Drug and Alcohol Dependence,
Elsevier Scientific Publishers
27 (1991) 51- 61 Ireland Ltd.
51
Lack of neurochemical evidence for neurotoxic effects of repeated cocaine administration in rats on brain monoamine neurons* S.Y. Yeh and E.B. De Souza** National Institute
On Drug Abuse, Addiction Research
Center, Baltimore,
Maryland (U.S.A.)
(Received June 27th. 1990) Rats were injected with cocaine (20 mg/kg, S.C.or i.p. twice daily for 8 days) or saline and killed at 1,8,15 or 48 days after the last injection. The concentrations of norepinephrine (NE), dopamine (DA), serotonin (5-HT) and their metabolites, assayed by HPLC-EC, in frontal cortex, hippocampus, striatum, hypothalamus, midbrain, pons-medulla and spinal cord were not significantly different from those in the saline-injected controls at any of the time points examined. These data suggest that the repeated cocaine administration in rats does not produce any long-term depletion in brain catecholamine and 5-HT content suggesting no neurotoxic effects of the drug. Key words: repeated
cocaine administration; acid; serotonin; 5hydroxyindoleaeetic acid
cocaine; norepinephrine;
Introduction
The abuse of cocaine has rapidly increased in the last decade in the United States. Cocaine, a central nervous system stimulant, shares some behavioural effects with amphetamine-like compounds. Amphetamine and its analogues, such as methamphetamine (MA), p-chloramphe3,4-methylenedioxyamphettamine (PCAL * A preliminary report on these experiments appeared in the Federation Proceedings, FASEB. 46, 404 and the Society for Neuroscience 19th annual meeting abstracts. 15,803 (abstract). **Present address: Dr. Errol B. DeSouza, CNS Research, E.I. duPont de Nemours and Co., Experimental Station E40014352, P.O. Box 80400, Willmington, DE 19880-0400, U.S.A. Correspondence to: Dr. S.Y. Yeh, NIDA Addiction Research Center, P.O. Box 5180. Baltimore, MD 21224, U.S.A. Abbreviations: NE, norepinephrine; DA, dopamine; DOPAC, dihydroxyphenylacetic acid; HVA, homovanillic acid; DHBA, dihydroxybenzylamine; 5-HT. 5_hydroxytryptamine (serotonin); 5-HIAA, 5-hydroxyindoleacetic acid; HPLC-EC, high-performance liquid chromatography-electrochemical detection. Printed and Published in Ireland
dopamine; dihydroxyphenylacetic
acid; homovanillic
amine (MPA) and 3,4-methylenedioxymethamphetamine (MMDA) have been shown to be neurotoxic to brain monoamine neurons in a variety of species including rodents and monkeys (Seiden et al., 1975; Fuller, 1978; Morgan and Gibb, 1980; Ricaurte et al., 1982; 1985; Schmidt et al., 1986; Commins et al., 1987; Schmidt, 1987; Battaglia et al., 1987; 1988; O’Hearn et al., 1988; Insel et al., 1989). The potential neurotoxic effects of cocaine on rat brain monoamine neurons, defined as depletion of catecholamines and serotonin (5-HT) and reduction of tyrosine and tryptophan hydroxyIase activity, is an ongoing controversy. Depletion of brain dopamine (DA) has been hypothesized as an important event. in cocaine reinforcement (Dackis and Gold, 1983), implicating depletion of DA by repeated cocaine injection. The effects of acute and chronic administration of cocaine in rats on brain monoamine function have been examined using a variety of treatment regimens. The concentrations of monoamines and their metabolites and activities of tryptophan and tyrosine hydroxylase in the brain of rats were not significantly
52
altered at 1- 60 days following administration of various doses of cocaine by different routes for varying periods of time (Gunne and Jonsson, 1964; Taylor and Ho, 1976; 1977; Yeh 1987; Hanson et al., 1987; Kalivas et al., 1988; Kleven et al., 19881. Similarly, the concentrations of 5hydroxyindoleacetic acid (5-HIAA) were not changed in the cerebrospinal fluid of monkeys administered cocaine daily for 6 months (Post et al., 1976). Histological data further demonstrate that continuous administration of high doses of cocaine in rats do not produce axonal terminal degeneration in the striatum and frontal cortex that is detectable by Fink-Heimer silver staining or tyrosine hydroxylase immunolabeling (Ryan et al., 19881. On the other hand, other neurochemical and immunocytochemical studies reported that chronic cocaine administration slightly decreased concentrations of 5-HT and 5-HIAA in the septum-caudate (Taylor and Ho, 19771, produced longlasting decreases in tyrosine hydroxylase-like immunoreactive neurons (Trulson el at., 1986; 19871 and decreased DA synthesis in several discrete areas of rat brain (Trulson and Ulissey, 19871. Wyatt and colleagues reported long-term neurochemical decreases in some, but not all, DA markers in the frontal cortex and hypothalamus (Wyatt et al., 1988). These conflicting observations relating to the potential neurotoxic effects of cocaine could be due to different experimental