- Email: [email protected]
Lack of long-term changes in cocaine and monoamine concentrations in rat CNS following chronic administration of cocaine
Pergamon
0197-0186(95)00061-5
Neurochem. Int. Vol. 28, No. 1, pp. 51-57, 1996 Copyright© 1996ElsevierScienceLtd Printedin Great Britain.All rights reserved 01974)186/96 $9.50+0.00
LACK OF LONG-TERM CHANGES IN COCAINE A N D M O N O A M I N E CONCENTRATIONS IN RAT CNS FOLLOWING CHRONIC ADMINISTRATION OF COCAINE MARIO E. ALBURGES, 1'2 DENNIS J. CROUCH, 1 DAVID M. A N D R E N Y A K l and JAMES K. WAMSLEY 3. ~Center for Human Toxicology, Pharmacology and Toxicology Department, University of Utah, Salt Lake City, UT 84108, U.S.A. 2C~itedra de Toxicologia, Escuela de Bioanhlisis,Facultad de Medicina, Universidad del Zulia, Maracaibo, Zulia, Venezuela 3Departments of Psychiatry and Pharmacology, New York Medical College, Valhalla, NY 10595, U.S.A. (Received 14 November 1994 ; accepted 4 April 1995)
Abstract--In previous studies, we reported time-dependent and dose-dependent changes in the rat dopaminergic receptor system followingchronic administration of cocaine. The aim of the present investigation was to monitor the concentration of monoamines (using HPLC-ECD) and cocaine (using GC-PCI/MS) in rat CNS following a dose schedule of 5, 10, 15, 20 and 25 mg/kg, i.p., b.i.d, for 21 days. 12 h after the last cocaine injection, cortical and striatal concentrations of monoamines and their metabolites were not significantly different in saline vs cocaine treated animals. In addition, the cocaine concentration in the brain regions examined did not change with the different doses used. Accumulation of a metabolite of cocaine (ecgonine methyl ester) was the only alteration found. These results indicate that alterations in the dopaminergic receptor system following chronic cocaine administration are not due to changes in neurotransmitter concentration or accumulation of cocaine in the brain.
COC in many organ systems has been attributed to the drug's potent vasoconstrictive properties (Gay, 1982; Coleman et al., 1982; Kaku and Lowenstein, 1990; Sloan et al., 1990; Levine et al., 1991 ; Goldfrank and Hoffman, 1991). The behavioral consequences of COC exposure have been credited to induced changes in neurotransmitters with the dopaminergic system represented as the most responsible for the adverse effects (Roberts et al., 1977 ; Dackis et al., 1986; Mattia et al., 1986; Kuhar et al., 1988; Bergman et al., 1989 ; Johanson and Fischman, 1989 ; *Author to whom all correspondence should be addressed. Abbreviations: Cocaine (COC); benzoylecgonine (BE); Carroll et al., 1992, 1993). Important research has been performed in an ecgonine methyl e s t e r (EME); 3, 4-dihydroxyphenylethylamine, dopamine (DA); 3, 4-dihy- attempt to uncover the mechanism of COC toxicity in droxyphenylacetic acid (DOPAC) ; 3-methoxy-4- the CNS (Taylor and Ho, 1978; Ritz et al., 1987; hydroxyphenylacetic acid (HVA); 1-(3,4-dihydroxyphenyl)-2-aminoethanol, norepinephrine (NE) ; 1- Roberts et al., 1977 ; Kuhar et al., 1988 ; Pettit et al., (3,4-dihydroxyphenyl)-2-(methylamino)ethanol, epi- 1990; Kalivas and Duffy, 1990). However, little is nephrine (E); 3-methoxy-4-hydroxyphenethyleneglycol known about the relationship between the neuro(MHPG); 5-hydroxytryptamine, serotonin (5-HT); 5- biology of COC and its euphorigenic and psyhydroxyindoleacetic acid (5-HIAA); high-performance chotropic effects. Investigations have provided liquid chromatography/electrochemical detection (HPLC-ECD) ; gas chromatography-positive chemical substantial evidence that one of the primary neurochemical consequences of cocaine administration ionization/mass spectrometry (GC-PCI/MS). 51
Drug addiction is one of the major health problems in our society. The incidence of abuse of behaviorally active drugs has increased tremendously in the last two decades. Drug abuse patterns have shifted from heroin to agents such as cocaine (COC), amphetamines and other central nervous system (CNS) stimulants (Tims and Leukefeld, 1993). Of these, COC has characteristically been one of the most abused sympathomimetic drugs. The widespread toxicity of
Mario E. Alburges el al.
52
is the blockade o f the d o p a m i n e transport system, resulting in an increase in brain concentrations o f the neurotransmitter (Ritz et al., 1987; Fowler et al., 1989; Roberts and K o o b , 1982; Woolverton and Johnson, 1992). Several reports indicate that the reinforcing properties of COC are also associated more with the dopaminergic system than other neurotransmitter systems ( K u h a r e t a l . , 1988, 1 9 9 1 : D a c k i s e t a l . , 1986: Roberts et al., 1977). In addition, previous experiments in rodents, have demonstrated that alterations occur in d o p a m i n e (DA) transporters, D~ receptor binding (Ritz et al., 1987: Alburges et al., 1993a; Wamsley and Alburges, 1993) and D~ receptor m R N A (Laurier et al., 1994) in response to chronic administration o f COC. These changes are associated with modulation o f D r r e c e p t o r s and D A - u p t a k e sites in distinct areas o f brain. One explanation for the receptor changes is that COC administered chronically reduces the neurotransmitter concentrations such that subsequent receptor upregulation occurs. A downregulation o f D A receptors might be expected to occur acutely in response to C O C because o f the induced increase in synaptic DA. However, long-term changes in the neurotransmitter (several hours after injection o f COC) have not been adequately addressed. In the present study, the effect o f chronic COC administration on neurotransmitter concentrations was investigated, and the accumulation of COC and its metabolites in brain tissue were determined. These parameters were analyzed following injection o f various doses o f C O C (5, 10, 15, 20 and 25 mg/kg, i.p., b.i.d.). The neurotransmitters and their metabolites were measured in striatum and cortex using H P L C ECD. COC and its metabolites (BE and E M E ) were measured using G C - P C 1 / M S .
EXPERIMENTAL PROCEDURES
Animal dosing and tissue preparation Male, Long-Evans rats weighing 18~220 g were maintained on a 12 h light/dark cycle with food and water available ad libitum. Based on results obtained in previous experiments (Alburges et al., 1993a ; Wamsley and Alburges, 1993), individual groups of rats (5 6 animals/group) were injected with saline or COC (5, 10, 15, 20 and 25 mg/kg, i.p., b.i.d.) for 21 days. 12 h after last injection, the animals were deeply anesthetized with carbon dioxide and perfused intracardially with an ice-cold isotonic saline solution. Cortices and striata were dissected, frozen and stored at - 7 O C until the monoamines, COC and their metabolites were analyzed. Brain regions from each animal were processed individually.
