Animals were injected i.m. with either 0.9% saline vehicle (1.0 ml/15 kg) or cocaine (4.0 mg/kg; 60 mg/ml), four times daily at 08.00, 13.00, 18.00 and 23.00 h for 14 days. Three animals began the injection regimen each day for three consecutive days. On the fourth day after the start of the injections, one monkey from the first group died, apparently after convulsing. Three days later another monkey convulsed after receiving cocaine, and was treated immediately with diazepam (15 mg as 5 mg/ml, i.m.). The next day the dose for all monkeys was reduced to 3.0 mg/kg/injection for the remainder of the experiment.

The monkeys were scored at least twice daily for the presence of cocaine-induced behaviors (see Table I) using a modified version of a checklist described by Post and co-workers 37. Animals were observed in their home cages approximately 15 rain after the injection. All ratings were performed by the same investigator (G.M.E), and each animal was observed for a minimum of 60 s. Statistical differences within and between groups were tested by Kullback's information statistic 28'~°, where the criterion for significance was P ~< 0.05. Two weeks after the last injection, the animals were anesthetized with Ketalar (ketamine HCI, 6.5-8.1 rng/kg; i.m.) followed by sodium pentobarbital (approximately 120-270 mg as 30 mg/ml; i.v.) and sacrificed by exsanguination. The brains were removed within 5 min and taken to a cold room (4°C) where regions were dissected from slices in a manner analogous to that described previously for rat brain dissection ~3. Sections comprising prefrontal cortex, caudate, nucleus accumbens and substantia nigra were wrapped in aluminum foil, frozen in liquid nitrogen and stored at -70°C until preparation for assay. Radioligand binding methods for DI and D z dopamine binding sites, dopamine transporter binding sites and fl adrenergic binding sites were adapted from original techniques 3"s'n'~6"43. To prepare tissue for binding, samples were first thawed over ice, then minced with a single-edged razor and mixed. A small aliquot of tissue (50100 mg) was removed from caudate and prefrontal cortex samples for assay of monoamine levels and metabolites (see below). Tissue samples were homogenized in ice-cold 50 mM Tris-HCl buffer (pH 7.7, 25°C) containing 120 mM NaCI and 5 mM EDTA. Aliquots were distributed into tubes and centrifuged at 43,000 x g for 15 rain at 4°C. The supernatant was discarded and the pellets were stored at -70°C until resuspended in assay buffer (50 mM Tris-HC1, pH 7.7, 25°C, with 120 mM NaCI) at time of assay. For each assay, the data represent the results of a single determination with each subject (n --- 4 saline-treated animals and n = 4 cocainetreated animals). D1 dopamine binding sites were labeled with [125I]SCH 23982 (NEN Dupont, 2200 Ci/mmol). Binding sites were quantified using saturation studies (six or eight concentrations ranging from 0.012.7 nM). In brief, aliquots of tissue (10-160 ag protein/ml) in assay buffer (50 mM Tris-HCl, pH 7.7, 25°C, with 120 mM NaCI) were incubated with increasing concentrations of radioligand and 0.5/~M ketanserin for 60 rain at 37°C in a volume of 320 al. Parallel incubations in the presence of 1.0 mM fluphenazine defined specific binding. D 2 dopamine binding sites were labeled with [3H]spiperone (NEN Dupont, 32.4 Ci/mmol) in saturation studies using six concentrations ranging from 0.01-2.7 nM. Aliquots of tissue (10-150/~g proteirdml) in assay buffer (50 mM Tris-HCl, pH 7.7, 25°C, with 120 mM NaCI) were incubated with increasing concentrations of radioligand and 10/~M mianserin for 45 min at 37°C in a volume of

TABLE I

Definitions of behaviors scored 15 minutes post-injection Activity

Description

Locomotor activity

Translocation in the cage, usually referring to leg or whole body movement but also used to describe large swings of the upper body

Grooming/Bug-picking

Repetitive petting or picking at hair or skin

Visual checking

Rapid, continuous shifts of visual field resulting from repetitive eye and/or head movements

Visual tracking

Continuous, slow searching of the visual field for apparently nonexistent objects, often accompanied by staring