paradigms. In most previous studies, levels of catecholamines and their metabolites as well as tyrosine hydroxylase activity were examined at only 1 or 2 time points (1 or 42 and 60 days) after eessation of cocaine treatment (Taylor and Ho, 1977; Trulson and Ulissey, 1987; Wyatt et al., 19881. In one experiment the time points (3 and 60 days) were far apart (Kleven et al., 19881. Another study using challenge doses of cocaine examined the contents of catecholamines only at early time points of one and 15 days (Gunne and Jonnson, 1964; Kalivas et al., 19881. We believe that if cocaine has neurotoxic effects on DA neurons with accompanying depletion of DA, then DA may be either decreased gradually (chronic effects) or decreased at early, then increased (recovery phase) gradually after
cessation of cocaine treatment. Accordingly we have reinvestigated in detail, the potential neurotoxic effects of cocaine on long-term alterations in DA, norepinephrine (NE) and 5-HT markers in discrete areas of the rat central nervous system (CNS) for periods of 1,8,15 and 48 days following a chronic high dose treatment regimen in rats. The results obtained in the present studies indicate that lack of alteration of the contents of DA, NE, 5-HT and their metabolites one to 48 days after cessation from the chronic cocaine administration, suggesting that chronic cocaine administration does not destroy adrenergic-, dopaminergicand serotonergic neurons. Methods Animal experiments
Seventy-six male Sprague - Dawley rats, 175- 200 g (Harlan Industries, Indianapolis, IN), were housed randomly in groups of three in propylene cages with corn chips as bedding (upon their arrival). The animals were kept in an air-conditioned room (22 + 1°C) with a 12-h light/dark cycle (lights on at 0700 hl for 1 week before being used in an experiment. Purina Lab Chow and water were available ad libitum. Experiment 1. The animals were given fifteen injections of cocaine HCl (20 mg/kg as base, dissolved in saline, S.C. or i.p.1 or equivalent volume of saline (2 ml/kg) at approximately 12-h intervals for 15 consecutive doses. The rats were killed on the 9th day, 1 day following the last injection, by decapitation. The brains were removed quickly and brain regions of interest were dissected, stored in cryotubes, frozen in liquid nitrogen and stored at - 70°C until assayed for monoamines and metabolites. Experiment 2. Rats were injected S.C. with cocaine (20 mglkgl or saline twice daily for 8 days as described in experiment 1 and sacrificed at 1, 8, 15, or 48 days following the last injection. Brain tissues were dissected and stored as described above for Experiment 1. Monoamine assay
NE, DA, DOPAC, HVA, DHBA, 5-HT, 5HIAA, sodium heptyl sulfonate and triethylam-
53
ine were purchased from Sigma Chem. Co. (St. Louis, MO). Organic solvents and other chemicals were obtained from J.T. Baker Co. (Phillipsburg, NJ). C,, radial-pak cartridge and C,,resolved column were obtained from Waters Associates (Milford, MA). NE, DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-HT and 5HIAA were assayed by HPLC (Waters Associates1 with an amperometric detector (Bioanalytic system1 (Chiueh and Kopin 19781. Tissues were homogenized in 0.1 N perchloric acid containing 0.1% of cysteine and dihydrobenzylamine (DHBA, 12.5 nglml, as internal standard). After centrifugation at 14 000 x g for 10 min, an aliquot of the supernatant was placed in 0.25 ml polyethylene tubes and frozen at - i’O°C until assay (less than 15 days). Samples were chromatographed on a C,, radial-pak cartridge (5-pm spheres, 5 mm x 100 mm) or a C,,-resolved column (5 pm spheres, 5 mm x 100 mm) and eluted with a mobile phase at a flow rate of 0.9 ml/min. Each liter of the mobile phase consisted 0.1 g EDTA, 1.3 g of sodium heptyl sulfonate, 8 ml of triethylamine and 45 ml of acetonitrile. The pH of the solution was adjusted to 2.55-2.65 with phosphoric acid (approximately 5.0 ml). Samples of cocaine- and saline-treated rats were injected alternately. Peak heights were measured with computing integrator (model LCI-100, Perkin-Elmer). Concentrations of monoamines and their metabolites in the homogenate were calculated from standard curves prepared daily from at least four concentrations containing dihydrobenzylamine (12.5 nglml as internal standard). The retention times of NE, DHBA, DA, DOPAC, 5HIAA, HVA and 5-HT were 3.15, 4.5, 6.45, 12.26,13.52,16.08 and 17.45 min, respectively. Data analysis
Data were analyzed for statistical significance using two-way ANOVA (route and drug in experiment 1; drug and time in experiment 2). Post hoc tests of significant effects were performed using separate variance t-test with Bonferroni-adjustment for multiple comparisons using the BMDP program (Dixon et al., 19881. The significance level was set at P < 0.05.