4nalvsis t~/ monoamines atut their metabolites Biogenic amines and their metabolites, were analyzed using the HPLC ECD method developed by Alburges et al. I 1993b). Brain tissue ( 10 20 mg) from each individual animal was homogenized in a 0.05 N perchloric acid solution containing dihydroxybenzylamine as an internal standard. Filtrates from brain homogenates were analyzed using a Waters 600E Multisolvent delivery unit, a WISP 700 automatic sample injector, a Nova-pack C~ column (3.9 x 300 mm, 4 I~m), and a model 464 pulsed electrochemical detector (Waters and Dynamic Solutions, division of Millipore, Milford, MA). Analvsi.s o[ cocaine and its metabolites COC. benzoylecgonine BE and ecognine methyl ester EME concentrations were obtained using a GC PCI/MS method (Crouch and Alburges, in preparation). A solidphase extraction device (Worldwide Monitoring Corporation, Horsham, PA) with vacuum manifold and vacuum gauge was employed. The solid-phase (SP) extraction cartridges used were 200 mg Clean Screen", ZSDAU020 (Worldwide Monitoring Corporation, Horsham, PA). Tissue samples (100 200 mg) were homogenized in 1 ml of 100 mM sodium phosphate buffer, pH 6.0, (sodium phosphate dibasic and sodium phosphate monobasic were obtained as analytical reagents from Mallinckrodt Chemical Works, St Louis, MO) containing an internal standard ( C O C - D 3 BE-D3 and EME-D3 ; 25 ng/ml each ; Radian Corporation, Austin, TX). Drug free ("blank") brain tissues (100 mg of tissue) were spiked with a standard mixture containing COC, BE and EME (0.5 50 ng/ml of each drug: Alltech Associates Inc., Deerfield, IL) and the internal standard. After homogenization, the samples were processed by solid-phase extraction (SPE). The extractions were carried out according to the Clean Screen 'j' procedure (CBB200DAUZ050191, Worldwide Monitoring Corp., Hotsham, PA) with some modifications. Prior to the sample extraction, the SP-columns were conditioned with a 3 ml portion of methanol followed by a 3 ml portion of deionized water and then a 1 ml portion of 100 mM sodium phosphate buffer (pH 6.0). After centrifugation, the supernatants from the tissue homogenates were loaded onto individual columns and the vacuum was maintained at ~<3 in. of Hg. After the samples were drawn through the columns, the cartridges were washed with one 2 ml portion of 100 mM HCI and one 3 ml portion of methanol. The columns were allowed to dry Jbr 5 rain under vacuum (>~ of Hg). The analytes were then eluted from the columns with 3 ml of solvent mixture (methylene chloride, isopropyl alcohol, ammonium hydroxide ; 78 : 20 : 2). The elutes were collected in 13 x 100 glass tubes and evaporated to dryness at ~<40'C in a water bath under a gentle stream of air. The residues were reconstituted in 60-80 #l of MTBSTFA (N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide; Price, Rockford, IL), and placed in a heating block at 80°C for 45 rain. After derivatization. the tubes were removed from the heating block, cooled to room temperature, and a 40-50 pl vol of the residue was transferred to an autosampler vial for analysis. The analysis was performed with a Finnigan 9610 gas chromatograph equipped with a 4500 mass detector and with a CTC A200S autosampler. A capillary column (DB5 15 m-1 /~m; J & W Scientific, Felsom, CA) was used. The temperatures of the injector port and transfer line were 265 and 250°C, respectively. The ions monitored in the analysis were m/z 304, 314 and 4(/4, for COC, EME, and BE, respectively. The ions t\~r
Cocaine : effects on brain monoamine concentrations the internal standards were m/z 307, 317 and 407 for COCD3, EME-D3 and BE-D3, respectively. Drug concentrations were calculated against a calibration curve based on peak height ratios of analytes and a deuterated internal standard, as defined by a linear regression equation. Data in all the experiments are expressed as the mean+ SEM. These data were initially examined by an ANOVA for multiple comparisons. The posteriori comparisons were made using a Dunnett's t-test. The degree of significancewas set at P ~<0.05.
53
A. Strlatal t i s s u e 80
• = ~, = E E gl.
60
40
20
RESULTS
In order to study the effect of chronic COC administration on central neurotransmitter systems, cortices and striata from animals injected with different doses of COC (5, 10, 15, 20 and 25 mg/kg) or saline for 21 days, were analyzed for monoamines and their metabolites, 12 h after last injection. HPLC-ECD analysis of DA and its metabolites in cortical and striatal tissues are shown in Figs 1 (A) and (B). The concentrations of DA, DOPAC, and HVA in tissues from animals injected with COC in the dose-dependent protocol, were not significantly different from the saline group. Striatal and cortical DA and its metabolites [Figs 1(A) and (B)] did not change, even with progressive increase of the dose of COC administered. Striatal DA, DOPAC, and HVA were in the range of 39.32 + 5.98~2.50 + 12.51 pmol/mg tissue, 18.62+2.20-28.99+5.66 pmol/mg tissue, and 15.22___2.01-22.79+4.01 pmol/mg tissue, respectively. Cortical DA, DOPAC, and HVA concentrations were 1.80 + 0.25-2.50 + 0.13, 4.52 _+1.0407.10+1.01, and 0.89+0.10-1.51+0.90 pmol/mg tissue, respectively. Figures 2 (A) and (B) show that chronic administration of higher doses of COC did not significantly change the concentration of NE, E, and MHPG in cortical and striatal tissues. The concentration of cortical NE and MHPG [Fig. 2(B)] did show an apparent dose-dependent trend that was not statistically significant. The striatal concentrations of NE, E, and MHPG were in the range 0.51__0.08-0.69+0.13, 1.49 + 0.18-2.08 _ 0.24, and 0.30 + 0.08-0.65 + .0.06 pmol/mg tissue, respectively. The cortical concentration values for NE, E, and MHPG were in the range 2.21 + 0.50-3.85 ___0.52, 0.20 + 0.80-0.52 + 0.75, and 2.22+0.31 3.10+0.42 pmol/mg tissue, respectively. Chronic administration of different doses of COC was without effect on the concentrations of 5-HT and 5-HIAA in striatal and cortical tissues (Fig. 3). Concentrations of 5-HT [Figs 3(A) and (B)] show slight dose-dependent increases, but there is no significant
Saline 5 10 15 20 25 Cocaine doses (mg/kg, i.p., b.i.d.)
B. Cortical
tissue
10
8 0 m
~
e
E O
E 2
0 Saline 5 10 15 20 25 Cocaine doses (mg/kg, I.p., b.i.d.)
Fig. 1. Effects of chronic COC administration on rat striatal (A) and cortical (B) concentrations of DA, DOPAC and HVA. Results are expressed as pmol/mg tissue and represent the mean ___SEM of 5 ~ animals per group. Concentrations of DA, DOPAC and HVA in cortices and striata from animals injected with COC were not significantlydifferent from control animals. difference between the saline group and COC treated animals at any of the different doses. The striatal concentrations of 5-HT and 5-HIAA in COC or saline injected animals varied from 1.65 _ 0.48 to 2.59 _+0.42 pmol/mg tissue and from 2.67+0.60 to 3.33+0.67 pmol/mg tissue. The cortical concentrations of 5-HT and 5-HIAA in these animals were in the range 1.78+0.51-2.89+0.52 and 1.98+0.34-2.99+0.68 pmol/mg tissue. The GC-PCI/MS method described herein was sensitive enough to detect 500 pg/ml or 5 pg/mg of tissue, and satisfactory separation of the three analytes under study was found (COC, BE and EME). Figures 4 (A) and (B), show the striatal and cortical concentrations of COC and EME, 12 h after the last administration of different doses of COC (5, 10, 15, 20 and 25 mg/kg)
Mario E. Alburges et al.
54
A. Striatal t i s s u e
Q
[]
NE-St
[] •
E-St MHPG-St
A. Striatal t 5
[]
5HT-St
•
5HIAA-St
tissue
°-4., O1 O
E
o.
1-
o
o
Saline 5 10 15 20 25 Cocaine doses (mg/kg, i.p., b.i.d.)
B. Cortical 54o
: ;iiiiii O.
[]
NE-Cx
[]
E-Cx.