Buccal movement

Repetitive movements of the tongue or lips

Splayed legs

Legs spread apart and turned outward, often accompanied by swaying

237 520 ~1. Parallel incubations in the presence of 1.0 mM haloperidol defined specific binding. Dopamine transporter sites were labeled with [3H]GBR 12935 (NEN Dupont, 20.7 Ci/mmol) in saturation studies using six concentrations ranging from 0.1-2.5 nM. Aliquots of tissue (10-200/~g protein/ml) in assay buffer (50 mM Tris-HCl, pH 7.7, 25°C, with 120 mM NaCl) were incubated with increasing concentrations of radioligand for 45 min at 25°C in a volume of 520 pl. Parallel incubations in the presence of 50/~M GBR 12909 defined specific binding. fl Adrenergic binding sites were labeled with [125I]iodocyanopindolol ([125I]ICYP; NEN Dupont, 2200 Ci/mmol) in saturation studies using six concentrations ranging from 0.005-0.22 nM. Aliquots of tissue (60-310/~g protein/ml) in assay buffer (50 mM Tris-HCl, pH 7.7, 25°C, with 120 mM NaC1) were incubated with increasing concentrations of radioligand for 60 rain at 37°C in a volume of 300 /A. Parallel incubations in the presence of 1.0 mM propranolol defined specific binding. For all assays, incubation was terminated by filtration under reduced pressure over Whatman GF/B filter paper using a Brandel Cell Harvester modified for radioligand binding assays. Filter paper was soaked in 0.01% polylysine to reduce non-specific binding. Filters were rinsed 3 times with 5.0 ml ice-cold "Iris HC1 buffer. For [3H]spiperone and [3H]GBR 12935, filters were placed in glass vials and after addition of 3.0 ml of CytoScint (ICN Biomedicals, Inc.), the samples were allowed to equilibrate for at least 12 h before counting by liquid scintillation spectrometry. For [x25I]SCH 23982 and [125I]ICYP the filters were placed in vials and counted on a Micromedic gamma counter. Raw counts from all assays were transformed using RADLIG 3°, a radioligand binding analysis software package which contains EBDA and a version of LIGAND derived from the original32. In all cases saturation isotherms were best fit by a single site model and final derivation of radioligand affinity (Kd) and the density of binding sites (Bmax) was made using weighted Eadie-Hofstee plots as described by Zivin and Waud 55 and adapted for Lotus 1-2-3. In all cases, computer-assisted analysis of binding isotherms indicated a single, saturable site of interaction, where specific binding varied as a function of the density of binding sites and concentration of radioligand, Statistical differences between groups were tested by one-way ANOVA, and the criterion for significance was P ~< 0.05. Protein content was determined using the commercially available Pierce assay (Pierce, Rockford, IL). Samples from the caudate and prefrontal cortex were assayed for DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT), and 5-hydroxyindole acetic acid (5HIAA) content using high performance liquid chromatography with electrochemical detection as described previously2"27. Briefly, samples were eluted on stainless steel RSIL 5 /~m C18-HL ODS reverse-phase columns at a flow rate of 1 ml/min. The aqueous mobile phase contained 0.34 mM EDTA, 0.09 mM octylsodium sulfate. 7.0 mM Na~HPO4, 68 mM citric acid and 10% methanol. Compounds were detected electrochemically by oxidation over a glassy carbon working electrode at a potential of +0.75 V relative

to the Ag/AgC1 reference electrode. Output from the detector (BAS LC-2a, Bioanalytical Systems, or EG & G Model 400, Princeton Applied Research) was channeled to an integrator (Shimadzu CR5-A) which monitored peak retention times and calculated peak areas and values based on a two point calibration of standards. Statistical differences between groups were tested by one-way ANOVA, and the criterion for significance was P ~< 0.05.

RESULTS

Effects o f repeated cocaine on binding site densities Repeated

cocaine

administration

significantly

sites in the c a u d a t e nucleus f r o m 228 + 33 f m o l / m g p r o tein ( m e a n + S . E . M . ) in s a l i n e - t r e a t e d animals to 117 + 15 ( F = 9.44; P < 0.05) in c o c a i n e - t r e a t e d m o n k e y s w h e n e x a m i n e d t w o w e e k s after the last i n j e c t i o n (Table II). D u e to b e t w e e n - s u b j e c t variability, the densities of [125I]SCH 23982-labeled binding sites in the p r e f r o n t a l c o r t e x w e r e not statistically d i f f e r e n t (26 + 16 for saline vs. 107 + 35 for c o c a i n e ; F = 3.46; P = 0.12), n o r was t h e r e any d i f f e r e n c e in [~25I]SCH 23982-labeled binding site densities in the nucleus a c c u m b e n s .

D e n s i t i e s of

[3H]spiperone b i n d i n g sites w e r e not significantly different b e t w e e n g r o u p s in c a u d a t e n u c l e u s , p r e f r o n t a l cortex, nucleus a c c u m b e n s o r substantia nigra t w o w e e k s after the last c o c a i n e injection. T h e a p p a r e n t increase in [3H]spiperone binding in nucleus a c c u m b e n s of c o c a i n e t r e a t e d animals was d u e to a large Bmax in o n e m o n k e y . A

significant d e c r e a s e

was f o u n d

in the

density

for saline vs. 170 + 96 for c o c a i n e ; F = 11.30; P < 0.05) but n o t p r e f r o n t a l c o r t e x nor nucleus a c c u m b e n s , again due to i n t e r - s u b j e c t variability. In addition, r e p e a t e d cocaine injections c a u s e d a small but statistically significant d e c r e a s e (31 + 2 for saline vs. 19 + 3 for c o c a i n e ; F = 12.77; P = 0.01) in [125I]ICYP b i n d i n g in c a u d a t e nucleus. T h e r e was no d i f f e r e n c e in [125I]ICYP b i n d i n g obs e r v e d in p r e f r o n t a l cortex. It is w o r t h w h i l e to n o t e that the variability in Brnax in the [125I]SCH 23982, [ 3 H ] G B R 12935 and [3H]spiperone assays was due to the s a m e o n e o r two animals in all re-

Average B,~ax values following repeated administration of saline or cocaine (3-4 mg/kg) Values are means ± S.E.M. expressed in fmol/mg protein. * Differs from Sal (P