Results
The effects of S.C.and i.p. repeated high-dose cocaine administration (20 mg/kg, twice daily, 8 days) on the concentrations of monoamines and their metabolites in four areas of brain were compared in the first experiment. No significant or consistent alterations were observed in any of the monoamine markers measured at 1 day following cessation of the treatment, regardless of the route of drug administration (Table Il. A significant increase in the HVA concentration was observed in the midbrain/ pons of rats that were chronically injected with cocaine using the subcutaneous, but not intraperitoneal route of administration. A second experiment was carried out to measure concentrations of monoamines and their metabolites at 1,8,15 or 48 days after high-dose S.C. cocaine treatment since the time interval after cessation of chronic cocaine treatment was short (i.e. 1 day) in Experiment 1 and inadequate for assessment of neurotoxic effects of cocaine and because of long lasting neurotoxic effects of cocaine reported in the literature (Trulson et al., 1986; 1987; Trulson and Ulissey, 19871. The concentrations of DA, DOPAC, HVA, NE, 5-HT and 5-HIAA in regions of striaturn, frontal cortex, hypothalamus, hippocampus, midbrain, ponslmedulla and spinal cord were not significantly different from those in saline-injected controls at any of the time points (Fig. 11. Some subtle differences were observed in the monoamine concentrations in some brain regions with respect to time intervals and hemispheres (Table II). These subtle differences appears to be due to either the electrochemical detector of the HPLC system or variation of standard curves obtained on varying days (see discussion below). No subtle difference was observed when the concentrations of monoamines and their metabolites in the samples of cocaine-treated rats were expressed as percent of that in the sample of salinetreated rats (Fig. 11. Discussion
The data of the present
study demonstrate
54
Table I.
Short-term effects of repeated high-dose cocaine administration on the concentrations of monoamines ites in the discrete regions of rat brain: comparison of subcutaneous and intraperitoneal routes of administration. Drug route
Striatum DA DOPAC HVA NE 5-HT 5-HIAA Frontal DA
Concentration
(pglmg tissue)
Cocaine, S.C. (n = 7)
Cocaine, i.p.(n = 6)
Saline, i.p. (n = 7)
12430 3347 996 98 563
10984 2499 1066 126 431
11490 3048 835 82 494
f -t k 2 f
470 246 45 25 53
and metabol-
f 2 2 + +
240 481 96 30 21
k k f f 2
297 393 99 14 39
1170 f 114
950 + 69
1048 f 96
186 e 55
136 f 15
133 f 9
149 113 483 773 722
123 93 393 798 655
148 95 369 783 643
Cortex
DOPAC HVA NE 5-HT 5-HIAA Hippocampus DA DOPAC HVA NE 5-HT 5-HIAA Midbrainlpons DA DOPAC HVA NE 5-HT 5-HIAA
f f f f 2
17 13 55 36 34
28 r 7 B.D.
23 B.D. B.D. 654 583 819
f * ” + %
10 8 36 36 36
2 3
+ 2 2 2 k
9 6 32 29 35
28 r 9 B.D.
f 54 2 39 f 36
B.D. 592 2 42 613 -c 58 726 f 29
233 f 20 338 f 51 74 f 21*
238 + 28 198 * 53 41 k 8
246 rt 21 182 f 54 21 2 6
233 f 20 274 +. 11 1860 2 135
238 + 28 263 -’ 24 1803 2 205
246 2 21 258 f 26 1481 f 210
B.D. 556 + 28 539 + 22 756 -c 64
Rats were injected with cocaine (20 mg/hg, S.C. or i.p.) or equivalent volume of saline (2 ml/kg), twice daily for 8 days (total of 15 doses) and were sacrificed one day after the last injection. Values represent the mean f S.E.M. Data were analyzed by twoway ANOVA and Duncan’s multiple range test. Post hoc tests of significant effects were performed using separate variance ttest with Bonferroni-adjustment for multiple comparisons using the BMDP program. *Significantly different at P < 0.05 from saline-injected controls. B.D. represents below detectability of the assay.
that repeated high-dose cocaine administration for 8 days does not significantly alter the concentrations of DA and its metabolites HVA or DOPAC, NE, 5-HT and 5-HIAA in several discrete areas of rat CNS killed at 1, 8, 15 or 48 days after the last injection when compared to saline-injected controls. These data demonstrate a lack of long-term effects of cocaine on dopaminergic, noradrenergic and serotonergic neurons of rat brain. While the data are in
agreement with the results of some investigators (Gunne & Jonsson, 1964; Taylor and Ho 1976; Hanson et al., 1978; Kalivas et al., 1988; Kleven et al., 19881, they conflict with other reports demonstrating long-term neurochemical decreases in dopamine markers in the frontal cortex and hypothalamus (Wyatt et al., 19881 and decreases in dopamine synthesis in several discrete areas of rat brain (Trulson and Ulissey, 19871. The reasons for the apparent discrepan-
55