Saline 5 10 15 20 25 Cocaine doses (mg/kg, i.p., b.i.d.)
tissue
B. Cortical
=~
3"
O1
S B
2"
O
1 0 Saline 5 10 15 20 25 Cocaine doses (mgJkg, i.p., b.i.d.) Fig. 2. Effects of chronic COC administration on rat striatal (A) and cortical (B) concentrations of NE, E and MHPG. Results are expressed as pmol/mg tissue and represent the mean+SEM of 5-6 animals per group. Concentrations of NE, E and MHPG in cortices and striata from animals injected with COC were not significantly different from control animals.
or saline. In both tissues, the concentrations of BE were ~< 5 pg/mg tissue. The striatal concentrations of COC and E M E were in the range 6.18+0.7(~ 10.29+5.37 and 17.29_+2.9340.86+4.59 pg/mg tissue. Cortical concentrations of C O C and E M E were 7.91 +_ 1.43-14.07 + 2.18 and 31.06 + 3.66-55.23 _+ 5.60 pg/mg tissue, respectively. Cortices and striata from animals injected with 15, 20 and 25 mg/kg of COC exhibited significant accumulations of E M E when compared to groups of animals injected with the lower dose of C O C (5 vs 15 mg/kg, P ~< 0.01 ; 5 vs 20 and 25 mg/kg, P ~< 0.05). Nevertheless, the concentration of striatal and cortical COC in animals injected with different doses of COC showed no dose-dependent differences.
5HT-Cx
"
5HIAA'Cx
5
¢D •,=
[]
tissue
41
]
:NIl1/ o
Saline 5 10 15 20 25 Cocaine doses (mg/kg, i.p., b.i.d.)
Fig. 3. Effects of chronic COC administration on rat striatal (A) and cortical (B) concentrations of 5-HT and 5-HIAA. Results are expressed as pmol/mg tissue and represent the mean + SEM of 5 ~ animals per group. Concentrations of 5HT and 5-HIAA in cortices and striata from animals injected with COC were not significantly different from control animals. DISCUSSION
Investigators have suggested that the primary action of C O C in the CNS is to inhibit D A uptake in dopaminergic neurons (Johanson and Fischman, 1989; Woolverton and Johnson, 1992). Thus, it is expected that many of the psychotropic effects of COC may be related to excess of D A neurotransmission. Previous results have provided evidence suggesting that increases in Dl-receptors, DA-uptake sites and COC binding accompany chronic administration of COC (Alburges et al., 1993a ; Wamsley and Alburges, 1993). COC-induced increases in binding associated with the D A transporter complex, might be expected since COC acts as an antagonist to the process and may cause upregulation. The increase in D,-receptor
Cocaine : effects on brain monoamine concentrations
A. Striatal tissue 50 40 Q
30 O1
20 OI Q.
10 0 Saline 5 10 15 Cocaine doses (mglkg,
20
25
i.p., b.i.d.)
B. Cortical tissue
70 60 Q Q0 ¢9
50 40
E
30
O.
20 10 0 Saline 5 10 Cocaine doses
15
20
25
(mg/kg, i.p., b.i.d.)
Fig. 4. Striatal (A) and cortical (B) concentrations of COC and EME, 12 h after the last administration of different doses of COC. Results are expressed as pg/mg tissue and represent the mean + SEM of 5-6 animals per group. Cortices and striata from animals injected with 15, 20 and 25 mg/kg COC exhibited a significant accumulation of EME when compared with the group of animals injected with a lower dose of COC, 5 mg/kg. (*P ~< 0.05 ; **PS <~ 0.01).
binding which accompanies chronic COC administration, is more perplexing. However, the increase could be due to a depletion of D A feasibly caused by the prolonged blockade of reuptake by COC. The D]-receptor upregulation could ultimately result as a consequence of diminished concentrations of DA. In the present study, an experiment was designed to provide information on the potential presence of longterm changes in the brain concentrations of COC, monoamines, and their metabolites, in response to injection of various doses of COC. Changes in these neurochemicals could play an important role in inducing the alterations in the dopaminergic receptor system seen after chronic administration of COC. Concentrations of DA, NE, serotonin and their
55
metabolites were measured in striatal and cortical brain regions from animals chronically injected with COC. None of the COC doses used were able to produce significant changes in the brain concentrations of monoamines or their metabolites. Reports from other investigators, relating the effect of COC to the concentration ofmonoamines, have shown short-term increases in monoamine levels. Studies where increased concentrations of brain monoamines were described (Bradberry and Roth, 1989; Pettit et al., 1990; Kalivas and Duffy, 1990; Pettit and Justice, 1989), involved the observation of high levels of D A 15-30 min following administration of the drug. However, these changes were transient and in 60-120 min the D A values returned to the base line concentration. It is very difficult to establish a direct comparison between these studies due to differences in treatment regimes, schedules used in the administration of the drug, and in the biological specimen collection time. On the other hand, our results are in accordance with previous reports (Hanson et al., 1987; Kleven et al., 1988; Yeh and DeSouza, 1991; Alburges and Wamsley, 1993) where 10-20 mg/kg of COC was used in the treatment, and the brain tissues were obtained at least 12-18 h after the last administration of COC. We selected a long postinjection time to examine, since Dl-receptor upregulation had been reported to be measurable 12 h after the last administration of COC (Alburges et al., 1993a). In order to account for this upregulation, the concentrations of dopamine should still be reduced at this time point. This was clearly not the case. The present study marks the first investigation to examine potential neurochemical changes following 21 days of treatment with COC. Hydrolysis (formation of BE and EME) and Ndemethylation (formation of norcocaine) are the main pathways of the metabolism of COC animals (including humans). Using a newly developed G C - P C I / M S method, we measured the concentrations of COC, BE and EME in striatal and cortical tissue from animals chronically treated with five different doses of COC. Our findings demonstrate that even with the lowest dose of COC administered (Smg/kg), tissues obtained 12 h after the last COC administration still contain COC at concentrations of approx. 10 pg/mg tissue. Even though COC did not show progressive accumulation with an increase in dose, it is not clear if the concentration of COC remaining in the brain could affect the threshold of the drug's behavioral toxicity. In addition, these results clearly demonstrate that 15, 20 and 25 mg/kg doses of COC lead to cortical and striatal tissue accumulation of one of the major metabolites of COC (EME).
Mario E. Alburges et al.
56
Investigators have l b u n d that only norcocaine has c o m p a r a b l e brain activity to C O C (Hawks et al.. 1975). Our study did not include m e a s u r e m e n t of norcocaine in the brain. However, an accumulation of E M E was noted. A n y relationship between the presence of these metabolites and the neurotoxicity of C O C is u n k n o w n . This potential relationship needs to be addressed in further studies, as well as determ i n a t i o n o f the c o n c e n t r a t i o n of norcocaine in striatal a n d cortical tissues after chronic a d m i n i s t r a t i o n o f COC. In summary, the psychotropic a n d reinforcing proprieties o f C O C have been related to the drugs' effects on m o n o a m i n e activity (Janowsky a n d Davis, 1976Snyder, 1972; Jaffe, 1990; L i e b e r m a n et al., 1990). These observations suggest that chronic administration o f C O C might cause long-term changes in the brain m o n o a m i n e systems. Nevertheless, the present results indicate that chronic C O C a d m i n i s t r a t i o n does not produce significant a n d persistent changes in the striatal a n d cortical c o n c e n t r a t i o n of DA, NE, serotonin or their metabolites. In addition, the same dose schedule failed to produce significant changes in the brain c o n c e n t r a t i o n of C O C in these animals. However, a n accumulation of E M E was observed with the three higher C O C doses used. This E M E accumulation m a y indicate a saturated t r a n s p o r t system or damage, that can affect the removal process of E M E out of the brain. It is possible, that the behavioral sensitization effects described d u r i n g chronic C O C use, may be associated with changes in the dopaminergic receptor system. This m a y involve the upregulation (possible supersensitivity) in D~-receptors, C D C binding and D A - u p t a k e sites, as reported previously ( K u h a r et al., 1991 ; Alburges et al., 1993a ; Wamsley and Alburges, 1993) and m a y also involve other alterations in brain t r a n s p o r t mechanisms responsible for the elimination of C O C and its metabolites from the brain. Acknowledgements The authors wish to express their appreciation to Candy Johnson and Alan Spanbauer for their technical assistance: and Estrella Urdaneta-Alburges for her secretarial skills applied to the manuscript. Dr Mario E. Alburges is an aggregate professor from the University of Zulia, School of Medicine, Maracaibo, Zulia, Venezuela, on leave of absence. Part of this research was supported by a grant from the Public Health Service (DA 05167).
REFERENCES
Alburges M. E. and Wamsley J. K. (1993) Effects on monoamine levels in rat CNS after chronic administration of cocaine. Invest. Clin. 34, 181 192. Alburges M. E., Narang N. and Wamsley J. K. (1993a)
Alterations in the dopaminergic receptor system alter chronic administration of cocaine. Synapse 14, 314-323. Alburges M. E. Narang N. and Wamsley J. K. (1993b) A sensitive and rapid HPLC ECD method for simultaneous analysis of norepinephrine, dopamine, serotonin and their metabolites in brain tissue. Blamed. Chromatogr. 7, 30(~ 310. Bergman J., Madras B. K., Johnson S. E. and Spealman R. D. (1989) Effects of cocaine and related drugs in nonhuman primates. II!. Self-administration by squirrel monkeys. J. Pharmac. exp.Ther. 219, 15(~155. Bradberry C. W. and Roth R. H. (1989) Cocaine increases extracellular dopamine in rat nucleus accumbens and ventral tegmetal area as shown by in vivo microdialysis. Neurosci. Lett. 103, 97 102. Carroll F. I., Lewin A. H., Boja J. W. and Kuhar M. J. (1992) Cocaine receptor : biochemical characterization and structure-activity relationships for the dopamine transporter. J. Med. Chem. 35, 969-981. Carroll F. 1., Gray J. L., Abraham P.. Kuzemko M. A., Lewin A. H., Boja J. W. and Kuhar M. J. (1993) 3-Aryl2(3'-substituted-l', 2', 4'-oxadiazol-5'-yl) tropane analogues of cocaine : Affinities at the cocaine binding site at the dopamine, serotonin, and norepinephrine transporters. J. Med. Chem. 36, 2886-2890. Cocaine: Clean Screen~-CBB200DAU050191. Worldwide Monitoring Corp., Horsham, P. A. Coleman D. L., Ross T. F. and Naughton J. L. (1982) Myocardial ischemia and infarction related to recreational cocaine use. West. J. Med. 136, 444-446. Dackis C. A., Gold M. S., Davies R. K. and Sweeney D. R. (1986) Bromocriptine treatment for cocaine abuse: The dopamine depletion hypothesis. Int. J. Psvchiat. Med. 15, 125 135. Fowler J. S., Volkow N. D., Wolf A. D., Dewey S. k., Schlyer D. J., MacGregor R. R., Hitzemann R., Logan J.. Bendriem B., Gatley S. J. and Christman D (1989) Mapping cocaine binding sites in human and baboon brain in l,it~o. Synapse 4, 371-377. Gay G, R. (1982) Clinical management of acute and chronic cocaine poisoning. Ann. Emerq. Med. 11, 562 572. Goldfrank L. R. and Hoffman R. S. (1991) The cardiovascular effects of cocaine. Ann. Emer#. Med. 20, 165-175. Hanson G. R., Matsuda L. A. and Gibb J. W. (1987) Effects of cocaine on methamphetamine-induced neurochemical changes: Characterization of cocaine as a monoamine uptake blocker. J. Pharmac. exp. Ther. 242, 507 513. Hawks R. L., Kopin I. J.. Colburn R. W. and Thoa N. B. (1975) Norcocaine: A pharmacologically active metabolite of cocaine found in brain. L(fe Sci. 15, 2189 2195. Jaffe J. H. (1990) Drug addition and drug abuse. In The Pharmacological Basis oJ'the Therapeutics (Gilman A. G., Rail T. W., Nies A. S. and Taylor P., eds), 8th ed., pp. 522 573. Pergamon Press, New York. Janowsky D. S. and Davis J. M. ([976) Methylphenidate, dextroamphetamine, and levamphetamine: Effect on schizophrenic symptoms. Arch. Gen. Psyehiat. 33, 304308. Johanson C. E. and Fischman M. W. (1989) The pharmacology of cocaine related to its abuse. Pharmac. Rev. 41, 3-52. Kaku D. A. and Lowenstein D. H. (1990) Emergence of recreational drug abuse as a major risk factor for stroke in young adults. Ann. Int. Med. 113, 821-827. Kalivas P. W. and Duffy P. (1990) Effect of acute and daily
Cocaine : effects on brain monoamine concentrations cocaine treatment on extracellular dopamine in the nucleus accumbens. Synapse 5, 48 58. Kleven M. S., Woolverton W. L. and Seiden L. S. (1988) Lack of long-term monoamine depletion following repeated or continuous exposure to cocaine. Brain Res. Bull. 21,233-237. Kuhar M. J., Ritz M. C. and Sharkey J. (1988) Cocaine receptors on dopamine transporters mediate cocaine reinforced behavior. NIDA Res. Monogr. 88, 14-22. Kuhar M. J., Ritz M. C. and Boj a J. W. ( 1991) The dopamine hypothesis of the reinforcing properties of cocaine. Trends Neurosci. 14, 299-302. Laurier L. G., Corrigall W. A. and George S. R. (1994) Dopamine receptor density, sensitivity and mRNA levels are altered following self-administration of cocaine in the rat. Brain Res. 634, 31~40. Levine S. R., Brust J. C. M., Futrell N., Brass L. M., Blake D., Fayad P., Schultz L. R., Millikan C. H., Ho K-L. and Welch K. M. A. (1991) A comparative study of the cerebrovascular complications of cocaine : alkaloidal versus hydrochloride. Neurology 41, 1173-1177. Lieberman J. A., Kinon B. J. and Loebel A. D. (1990) Dopaminergic mechanism in idiopathic and drug-induced psychoses. Schizophrenia Bull. 16, 97-110. Mattia A., Leccesse A. P. and Morton J. E. (1986) Effects of quizazine on cocaine self-administration in rats. The Pharmacologist 28, 151. Pettit H. O. and Justice J. B. Jr (1989) Dopamine in the nucleus accumbens during cocaine self-administration as studied by in vivo microdialysis. Pharmac. Biochem. Behav. 34, 899 904, Pettit H. O., Pan H. T., Parsons L. H. and Justice J. B. Jr (1990) Extracellular concentrations of cocaine and dopamine are enhanced during chronic cocaine administration. J. Neurochem. 55, 798-804.
57
Ritz M. C., Lamb R. J., Goldberg S. R. and Kuhar M. J. (1987) Cocaine receptors in dopamine transporters are related to self-administration of cocaine. Science 237, 1219-1223. Roberts D. C. and Koob G. F. (1982) Disruption of cocaine self-administration following 6-hydroxy-dopamine lesions of ventral tegemental area in rats. Pharmac. Biochem. Behav. 17, 901-904. Roberts D. C., Corcoran M. E. and Fibiger H. C. (1977) On the role of ascending catecholamine systems in intravenous self-administration of cocaine. Pharmac. Biochem. Behav. 6, 615~520. Sloan M. A., Kittner S. J., Rigamonti D. and Price T. R. (1990). Incidence of stroke associated with use/abuse of drugs. Neurology 40(Suppl, 1), 251 252. Snyder S. H. (1972) Catecholamines in the brain as mediators of amphetamine psychosis. Arch. Gen. Psychiat. 27, 169 179. Taylor D. and Ho B. T. (1978) Comparison of inhibition of monoamine uptake by cocaine, methylphenidate and amphetamine. Res. Commun. Chem. Path. Pharmac. 21, 67 75. Tiros F. M. and Leukefeld C. G. (1993) Treatment of cocaine abusers : issues and perspectives. NIDA Res. Monogr. 135, 1 14. Wamsley J. K. and Alburges M. E. (1993) Cocaine causes a time dependent and dose dependent increase in D~ receptors and dopamine transporters. Proc. West. Pharmac. Soc. 36, 277~82. Woolverton W. L. and Johnson K. M. (1992) Neurobiology of cocaine abuse. Trends Pharmac. Sci. 13, 193 200. Yeh S. Y. and De Souza E. B. (1991) Lack of neurochemical evidence for neurotoxic effects of repeated cocaine administration in rats of brain monoamine neurons. Dru9 Alcohol Depend. 27, 51~1.