ties between the studies described above are not clear. The studies of Wyatt et al., (19881 and Trulson and Ulissey (19871 only show relatively small (15-28%) decreases in dopamine synthesis or steady state levels of dopamine and its metabolites following repeated cocaine administration. Furthermore, there were no consistent changes in all dopaminergic markers measured within a given brain region (Wyatt et al., 19881. For example, in hypothalamus only decreases in DOPAC and HVA were reported and only decreases in dopamine and DOPAC were observed in the frontal cortex without any alterations in HVA (Wyatt et al., 19881. Neurotoxicity usually results in changes in all neuronal markers measured. Additional support for a lack of neurotoxic effects of cocaine administration on dopamine neurons comes from histological studies. These studies demonstrated that continuous 3-day administration via implanted mini-pumps of cocaine hydrochloride (50 - 450 mglkglday, S.C. and loo-250 mglkglday iv.1 did not produce axonal degeneration in the frontal cortex or neostriatum that was detectable by either Fink-Heimer silver staining or tyrosine hydroxylase immunolabeling (Ryan et al., 19881. These data, however, contrast with other immunocytochemical studies by Trulson and colleagues who reported long-lasting (i.e. 60 days after the last injection of 10 mg/kg of cocaine daily for 10 days) decreases in tyrosine hydroxylase-like immunoreactive neurons in discrete areas of rat brain including striatum, nucleus accumbens, frontal cortex and ventral tegmental area (Trulson et al., 1986.19871. The studies by Trulson and colleagues utilized immunocytochemical techniques to ‘quantify’ reductions in tyrosine hydroxylase-like immunoreactivity in various brain areas. While immunocytochemistry has been successfully utilized to assess the neurodegenerative effects of amphetamines and other neurotoxins (O’Hearn et al., 1988; Appel et al., 19891, it provides qualitative data since changes in staining intensity are not reliably quantifiable. Furthermore, most studies utilizing immunocytochemistry to assess the neurodegenerative effects of neurotoxins rely heavily on the demonstration of the pres-
enee of cytopathology characteristic of degeneration in neurons (i.e. swollen and fragmented fibers). This type of cytopathologic evidence of degeneration is clearly evident after MDA, MDMA (O’Hearn et al., 19881 or fenfluramine (Appel et al., 19891 administration but notably absent following high-dose chronic cocaine treatment (Ryan et al., 19881. The discrepancy does not appear to relate to dosing regimens or routes of administration since a variety of dosing regimens including repeated subcutaneous, intraperitoneal or continued infusion with minipumps of doses of cocaine up to 450 mg/kg per day S.C.for periods of 3 days or 100 mgkg per day i.v. up to 21 days did not produce any neurochemical or neuroanatomical evidence for neurotoxicity (Yeh, 1987, Eleven et al., 1988; Ryan et al., 19881. Most recently, radioligand binding techniques have also been utilized to examine potential neurotoxic effects of cocaine administration by measuring the decreases in the density of [3H]mazindol-labeled dopamine uptake sites in brain areas containing DA terminals. No significant alterations (Terry et al., 19891 or decrease in DA uptake sites (Goeders et al., 19891 in mesolimbic or striatal terminals were noted following chronic cocaine administration. It is well known that cocaine is a NE, DA and 5-HT uptake inhibitor while amphetamine analogues alter monoamine function primarily by affecting their release. These differences may explain the effects of amphetamine analogues, e.g. PCA, MA, MDA and MDMA to induce neurotoxicity and the lack of neurotoxic effects of cocaine. PCA and MA induced neurotoxicity have been attributed to the presence of 5-HT and DA. Depletion of 5-HT using p-chlorphenylalanine (PCPA) and reserpine has shown to protect against the neurotoxic effects of PCA in the brain (Berger et al., 19891. Multiple administrations of high doses of MA to rats cause long-term depression of both DA and 5HT synthesis. Coadministration of catecholamine synthesis inhibitor, alpha-methyl-p-tyrosine has shown to antagonize this effect of MA on both neurotransmitter systems (Schmidt et al., 1985). Some concentrations of DA, DOPAC, HVA,
56 FRONTAL
STRIATUM
.C ORTEX
T
I _ 8
15
DAYS AFTER COCAlNE TREATMENT
48
I
DAYS AFTER COCAINE TREATMENT
HYPOTHALAMUS
L 0
DA
q OOPAC .
DAYS AFTER COCAINE TREATMENT
Fig. 1. continued on facing page.
DAYS AFTER COCAINE TREATMENI
WA
57
PONS-MEDULLA
MIDBRAIN
SPINAL CORD
-
k
T
I-_ll .IIE
Time course of effects of repeated high-dose Fig. 1. cocaine administration on the contents of monoamines and their metabolites in rat striatum, frontal cortex, hypothalamus, hippocampus, midbrain, pons-medulla and spinal cord. Rats were injected with cocaine (20 mg/kg, s.c.1 or equivalent volume of saline (2 ml/kg), twice daily for 8 days (total of 15 doses) and were sacrificed l-48 days after the last injection. Values are mean + S.E.M. N = 5- 7. The results are
li_riIi q m-l n 5aAA
8
15
48
expressed as percentage of mean control value of 5-7 rats at each interval. The concentrations (mean k SE., pglmg tissue) of DA, DOPAC, HVA, NE, 5-HT and 5-HIAA of saline control rats are in the ranges of the values reported in the literature.
10064 1967 918 115 480 139
+- 46W f 193 f 37a k 9’ 2 20 k ll(5P
1932 k 103 1125 + 103 141 (WC 884 + 57a-d 862 2 27”,d
13671 + 224b.d
1261 f 58* 771 2 27
60’ 12b,e 26(5) 63
5-HT 5-HIAA
f f f 2
530 96 171 3190
DA DOPAC HVA NE
NE 2925 + 125 5-HT 748 * 44d 5-HIAA 791 f 43’ Hypothalamus-right + + f r
260 172 125 2403
8(4F 25(4) 9(4) 239(4)
28 32 60(2) 85’ 51c.d 39’
43W 224 35’ 23(2) 26 30(3)=
167b 122 27(3) 49=d 61d
515 f 46(4F’.< 920 2 76(4jb-
Elsevier Scientific Publishers
27 (1991) 51- 61 Ireland Ltd.