0197-0186(95)00061-5
Neurochem. Int. Vol. 28, No. 1, pp. 51-57, 1996 Copyright© 1996ElsevierScienceLtd Printedin Great Britain.All rights reserved 01974)186/96 $9.50+0.00
LACK OF LONG-TERM CHANGES IN COCAINE A N D M O N O A M I N E CONCENTRATIONS IN RAT CNS FOLLOWING CHRONIC ADMINISTRATION OF COCAINE MARIO E. ALBURGES, 1'2 DENNIS J. CROUCH, 1 DAVID M. A N D R E N Y A K l and JAMES K. WAMSLEY 3. ~Center for Human Toxicology, Pharmacology and Toxicology Department, University of Utah, Salt Lake City, UT 84108, U.S.A. 2C~itedra de Toxicologia, Escuela de Bioanhlisis,Facultad de Medicina, Universidad del Zulia, Maracaibo, Zulia, Venezuela 3Departments of Psychiatry and Pharmacology, New York Medical College, Valhalla, NY 10595, U.S.A. (Received 14 November 1994 ; accepted 4 April 1995)
Abstract--In previous studies, we reported time-dependent and dose-dependent changes in the rat dopaminergic receptor system followingchronic administration of cocaine. The aim of the present investigation was to monitor the concentration of monoamines (using HPLC-ECD) and cocaine (using GC-PCI/MS) in rat CNS following a dose schedule of 5, 10, 15, 20 and 25 mg/kg, i.p., b.i.d, for 21 days. 12 h after the last cocaine injection, cortical and striatal concentrations of monoamines and their metabolites were not significantly different in saline vs cocaine treated animals. In addition, the cocaine concentration in the brain regions examined did not change with the different doses used. Accumulation of a metabolite of cocaine (ecgonine methyl ester) was the only alteration found. These results indicate that alterations in the dopaminergic receptor system following chronic cocaine administration are not due to changes in neurotransmitter concentration or accumulation of cocaine in the brain.
COC in many organ systems has been attributed to the drug's potent vasoconstrictive properties (Gay, 1982; Coleman et al., 1982; Kaku and Lowenstein, 1990; Sloan et al., 1990; Levine et al., 1991 ; Goldfrank and Hoffman, 1991). The behavioral consequences of COC exposure have been credited to induced changes in neurotransmitters with the dopaminergic system represented as the most responsible for the adverse effects (Roberts et al., 1977 ; Dackis et al., 1986; Mattia et al., 1986; Kuhar et al., 1988; Bergman et al., 1989 ; Johanson and Fischman, 1989 ; *Author to whom all correspondence should be addressed. Abbreviations: Cocaine (COC); benzoylecgonine (BE); Carroll et al., 1992, 1993). Important research has been performed in an ecgonine methyl e s t e r (EME); 3, 4-dihydroxyphenylethylamine, dopamine (DA); 3, 4-dihy- attempt to uncover the mechanism of COC toxicity in droxyphenylacetic acid (DOPAC) ; 3-methoxy-4- the CNS (Taylor and Ho, 1978; Ritz et al., 1987; hydroxyphenylacetic acid (HVA); 1-(3,4-dihydroxyphenyl)-2-aminoethanol, norepinephrine (NE) ; 1- Roberts et al., 1977 ; Kuhar et al., 1988 ; Pettit et al., (3,4-dihydroxyphenyl)-2-(methylamino)ethanol, epi- 1990; Kalivas and Duffy, 1990). However, little is nephrine (E); 3-methoxy-4-hydroxyphenethyleneglycol known about the relationship between the neuro(MHPG); 5-hydroxytryptamine, serotonin (5-HT); 5- biology of COC and its euphorigenic and psyhydroxyindoleacetic acid (5-HIAA); high-performance chotropic effects. Investigations have provided liquid chromatography/electrochemical detection (HPLC-ECD) ; gas chromatography-positive chemical substantial evidence that one of the primary neurochemical consequences of cocaine administration ionization/mass spectrometry (GC-PCI/MS). 51
Drug addiction is one of the major health problems in our society. The incidence of abuse of behaviorally active drugs has increased tremendously in the last two decades. Drug abuse patterns have shifted from heroin to agents such as cocaine (COC), amphetamines and other central nervous system (CNS) stimulants (Tims and Leukefeld, 1993). Of these, COC has characteristically been one of the most abused sympathomimetic drugs. The widespread toxicity of
Mario E. Alburges el al.
52
is the blockade o f the d o p a m i n e transport system, resulting in an increase in brain concentrations o f the neurotransmitter (Ritz et al., 1987; Fowler et al., 1989; Roberts and K o o b , 1982; Woolverton and Johnson, 1992). Several reports indicate that the reinforcing properties of COC are also associated more with the dopaminergic system than other neurotransmitter systems ( K u h a r e t a l . , 1988, 1 9 9 1 : D a c k i s e t a l . , 1986: Roberts et al., 1977). In addition, previous experiments in rodents, have demonstrated that alterations occur in d o p a m i n e (DA) transporters, D~ receptor binding (Ritz et al., 1987: Alburges et al., 1993a; Wamsley and Alburges, 1993) and D~ receptor m R N A (Laurier et al., 1994) in response to chronic administration o f COC. These changes are associated with modulation o f D r r e c e p t o r s and D A - u p t a k e sites in distinct areas o f brain. One explanation for the receptor changes is that COC administered chronically reduces the neurotransmitter concentrations such that subsequent receptor upregulation occurs. A downregulation o f D A receptors might be expected to occur acutely in response to C O C because o f the induced increase in synaptic DA. However, long-term changes in the neurotransmitter (several hours after injection o f COC) have not been adequately addressed. In the present study, the effect o f chronic COC administration on neurotransmitter concentrations was investigated, and the accumulation of COC and its metabolites in brain tissue were determined. These parameters were analyzed following injection o f various doses o f C O C (5, 10, 15, 20 and 25 mg/kg, i.p., b.i.d.). The neurotransmitters and their metabolites were measured in striatum and cortex using H P L C ECD. COC and its metabolites (BE and E M E ) were measured using G C - P C 1 / M S .
EXPERIMENTAL PROCEDURES
Animal dosing and tissue preparation Male, Long-Evans rats weighing 18~220 g were maintained on a 12 h light/dark cycle with food and water available ad libitum. Based on results obtained in previous experiments (Alburges et al., 1993a ; Wamsley and Alburges, 1993), individual groups of rats (5 6 animals/group) were injected with saline or COC (5, 10, 15, 20 and 25 mg/kg, i.p., b.i.d.) for 21 days. 12 h after last injection, the animals were deeply anesthetized with carbon dioxide and perfused intracardially with an ice-cold isotonic saline solution. Cortices and striata were dissected, frozen and stored at - 7 O C until the monoamines, COC and their metabolites were analyzed. Brain regions from each animal were processed individually.
4nalvsis t~/ monoamines atut their metabolites Biogenic amines and their metabolites, were analyzed using the HPLC ECD method developed by Alburges et al. I 1993b). Brain tissue ( 10 20 mg) from each individual animal was homogenized in a 0.05 N perchloric acid solution containing dihydroxybenzylamine as an internal standard. Filtrates from brain homogenates were analyzed using a Waters 600E Multisolvent delivery unit, a WISP 700 automatic sample injector, a Nova-pack C~ column (3.9 x 300 mm, 4 I~m), and a model 464 pulsed electrochemical detector (Waters and Dynamic Solutions, division of Millipore, Milford, MA). Analvsi.s o[ cocaine and its metabolites COC. benzoylecgonine BE and ecognine methyl ester EME concentrations were obtained using a GC PCI/MS method (Crouch and Alburges, in preparation). A solidphase extraction device (Worldwide Monitoring Corporation, Horsham, PA) with vacuum manifold and vacuum gauge was employed. The solid-phase (SP) extraction cartridges used were 200 mg Clean Screen", ZSDAU020 (Worldwide Monitoring Corporation, Horsham, PA). Tissue samples (100 200 mg) were homogenized in 1 ml of 100 mM sodium phosphate buffer, pH 6.0, (sodium phosphate dibasic and sodium phosphate monobasic were obtained as analytical reagents from Mallinckrodt Chemical Works, St Louis, MO) containing an internal standard ( C O C - D 3 BE-D3 and EME-D3 ; 25 ng/ml each ; Radian Corporation, Austin, TX). Drug free ("blank") brain tissues (100 mg of tissue) were spiked with a standard mixture containing COC, BE and EME (0.5 50 ng/ml of each drug: Alltech Associates Inc., Deerfield, IL) and the internal standard. After homogenization, the samples were processed by solid-phase extraction (SPE). The extractions were carried out according to the Clean Screen 'j' procedure (CBB200DAUZ050191, Worldwide Monitoring Corp., Hotsham, PA) with some modifications. Prior to the sample extraction, the SP-columns were conditioned with a 3 ml portion of methanol followed by a 3 ml portion of deionized water and then a 1 ml portion of 100 mM sodium phosphate buffer (pH 6.0). After centrifugation, the supernatants from the tissue homogenates were loaded onto individual columns and the vacuum was maintained at ~<3 in. of Hg. After the samples were drawn through the columns, the cartridges were washed with one 2 ml portion of 100 mM HCI and one 3 ml portion of methanol. The columns were allowed to dry Jbr 5 rain under vacuum (>~ of Hg). The analytes were then eluted from the columns with 3 ml of solvent mixture (methylene chloride, isopropyl alcohol, ammonium hydroxide ; 78 : 20 : 2). The elutes were collected in 13 x 100 glass tubes and evaporated to dryness at ~<40'C in a water bath under a gentle stream of air. The residues were reconstituted in 60-80 #l of MTBSTFA (N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide; Price, Rockford, IL), and placed in a heating block at 80°C for 45 rain. After derivatization. the tubes were removed from the heating block, cooled to room temperature, and a 40-50 pl vol of the residue was transferred to an autosampler vial for analysis. The analysis was performed with a Finnigan 9610 gas chromatograph equipped with a 4500 mass detector and with a CTC A200S autosampler. A capillary column (DB5 15 m-1 /~m; J & W Scientific, Felsom, CA) was used. The temperatures of the injector port and transfer line were 265 and 250°C, respectively. The ions monitored in the analysis were m/z 304, 314 and 4(/4, for COC, EME, and BE, respectively. The ions t\~r
Cocaine : effects on brain monoamine concentrations the internal standards were m/z 307, 317 and 407 for COCD3, EME-D3 and BE-D3, respectively. Drug concentrations were calculated against a calibration curve based on peak height ratios of analytes and a deuterated internal standard, as defined by a linear regression equation. Data in all the experiments are expressed as the mean+ SEM. These data were initially examined by an ANOVA for multiple comparisons. The posteriori comparisons were made using a Dunnett's t-test. The degree of significancewas set at P ~<0.05.
53
A. Strlatal t i s s u e 80
• = ~, = E E gl.
60
40
20
RESULTS
In order to study the effect of chronic COC administration on central neurotransmitter systems, cortices and striata from animals injected with different doses of COC (5, 10, 15, 20 and 25 mg/kg) or saline for 21 days, were analyzed for monoamines and their metabolites, 12 h after last injection. HPLC-ECD analysis of DA and its metabolites in cortical and striatal tissues are shown in Figs 1 (A) and (B). The concentrations of DA, DOPAC, and HVA in tissues from animals injected with COC in the dose-dependent protocol, were not significantly different from the saline group. Striatal and cortical DA and its metabolites [Figs 1(A) and (B)] did not change, even with progressive increase of the dose of COC administered. Striatal DA, DOPAC, and HVA were in the range of 39.32 + 5.98~2.50 + 12.51 pmol/mg tissue, 18.62+2.20-28.99+5.66 pmol/mg tissue, and 15.22___2.01-22.79+4.01 pmol/mg tissue, respectively. Cortical DA, DOPAC, and HVA concentrations were 1.80 + 0.25-2.50 + 0.13, 4.52 _+1.0407.10+1.01, and 0.89+0.10-1.51+0.90 pmol/mg tissue, respectively. Figures 2 (A) and (B) show that chronic administration of higher doses of COC did not significantly change the concentration of NE, E, and MHPG in cortical and striatal tissues. The concentration of cortical NE and MHPG [Fig. 2(B)] did show an apparent dose-dependent trend that was not statistically significant. The striatal concentrations of NE, E, and MHPG were in the range 0.51__0.08-0.69+0.13, 1.49 + 0.18-2.08 _ 0.24, and 0.30 + 0.08-0.65 + .0.06 pmol/mg tissue, respectively. The cortical concentration values for NE, E, and MHPG were in the range 2.21 + 0.50-3.85 ___0.52, 0.20 + 0.80-0.52 + 0.75, and 2.22+0.31 3.10+0.42 pmol/mg tissue, respectively. Chronic administration of different doses of COC was without effect on the concentrations of 5-HT and 5-HIAA in striatal and cortical tissues (Fig. 3). Concentrations of 5-HT [Figs 3(A) and (B)] show slight dose-dependent increases, but there is no significant
Saline 5 10 15 20 25 Cocaine doses (mg/kg, i.p., b.i.d.)
B. Cortical
tissue
10
8 0 m
~
e
E O
E 2
0 Saline 5 10 15 20 25 Cocaine doses (mg/kg, I.p., b.i.d.)
Fig. 1. Effects of chronic COC administration on rat striatal (A) and cortical (B) concentrations of DA, DOPAC and HVA. Results are expressed as pmol/mg tissue and represent the mean ___SEM of 5 ~ animals per group. Concentrations of DA, DOPAC and HVA in cortices and striata from animals injected with COC were not significantlydifferent from control animals. difference between the saline group and COC treated animals at any of the different doses. The striatal concentrations of 5-HT and 5-HIAA in COC or saline injected animals varied from 1.65 _ 0.48 to 2.59 _+0.42 pmol/mg tissue and from 2.67+0.60 to 3.33+0.67 pmol/mg tissue. The cortical concentrations of 5-HT and 5-HIAA in these animals were in the range 1.78+0.51-2.89+0.52 and 1.98+0.34-2.99+0.68 pmol/mg tissue. The GC-PCI/MS method described herein was sensitive enough to detect 500 pg/ml or 5 pg/mg of tissue, and satisfactory separation of the three analytes under study was found (COC, BE and EME). Figures 4 (A) and (B), show the striatal and cortical concentrations of COC and EME, 12 h after the last administration of different doses of COC (5, 10, 15, 20 and 25 mg/kg)
Mario E. Alburges et al.
54
A. Striatal t i s s u e
Q
[]
NE-St
[] •
E-St MHPG-St
A. Striatal t 5
[]
5HT-St
•
5HIAA-St
tissue
°-4., O1 O
E
o.
1-
o
o
Saline 5 10 15 20 25 Cocaine doses (mg/kg, i.p., b.i.d.)
B. Cortical 54o
: ;iiiiii O.
[]
NE-Cx
[]
E-Cx.
Saline 5 10 15 20 25 Cocaine doses (mg/kg, i.p., b.i.d.)
tissue
B. Cortical
=~
3"
O1
S B
2"
O
1 0 Saline 5 10 15 20 25 Cocaine doses (mgJkg, i.p., b.i.d.) Fig. 2. Effects of chronic COC administration on rat striatal (A) and cortical (B) concentrations of NE, E and MHPG. Results are expressed as pmol/mg tissue and represent the mean+SEM of 5-6 animals per group. Concentrations of NE, E and MHPG in cortices and striata from animals injected with COC were not significantly different from control animals.
or saline. In both tissues, the concentrations of BE were ~< 5 pg/mg tissue. The striatal concentrations of COC and E M E were in the range 6.18+0.7(~ 10.29+5.37 and 17.29_+2.9340.86+4.59 pg/mg tissue. Cortical concentrations of C O C and E M E were 7.91 +_ 1.43-14.07 + 2.18 and 31.06 + 3.66-55.23 _+ 5.60 pg/mg tissue, respectively. Cortices and striata from animals injected with 15, 20 and 25 mg/kg of COC exhibited significant accumulations of E M E when compared to groups of animals injected with the lower dose of C O C (5 vs 15 mg/kg, P ~< 0.01 ; 5 vs 20 and 25 mg/kg, P ~< 0.05). Nevertheless, the concentration of striatal and cortical COC in animals injected with different doses of COC showed no dose-dependent differences.