51
Lack of neurochemical evidence for neurotoxic effects of repeated cocaine administration in rats on brain monoamine neurons* S.Y. Yeh and E.B. De Souza** National Institute
On Drug Abuse, Addiction Research
Center, Baltimore,
Maryland (U.S.A.)
(Received June 27th. 1990) Rats were injected with cocaine (20 mg/kg, S.C.or i.p. twice daily for 8 days) or saline and killed at 1,8,15 or 48 days after the last injection. The concentrations of norepinephrine (NE), dopamine (DA), serotonin (5-HT) and their metabolites, assayed by HPLC-EC, in frontal cortex, hippocampus, striatum, hypothalamus, midbrain, pons-medulla and spinal cord were not significantly different from those in the saline-injected controls at any of the time points examined. These data suggest that the repeated cocaine administration in rats does not produce any long-term depletion in brain catecholamine and 5-HT content suggesting no neurotoxic effects of the drug. Key words: repeated
cocaine administration; acid; serotonin; 5hydroxyindoleaeetic acid
cocaine; norepinephrine;
Introduction
The abuse of cocaine has rapidly increased in the last decade in the United States. Cocaine, a central nervous system stimulant, shares some behavioural effects with amphetamine-like compounds. Amphetamine and its analogues, such as methamphetamine (MA), p-chloramphe3,4-methylenedioxyamphettamine (PCAL * A preliminary report on these experiments appeared in the Federation Proceedings, FASEB. 46, 404 and the Society for Neuroscience 19th annual meeting abstracts. 15,803 (abstract). **Present address: Dr. Errol B. DeSouza, CNS Research, E.I. duPont de Nemours and Co., Experimental Station E40014352, P.O. Box 80400, Willmington, DE 19880-0400, U.S.A. Correspondence to: Dr. S.Y. Yeh, NIDA Addiction Research Center, P.O. Box 5180. Baltimore, MD 21224, U.S.A. Abbreviations: NE, norepinephrine; DA, dopamine; DOPAC, dihydroxyphenylacetic acid; HVA, homovanillic acid; DHBA, dihydroxybenzylamine; 5-HT. 5_hydroxytryptamine (serotonin); 5-HIAA, 5-hydroxyindoleacetic acid; HPLC-EC, high-performance liquid chromatography-electrochemical detection. Printed and Published in Ireland
dopamine; dihydroxyphenylacetic
acid; homovanillic
amine (MPA) and 3,4-methylenedioxymethamphetamine (MMDA) have been shown to be neurotoxic to brain monoamine neurons in a variety of species including rodents and monkeys (Seiden et al., 1975; Fuller, 1978; Morgan and Gibb, 1980; Ricaurte et al., 1982; 1985; Schmidt et al., 1986; Commins et al., 1987; Schmidt, 1987; Battaglia et al., 1987; 1988; O’Hearn et al., 1988; Insel et al., 1989). The potential neurotoxic effects of cocaine on rat brain monoamine neurons, defined as depletion of catecholamines and serotonin (5-HT) and reduction of tyrosine and tryptophan hydroxyIase activity, is an ongoing controversy. Depletion of brain dopamine (DA) has been hypothesized as an important event. in cocaine reinforcement (Dackis and Gold, 1983), implicating depletion of DA by repeated cocaine injection. The effects of acute and chronic administration of cocaine in rats on brain monoamine function have been examined using a variety of treatment regimens. The concentrations of monoamines and their metabolites and activities of tryptophan and tyrosine hydroxylase in the brain of rats were not significantly
52
altered at 1- 60 days following administration of various doses of cocaine by different routes for varying periods of time (Gunne and Jonsson, 1964; Taylor and Ho, 1976; 1977; Yeh 1987; Hanson et al., 1987; Kalivas et al., 1988; Kleven et al., 19881. Similarly, the concentrations of 5hydroxyindoleacetic acid (5-HIAA) were not changed in the cerebrospinal fluid of monkeys administered cocaine daily for 6 months (Post et al., 1976). Histological data further demonstrate that continuous administration of high doses of cocaine in rats do not produce axonal terminal degeneration in the striatum and frontal cortex that is detectable by Fink-Heimer silver staining or tyrosine hydroxylase immunolabeling (Ryan et al., 19881. On the other hand, other neurochemical and immunocytochemical studies reported that chronic cocaine administration slightly decreased concentrations of 5-HT and 5-HIAA in the septum-caudate (Taylor and Ho, 19771, produced longlasting decreases in tyrosine hydroxylase-like immunoreactive neurons (Trulson el at., 1986; 19871 and decreased DA synthesis in several discrete areas of rat brain (Trulson and Ulissey, 19871. Wyatt and colleagues reported long-term neurochemical decreases in some, but not all, DA markers in the frontal cortex and hypothalamus (Wyatt et al., 1988). These conflicting observations relating to the potential neurotoxic effects of cocaine could be due to different experimental paradigms. In most previous studies, levels