5HT-Cx
"
5HIAA'Cx
5
¢D •,=
[]
tissue
41
]
:NIl1/ o
Saline 5 10 15 20 25 Cocaine doses (mg/kg, i.p., b.i.d.)
Fig. 3. Effects of chronic COC administration on rat striatal (A) and cortical (B) concentrations of 5-HT and 5-HIAA. Results are expressed as pmol/mg tissue and represent the mean + SEM of 5 ~ animals per group. Concentrations of 5HT and 5-HIAA in cortices and striata from animals injected with COC were not significantly different from control animals. DISCUSSION
Investigators have suggested that the primary action of C O C in the CNS is to inhibit D A uptake in dopaminergic neurons (Johanson and Fischman, 1989; Woolverton and Johnson, 1992). Thus, it is expected that many of the psychotropic effects of COC may be related to excess of D A neurotransmission. Previous results have provided evidence suggesting that increases in Dl-receptors, DA-uptake sites and COC binding accompany chronic administration of COC (Alburges et al., 1993a ; Wamsley and Alburges, 1993). COC-induced increases in binding associated with the D A transporter complex, might be expected since COC acts as an antagonist to the process and may cause upregulation. The increase in D,-receptor
Cocaine : effects on brain monoamine concentrations
A. Striatal tissue 50 40 Q
30 O1
20 OI Q.
10 0 Saline 5 10 15 Cocaine doses (mglkg,
20
25
i.p., b.i.d.)
B. Cortical tissue
70 60 Q Q0 ¢9
50 40
E
30
O.
20 10 0 Saline 5 10 Cocaine doses
15
20
25
(mg/kg, i.p., b.i.d.)
Fig. 4. Striatal (A) and cortical (B) concentrations of COC and EME, 12 h after the last administration of different doses of COC. Results are expressed as pg/mg tissue and represent the mean + SEM of 5-6 animals per group. Cortices and striata from animals injected with 15, 20 and 25 mg/kg COC exhibited a significant accumulation of EME when compared with the group of animals injected with a lower dose of COC, 5 mg/kg. (*P ~< 0.05 ; **PS <~ 0.01).
binding which accompanies chronic COC administration, is more perplexing. However, the increase could be due to a depletion of D A feasibly caused by the prolonged blockade of reuptake by COC. The D]-receptor upregulation could ultimately result as a consequence of diminished concentrations of DA. In the present study, an experiment was designed to provide information on the potential presence of longterm changes in the brain concentrations of COC, monoamines, and their metabolites, in response to injection of various doses of COC. Changes in these neurochemicals could play an important role in inducing the alterations in the dopaminergic receptor system seen after chronic administration of COC. Concentrations of DA, NE, serotonin and their
55
metabolites were measured in striatal and cortical brain regions from animals chronically injected with COC. None of the COC doses used were able to produce significant changes in the brain concentrations of monoamines or their metabolites. Reports from other investigators, relating the effect of COC to the concentration ofmonoamines, have shown short-term increases in monoamine levels. Studies where increased concentrations of brain monoamines were described (Bradberry and Roth, 1989; Pettit et al., 1990; Kalivas and Duffy, 1990; Pettit and Justice, 1989), involved the observation of high levels of D A 15-30 min following administration of the drug. However, these changes were transient and in 60-120 min the D A values returned to the base line concentration. It is very difficult to establish a direct comparison between these studies due to differences in treatment regimes, schedules used in the administration of the drug, and in the biological specimen collection time. On the other hand, our results are in accordance with previous reports (Hanson et al., 1987; Kleven et al., 1988; Yeh and DeSouza, 1991; Alburges and Wamsley, 1993) where 10-20 mg/kg of COC was used in the treatment, and the brain tissues were obtained at least 12-18 h after the last administration of COC. We selected a long postinjection time to examine, since Dl-receptor upregulation had been reported to be measurable 12 h after the last administration of COC (Alburges et al., 1993a). In order to account for this upregulation, the concentrations of dopamine should still be reduced at this time point. This was clearly not the case. The present study marks the first investigation to examine potential neurochemical changes following 21 days of treatment with COC. Hydrolysis (formation of BE and EME) and Ndemethylation (formation of norcocaine) are the main pathways of the metabolism of COC animals (including humans). Using a newly developed G C - P C I / M S method, we measured the concentrations of COC, BE and EME in striatal and cortical tissue from animals chronically treated with five different doses of COC. Our findings demonstrate that even with the lowest dose of COC administered (Smg/kg), tissues obtained 12 h after the last COC administration still contain COC at concentrations of approx. 10 pg/mg tissue. Even though COC did not show progressive accumulation with an increase in dose, it is not clear if the concentration of COC remaining in the brain could affect the threshold of the drug's behavioral toxicity. In addition, these results clearly demonstrate that 15, 20 and 25 mg/kg doses of COC lead to cortical and striatal tissue accumulation of one of the major metabolites of COC (EME).
Mario E. Alburges et al.
56
Investigators have l b u n d that only norcocaine has c o m p a r a b l e brain activity to C O C (Hawks et al.. 1975). Our study did not include m e a s u r e m e n t of norcocaine in the brain. However, an accumulation of E M E was noted. A n y relationship between the presence of these metabolites and the neurotoxicity of C O C is u n k n o w n . This potential relationship needs to be addressed in further studies, as well as determ i n a t i o n o f the c o n c e n t r a t i o n of norcocaine in striatal a n d cortical tissues after chronic a d m i n i s t r a t i o n o f COC. In summary, the psychotropic a n d reinforcing proprieties o f C O C have been related to the drugs' effects on m o n o a m i n e activity (Janowsky a n d Davis, 1976Snyder, 1972; Jaffe, 1990; L i e b e r m a n et al., 1990). These observations suggest that chronic administration o f C O C might cause long-term changes in the brain m o n o a m i n e systems. Nevertheless, the present results indicate that chronic C O C a d m i n i s t r a t i o n does not produce significant a n d persistent changes in the striatal a n d cortical c o n c e n t r a t i o n of DA, NE, serotonin or their metabolites. In addition, the same dose schedule failed to produce significant changes in the brain c o n c e n t r a t i o n of C O C in these animals. However, a n accumulation of E M E was observed with the three higher C O C doses used. This E M E accumulation m a y indicate a saturated t r a n s p o r t system or damage, that can affect the removal process of E M E out of the brain. It is possible, that the behavioral sensitization effects described d u r i n g chronic C O C use, may be associated with changes in the dopaminergic receptor system. This m a y involve the upregulation (possible supersensitivity) in D~-receptors, C D C binding and D A - u p t a k e sites, as reported previously ( K u h a r et al., 1991 ; Alburges et al., 1993a ; Wamsley and Alburges, 1993) and m a y also involve other alterations in brain t r a n s p o r t mechanisms responsible for the elimination of C O C and its metabolites from the brain. Acknowledgements The authors wish to express their appreciation to Candy Johnson and Alan Spanbauer for their technical assistance: and Estrella Urdaneta-Alburges for her secretarial skills applied to the manuscript. Dr Mario E. Alburges is an aggregate professor from the University of Zulia, School of Medicine, Maracaibo, Zulia, Venezuela, on leave of absence. Part of this research was supported by a grant from the Public Health Service (DA 05167).