of catecholamines and their metabolites as well as tyrosine hydroxylase activity were examined at only 1 or 2 time points (1 or 42 and 60 days) after eessation of cocaine treatment (Taylor and Ho, 1977; Trulson and Ulissey, 1987; Wyatt et al., 19881. In one experiment the time points (3 and 60 days) were far apart (Kleven et al., 19881. Another study using challenge doses of cocaine examined the contents of catecholamines only at early time points of one and 15 days (Gunne and Jonnson, 1964; Kalivas et al., 19881. We believe that if cocaine has neurotoxic effects on DA neurons with accompanying depletion of DA, then DA may be either decreased gradually (chronic effects) or decreased at early, then increased (recovery phase) gradually after
cessation of cocaine treatment. Accordingly we have reinvestigated in detail, the potential neurotoxic effects of cocaine on long-term alterations in DA, norepinephrine (NE) and 5-HT markers in discrete areas of the rat central nervous system (CNS) for periods of 1,8,15 and 48 days following a chronic high dose treatment regimen in rats. The results obtained in the present studies indicate that lack of alteration of the contents of DA, NE, 5-HT and their metabolites one to 48 days after cessation from the chronic cocaine administration, suggesting that chronic cocaine administration does not destroy adrenergic-, dopaminergicand serotonergic neurons. Methods Animal experiments
Seventy-six male Sprague - Dawley rats, 175- 200 g (Harlan Industries, Indianapolis, IN), were housed randomly in groups of three in propylene cages with corn chips as bedding (upon their arrival). The animals were kept in an air-conditioned room (22 + 1°C) with a 12-h light/dark cycle (lights on at 0700 hl for 1 week before being used in an experiment. Purina Lab Chow and water were available ad libitum. Experiment 1. The animals were given fifteen injections of cocaine HCl (20 mg/kg as base, dissolved in saline, S.C. or i.p.1 or equivalent volume of saline (2 ml/kg) at approximately 12-h intervals for 15 consecutive doses. The rats were killed on the 9th day, 1 day following the last injection, by decapitation. The brains were removed quickly and brain regions of interest were dissected, stored in cryotubes, frozen in liquid nitrogen and stored at - 70°C until assayed for monoamines and metabolites. Experiment 2. Rats were injected S.C. with cocaine (20 mglkgl or saline twice daily for 8 days as described in experiment 1 and sacrificed at 1, 8, 15, or 48 days following the last injection. Brain tissues were dissected and stored as described above for Experiment 1. Monoamine assay
NE, DA, DOPAC, HVA, DHBA, 5-HT, 5HIAA, sodium heptyl sulfonate and triethylam-
53
ine were purchased from Sigma Chem. Co. (St. Louis, MO). Organic solvents and other chemicals were obtained from J.T. Baker Co. (Phillipsburg, NJ). C,, radial-pak cartridge and C,,resolved column were obtained from Waters Associates (Milford, MA). NE, DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-HT and 5HIAA were assayed by HPLC (Waters Associates1 with an amperometric detector (Bioanalytic system1 (Chiueh and Kopin 19781. Tissues were homogenized in 0.1 N perchloric acid containing 0.1% of cysteine and dihydrobenzylamine (DHBA, 12.5 nglml, as internal standard). After centrifugation at 14 000 x g for 10 min, an aliquot of the supernatant was placed in 0.25 ml polyethylene tubes and frozen at - i’O°C until assay (less than 15 days). Samples were chromatographed on a C,, radial-pak cartridge (5-pm spheres, 5 mm x 100 mm) or a C,,-resolved column (5 pm spheres, 5 mm x 100 mm) and eluted with a mobile phase at a flow rate of 0.9 ml/min. Each liter of the mobile phase consisted 0.1 g EDTA, 1.3 g of sodium heptyl sulfonate, 8 ml of triethylamine and 45 ml of acetonitrile. The pH of the solution was adjusted to 2.55-2.65 with phosphoric acid (approximately 5.0 ml). Samples of cocaine- and saline-treated rats were injected alternately. Peak heights were measured with computing integrator (model LCI-100, Perkin-Elmer). Concentrations of monoamines and their metabolites in the homogenate were calculated from standard curves prepared daily from at least four concentrations containing dihydrobenzylamine (12.5 nglml as internal standard). The retention times of NE, DHBA, DA, DOPAC, 5HIAA, HVA and 5-HT were 3.15, 4.5, 6.45, 12.26,13.52,16.08 and 17.45 min, respectively. Data analysis
Data were analyzed for statistical significance using two-way ANOVA (route and drug in experiment 1; drug and time in experiment 2). Post hoc tests of significant effects were performed using separate variance t-test with Bonferroni-adjustment for multiple comparisons using the BMDP program (Dixon et al., 19881. The significance level was set at P < 0.05.
Results
The effects of S.C.and i.p. repeated high-dose cocaine administration (20 mg/kg, twice daily, 8 days) on the concentrations of monoamines and their metabolites in four areas of brain were compared in the first experiment. No significant or consistent alterations were observed in any of the monoamine markers measured at 1 day following cessation of the treatment, regardless of the route of drug administration (Table Il. A significant increase in the HVA concentration was observed in the midbrain/ pons of rats that were chronically injected with cocaine using the subcutaneous, but not intraperitoneal route of administration. A second experiment was carried out to measure concentrations of monoamines and their metabolites at 1,8,15 or 48 days after high-dose S.C. cocaine treatment since the time interval after cessation of chronic cocaine treatment was short (i.e. 1 day) in Experiment 1 and inadequate for assessment of neurotoxic effects of cocaine and because of long lasting neurotoxic effects of cocaine reported in the literature (Trulson et al., 1986; 1987; Trulson and Ulissey, 19871. The concentrations of DA, DOPAC, HVA, NE, 5-HT and 5-HIAA in regions of striaturn, frontal cortex, hypothalamus, hippocampus, midbrain, ponslmedulla and spinal cord were not significantly different from those in saline-injected controls at any of the time points (Fig. 11. Some subtle differences were observed in the monoamine concentrations in some brain regions with respect to time intervals and hemispheres (Table II). These subtle differences appears to be due to either the electrochemical detector of the HPLC system or variation of standard curves obtained on varying days (see discussion below). No subtle difference was observed when the concentrations of monoamines and their metabolites in the samples of cocaine-treated rats were expressed as percent of that in the sample of salinetreated rats (Fig. 11. Discussion
The data of the present
study demonstrate
54
Table I.
Short-term effects of repeated high-dose cocaine administration on the concentrations of monoamines ites in the discrete regions of rat brain: comparison of subcutaneous and intraperitoneal routes of administration. Drug route
Striatum DA DOPAC HVA NE 5-HT 5-HIAA Frontal DA
Concentration
(pglmg tissue)
Cocaine, S.C. (n = 7)
Cocaine, i.p.(n = 6)
Saline, i.p. (n = 7)
12430 3347 996 98 563
10984 2499 1066 126 431
11490 3048 835 82 494
f -t k 2 f
470 246 45 25 53
and metabol-
f 2 2 + +
240 481 96 30 21
k k f f 2
297 393 99 14 39
1170 f 114
950 + 69
1048 f 96
186 e 55
136 f 15
133 f 9
149 113 483 773 722
123 93 393 798 655
148 95 369 783 643
Cortex
DOPAC HVA NE 5-HT 5-HIAA Hippocampus DA DOPAC HVA NE 5-HT 5-HIAA Midbrainlpons DA DOPAC HVA NE 5-HT 5-HIAA
f f f f 2
17 13 55 36 34
28 r 7 B.D.
23 B.D. B.D. 654 583 819
f * ” + %
10 8 36 36 36
2 3
+ 2 2 2 k
9 6 32 29 35
28 r 9 B.D.
f 54 2 39 f 36
B.D. 592 2 42 613 -c 58 726 f 29
233 f 20 338 f 51 74 f 21*
238 + 28 198 * 53 41 k 8
246 rt 21 182 f 54 21 2 6
233 f 20 274 +. 11 1860 2 135
238 + 28 263 -’ 24 1803 2 205
246 2 21 258 f 26 1481 f 210
B.D. 556 + 28 539 + 22 756 -c 64
Rats were injected with cocaine (20 mg/hg, S.C. or i.p.) or equivalent volume of saline (2 ml/kg), twice daily for 8 days (total of 15 doses) and were sacrificed one day after the last injection. Values represent the mean f S.E.M. Data were analyzed by twoway ANOVA and Duncan’s multiple range test. Post hoc tests of significant effects were performed using separate variance ttest with Bonferroni-adjustment for multiple comparisons using the BMDP program. *Significantly different at P < 0.05 from saline-injected controls. B.D. represents below detectability of the assay.
that repeated high-dose cocaine administration for 8 days does not significantly alter the concentrations of DA and its metabolites HVA or DOPAC, NE, 5-HT and 5-HIAA in several discrete areas of rat CNS killed at 1, 8, 15 or 48 days after the last injection when compared to saline-injected controls. These data demonstrate a lack of long-term effects of cocaine on dopaminergic, noradrenergic and serotonergic neurons of rat brain. While the data are in
agreement with the results of some investigators (Gunne & Jonsson, 1964; Taylor and Ho 1976; Hanson et al., 1978; Kalivas et al., 1988; Kleven et al., 19881, they conflict with other reports demonstrating long-term neurochemical decreases in dopamine markers in the frontal cortex and hypothalamus (Wyatt et al., 19881 and decreases in dopamine synthesis in several discrete areas of rat brain (Trulson and Ulissey, 19871. The reasons for the apparent discrepan-
55