REFERENCES
Alburges M. E. and Wamsley J. K. (1993) Effects on monoamine levels in rat CNS after chronic administration of cocaine. Invest. Clin. 34, 181 192. Alburges M. E., Narang N. and Wamsley J. K. (1993a)
Alterations in the dopaminergic receptor system alter chronic administration of cocaine. Synapse 14, 314-323. Alburges M. E. Narang N. and Wamsley J. K. (1993b) A sensitive and rapid HPLC ECD method for simultaneous analysis of norepinephrine, dopamine, serotonin and their metabolites in brain tissue. Blamed. Chromatogr. 7, 30(~ 310. Bergman J., Madras B. K., Johnson S. E. and Spealman R. D. (1989) Effects of cocaine and related drugs in nonhuman primates. II!. Self-administration by squirrel monkeys. J. Pharmac. exp.Ther. 219, 15(~155. Bradberry C. W. and Roth R. H. (1989) Cocaine increases extracellular dopamine in rat nucleus accumbens and ventral tegmetal area as shown by in vivo microdialysis. Neurosci. Lett. 103, 97 102. Carroll F. I., Lewin A. H., Boja J. W. and Kuhar M. J. (1992) Cocaine receptor : biochemical characterization and structure-activity relationships for the dopamine transporter. J. Med. Chem. 35, 969-981. Carroll F. 1., Gray J. L., Abraham P.. Kuzemko M. A., Lewin A. H., Boja J. W. and Kuhar M. J. (1993) 3-Aryl2(3'-substituted-l', 2', 4'-oxadiazol-5'-yl) tropane analogues of cocaine : Affinities at the cocaine binding site at the dopamine, serotonin, and norepinephrine transporters. J. Med. Chem. 36, 2886-2890. Cocaine: Clean Screen~-CBB200DAU050191. Worldwide Monitoring Corp., Horsham, P. A. Coleman D. L., Ross T. F. and Naughton J. L. (1982) Myocardial ischemia and infarction related to recreational cocaine use. West. J. Med. 136, 444-446. Dackis C. A., Gold M. S., Davies R. K. and Sweeney D. R. (1986) Bromocriptine treatment for cocaine abuse: The dopamine depletion hypothesis. Int. J. Psvchiat. Med. 15, 125 135. Fowler J. S., Volkow N. D., Wolf A. D., Dewey S. k., Schlyer D. J., MacGregor R. R., Hitzemann R., Logan J.. Bendriem B., Gatley S. J. and Christman D (1989) Mapping cocaine binding sites in human and baboon brain in l,it~o. Synapse 4, 371-377. Gay G, R. (1982) Clinical management of acute and chronic cocaine poisoning. Ann. Emerq. Med. 11, 562 572. Goldfrank L. R. and Hoffman R. S. (1991) The cardiovascular effects of cocaine. Ann. Emer#. Med. 20, 165-175. Hanson G. R., Matsuda L. A. and Gibb J. W. (1987) Effects of cocaine on methamphetamine-induced neurochemical changes: Characterization of cocaine as a monoamine uptake blocker. J. Pharmac. exp. Ther. 242, 507 513. Hawks R. L., Kopin I. J.. Colburn R. W. and Thoa N. B. (1975) Norcocaine: A pharmacologically active metabolite of cocaine found in brain. L(fe Sci. 15, 2189 2195. Jaffe J. H. (1990) Drug addition and drug abuse. In The Pharmacological Basis oJ'the Therapeutics (Gilman A. G., Rail T. W., Nies A. S. and Taylor P., eds), 8th ed., pp. 522 573. Pergamon Press, New York. Janowsky D. S. and Davis J. M. ([976) Methylphenidate, dextroamphetamine, and levamphetamine: Effect on schizophrenic symptoms. Arch. Gen. Psyehiat. 33, 304308. Johanson C. E. and Fischman M. W. (1989) The pharmacology of cocaine related to its abuse. Pharmac. Rev. 41, 3-52. Kaku D. A. and Lowenstein D. H. (1990) Emergence of recreational drug abuse as a major risk factor for stroke in young adults. Ann. Int. Med. 113, 821-827. Kalivas P. W. and Duffy P. (1990) Effect of acute and daily
Cocaine : effects on brain monoamine concentrations cocaine treatment on extracellular dopamine in the nucleus accumbens. Synapse 5, 48 58. Kleven M. S., Woolverton W. L. and Seiden L. S. (1988) Lack of long-term monoamine depletion following repeated or continuous exposure to cocaine. Brain Res. Bull. 21,233-237. Kuhar M. J., Ritz M. C. and Sharkey J. (1988) Cocaine receptors on dopamine transporters mediate cocaine reinforced behavior. NIDA Res. Monogr. 88, 14-22. Kuhar M. J., Ritz M. C. and Boj a J. W. ( 1991) The dopamine hypothesis of the reinforcing properties of cocaine. Trends Neurosci. 14, 299-302. Laurier L. G., Corrigall W. A. and George S. R. (1994) Dopamine receptor density, sensitivity and mRNA levels are altered following self-administration of cocaine in the rat. Brain Res. 634, 31~40. Levine S. R., Brust J. C. M., Futrell N., Brass L. M., Blake D., Fayad P., Schultz L. R., Millikan C. H., Ho K-L. and Welch K. M. A. (1991) A comparative study of the cerebrovascular complications of cocaine : alkaloidal versus hydrochloride. Neurology 41, 1173-1177. Lieberman J. A., Kinon B. J. and Loebel A. D. (1990) Dopaminergic mechanism in idiopathic and drug-induced psychoses. Schizophrenia Bull. 16, 97-110. Mattia A., Leccesse A. P. and Morton J. E. (1986) Effects of quizazine on cocaine self-administration in rats. The Pharmacologist 28, 151. Pettit H. O. and Justice J. B. Jr (1989) Dopamine in the nucleus accumbens during cocaine self-administration as studied by in vivo microdialysis. Pharmac. Biochem. Behav. 34, 899 904, Pettit H. O., Pan H. T., Parsons L. H. and Justice J. B. Jr (1990) Extracellular concentrations of cocaine and dopamine are enhanced during chronic cocaine administration. J. Neurochem. 55, 798-804.
57
Ritz M. C., Lamb R. J., Goldberg S. R. and Kuhar M. J. (1987) Cocaine receptors in dopamine transporters are related to self-administration of cocaine. Science 237, 1219-1223. Roberts D. C. and Koob G. F. (1982) Disruption of cocaine self-administration following 6-hydroxy-dopamine lesions of ventral tegemental area in rats. Pharmac. Biochem. Behav. 17, 901-904. Roberts D. C., Corcoran M. E. and Fibiger H. C. (1977) On the role of ascending catecholamine systems in intravenous self-administration of cocaine. Pharmac. Biochem. Behav. 6, 615~520. Sloan M. A., Kittner S. J., Rigamonti D. and Price T. R. (1990). Incidence of stroke associated with use/abuse of drugs. Neurology 40(Suppl, 1), 251 252. Snyder S. H. (1972) Catecholamines in the brain as mediators of amphetamine psychosis. Arch. Gen. Psychiat. 27, 169 179. Taylor D. and Ho B. T. (1978) Comparison of inhibition of monoamine uptake by cocaine, methylphenidate and amphetamine. Res. Commun. Chem. Path. Pharmac. 21, 67 75. Tiros F. M. and Leukefeld C. G. (1993) Treatment of cocaine abusers : issues and perspectives. NIDA Res. Monogr. 135, 1 14. Wamsley J. K. and Alburges M. E. (1993) Cocaine causes a time dependent and dose dependent increase in D~ receptors and dopamine transporters. Proc. West. Pharmac. Soc. 36, 277~82. Woolverton W. L. and Johnson K. M. (1992) Neurobiology of cocaine abuse. Trends Pharmac. Sci. 13, 193 200. Yeh S. Y. and De Souza E. B. (1991) Lack of neurochemical evidence for neurotoxic effects of repeated cocaine administration in rats of brain monoamine neurons. Dru9 Alcohol Depend. 27, 51~1.