ties between the studies described above are not clear. The studies of Wyatt et al., (19881 and Trulson and Ulissey (19871 only show relatively small (15-28%) decreases in dopamine synthesis or steady state levels of dopamine and its metabolites following repeated cocaine administration. Furthermore, there were no consistent changes in all dopaminergic markers measured within a given brain region (Wyatt et al., 19881. For example, in hypothalamus only decreases in DOPAC and HVA were reported and only decreases in dopamine and DOPAC were observed in the frontal cortex without any alterations in HVA (Wyatt et al., 19881. Neurotoxicity usually results in changes in all neuronal markers measured. Additional support for a lack of neurotoxic effects of cocaine administration on dopamine neurons comes from histological studies. These studies demonstrated that continuous 3-day administration via implanted mini-pumps of cocaine hydrochloride (50 - 450 mglkglday, S.C. and loo-250 mglkglday iv.1 did not produce axonal degeneration in the frontal cortex or neostriatum that was detectable by either Fink-Heimer silver staining or tyrosine hydroxylase immunolabeling (Ryan et al., 19881. These data, however, contrast with other immunocytochemical studies by Trulson and colleagues who reported long-lasting (i.e. 60 days after the last injection of 10 mg/kg of cocaine daily for 10 days) decreases in tyrosine hydroxylase-like immunoreactive neurons in discrete areas of rat brain including striatum, nucleus accumbens, frontal cortex and ventral tegmental area (Trulson et al., 1986.19871. The studies by Trulson and colleagues utilized immunocytochemical techniques to ‘quantify’ reductions in tyrosine hydroxylase-like immunoreactivity in various brain areas. While immunocytochemistry has been successfully utilized to assess the neurodegenerative effects of amphetamines and other neurotoxins (O’Hearn et al., 1988; Appel et al., 19891, it provides qualitative data since changes in staining intensity are not reliably quantifiable. Furthermore, most studies utilizing immunocytochemistry to assess the neurodegenerative effects of neurotoxins rely heavily on the demonstration of the pres-
enee of cytopathology characteristic of degeneration in neurons (i.e. swollen and fragmented fibers). This type of cytopathologic evidence of degeneration is clearly evident after MDA, MDMA (O’Hearn et al., 19881 or fenfluramine (Appel et al., 19891 administration but notably absent following high-dose chronic cocaine treatment (Ryan et al., 19881. The discrepancy does not appear to relate to dosing regimens or routes of administration since a variety of dosing regimens including repeated subcutaneous, intraperitoneal or continued infusion with minipumps of doses of cocaine up to 450 mg/kg per day S.C.for periods of 3 days or 100 mgkg per day i.v. up to 21 days did not produce any neurochemical or neuroanatomical evidence for neurotoxicity (Yeh, 1987, Eleven et al., 1988; Ryan et al., 19881. Most recently, radioligand binding techniques have also been utilized to examine potential neurotoxic effects of cocaine administration by measuring the decreases in the density of [3H]mazindol-labeled dopamine uptake sites in brain areas containing DA terminals. No significant alterations (Terry et al., 19891 or decrease in DA uptake sites (Goeders et al., 19891 in mesolimbic or striatal terminals were noted following chronic cocaine administration. It is well known that cocaine is a NE, DA and 5-HT uptake inhibitor while amphetamine analogues alter monoamine function primarily by affecting their release. These differences may explain the effects of amphetamine analogues, e.g. PCA, MA, MDA and MDMA to induce neurotoxicity and the lack of neurotoxic effects of cocaine. PCA and MA induced neurotoxicity have been attributed to the presence of 5-HT and DA. Depletion of 5-HT using p-chlorphenylalanine (PCPA) and reserpine has shown to protect against the neurotoxic effects of PCA in the brain (Berger et al., 19891. Multiple administrations of high doses of MA to rats cause long-term depression of both DA and 5HT synthesis. Coadministration of catecholamine synthesis inhibitor, alpha-methyl-p-tyrosine has shown to antagonize this effect of MA on both neurotransmitter systems (Schmidt et al., 1985). Some concentrations of DA, DOPAC, HVA,
56 FRONTAL
STRIATUM
.C ORTEX
T
I _ 8
15
DAYS AFTER COCAlNE TREATMENT
48
I
DAYS AFTER COCAINE TREATMENT
HYPOTHALAMUS
L 0
DA
q OOPAC .
DAYS AFTER COCAINE TREATMENT
Fig. 1. continued on facing page.
DAYS AFTER COCAINE TREATMENI
WA
57
PONS-MEDULLA
MIDBRAIN
SPINAL CORD
-
k
T
I-_ll .IIE
Time course of effects of repeated high-dose Fig. 1. cocaine administration on the contents of monoamines and their metabolites in rat striatum, frontal cortex, hypothalamus, hippocampus, midbrain, pons-medulla and spinal cord. Rats were injected with cocaine (20 mg/kg, s.c.1 or equivalent volume of saline (2 ml/kg), twice daily for 8 days (total of 15 doses) and were sacrificed l-48 days after the last injection. Values are mean + S.E.M. N = 5- 7. The results are
li_riIi q m-l n 5aAA
8
15
48
expressed as percentage of mean control value of 5-7 rats at each interval. The concentrations (mean k SE., pglmg tissue) of DA, DOPAC, HVA, NE, 5-HT and 5-HIAA of saline control rats are in the ranges of the values reported in the literature.
10064 1967 918 115 480 139
+- 46W f 193 f 37a k 9’ 2 20 k ll(5P
1932 k 103 1125 + 103 141 (WC 884 + 57a-d 862 2 27”,d
13671 + 224b.d
1261 f 58* 771 2 27
60’ 12b,e 26(5) 63
5-HT 5-HIAA
f f f 2
530 96 171 3190
DA DOPAC HVA NE
NE 2925 + 125 5-HT 748 * 44d 5-HIAA 791 f 43’ Hypothalamus-right + + f r
260 172 125 2403
8(4F 25(4) 9(4) 239(4)
28 32 60(2) 85’ 51c.d 39’
43W 224 35’ 23(2) 26 30(3)=
167b 122 27(3) 49=d 61d
515 f 46(4F’.< 920 2 76(4jb-
