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Increases in the locomotor activity of rats after intracerebral administration of cathinone
BrainResearchBulletin.Vol. 29, pp. 843-846, 1992 Printed in the USA. All rights reserved.
0361-9230/92$5.00 + .W Copyright0 1992Pergamon Press Ltd.
Increases in the Locomotor Activity of Rats After Intracerebral Administration of Cathinone DANIEL J. CALCAGNETTI’
AND MARTIN D. SCHECHTER
Department of Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272-9989 Received 19 February 1992; Accepted 12 May 1992 CALCAGNETTI, D. J. AND M. D. SCHECHTER. Increases in the locomotoractivityof rats after intracerebraladministration of cathinone. BRAIN RES BULL 29(6) 843-846, 1992.-There is a widespread practice among people living in Eastern Africa and Southern Arabia of chewing the leaves of the Rhat shrub so as to produce pharmacological effects that are practically indistinguishable from those produced by amphetamine (AMPH). Cathinone (CATH) has been identified as the psychostimulant constituent of this plant and, although the locomotor elevating effects of centrally administered AMPH and cocaine (COC) in rats are well known, there is a paucity of data regarding CATH. Three experiments were, therefore, conducted to measure locomotor activity following central administeration of CATH in rats. The first experiment determined the dose-dependent effects of CATH on activity following intracerebroventricular (ICV) administration. As psychostimulant effects are believed, at least in part, to be mediated by dopaminergic systems, in Experiment 2 CATH was injected into the dopamine nerve terminals of the nucleus accumbens. Experiment 3 examined the effects of CATH injection into the dopamine cell body region of the substantia nigra, and activity was measured. Results of the ICV injection of CATH revealed a dose-dependent increase of activity. The highest dose tested (64 rg) yielded a 117% increase in activity when compared to baseline, whereas a 20 pg bilateral nucleus accumbens (NA) injection of CATH increased activity fivefold. These findings evidence the hypothesis that the effects of CATH are dopaminergically mediated. Substantia nigra (SN) injections of CATH were without effect. Dopamine
Cathinone
Activity
lntracerebroventricular
THERE is evidence that people have been cultivating the Khat shrub (Cutha edulis Forsk., family Celastraceae) for its psy-
chostimulant
Nucleus accumbens
Substantia nigra
Rats
( 11,16,35) have characterized peripherally administered doses of CATH in rats. Most recently, the locomotor elevating effects of the psychostimulants AMPH and cocaine (COC) have been shown following both central (1,2,14,20) and peripheral (8,22,36) administration in rats. However, there are no reports concerning the activity stimulating effects of CATH in rats following central administration. Therefore, Experiment 1 investigated the doseresponse effect on general activity in rats administered CATH by the intracerebroventricular (ICV) route. The doses of CATH for ICV injection were selected on the basis of data demonstrating that ICV CATH (16-32 pg) produces conditioned place preference (4). Support for the hypothesis that psychostimulants require an intact dopaminergic system to exert their effects has been evidenced by reports that dopaminergic antagonists (15), or pretreatment with the relatively DA-selective neurotoxin 6-hydroxydopamine, into mesolimbic (i.e., nucleus accumbens) pathways significantly attenuate stimulant-induced activity (2 1). CATH and AMPH are believed to exert their effects by penetrating intraneural sites (13,17,18) and, in turn, promoting the presynaptic release of neural DA (23,33). In contrast, COC is believed to exert its effects primarily by binding to a site in the DA reuptake transporter (29) that, subsequently, results in reduced DA reuptake from the extracellular space (5,7). Thus,
effects longer than they have grown coffee’(12).
Local inhabitants of Eastern Africa and the Southern Arabian Peninsula (where the plant grows indigenously) gather in social settings to chew Kbat leaves. Consuming the juice from the
young leaves produces effects that are practically indistinguishable from amphetamine (AMPH); these include euphoria, allayed fatigue, insomnia, and excitation [for review see (18,24,25)]. The spread of Khat use was once limited because its psychoactive ingredients degrade rapidly once the leaves are picked (19). With the advent of modem rapid transportation, the growth of Khat use and abuse now seems to be spreading, as evidenced by its recent popularity in the Somalian community of Rome, Italy (25). Customs authorities in Great Britain, France, and the United States have also observed Khat shipments (19), and a toxic reaction to Khat chewing has been reported to have occurred in a patient living less than 35 miles from the site of the present studies (9). (-)Cathinone (CATH) is the major psychoactive alkaloid product of the Khat shrub (19). Several reports, using mice as subjects, have characterized the locomotor increases (fivefold above baseline) following peripherally administered doses (0.345 mg/kg) of CATH (10,33,37). In addition, three reports
’ Requests for reprints should be addressed to Daniel J. Calcagnetti, Ph.D., Department of Pharmacology, Northeastern Ohio Universities College of Medicine, P.O. Box 95, Rootstown, OH 44272-9989.
843
844
C/ZL~CAGNETTI
following administration of CATH. AMPH, or COC. the common end result is an increase in extracellular DA levels despite differing sites/mechanisms of action. As psychostimulants exert their effects via neural dopaminergic pathways, it was of interest to determine whether site-specific delivery of CATH would also result in elevated activity. Therefore, Experiment 2 examined the activity elevating effects of a single dose of CATH (20 pg) administered into the dopaminergic nerve terminal site of the nucleus accumbens (NA), and Experiment 3 determined the effects of CATH injected into the substantia nigra (SN), a site plentiful in dopamine cell bodies. METHOD
Male rats (purchased from Zivic-Miller Laboratories Inc., Allison Park, PA) of Sprague-Dawley descent served as subjects. Rats were individually housed in stainless steel hanging cages and allowed ad lib access to food (Purina 5008) and water. Housing took place in a colony room maintained at a constant temperature and humidity on a 12L: 12D h cycle (dark onset at 1800 h). Activity measurements took place in a room separate from the colony room during the latter half of the light phase (I 100-1600 h). A total of 28, 7, and 7 subjects were prepared for Experiments 1, 2, and 3. respectively. Surgcq~, Injection, and Drug
Rats (426-582 g) were anesthetized using 100 mg/kg of ketamine hydrochloride plus a 0.15 ml injection of xylazine ( 10 mg/ml, Sigma Chemical Co., St Louis, MO). For ICV implants, a single stainless steel outer guide cannula (22 gauge; Plastics One, Roanoke, VA) was stereotaxically implanted into the right lateral ventricle using the coordinates: 0.5 mm posterior to bregma, 1.5 mm lateral to midline, and 3.2 mm ventral to the surface of the dura, with the skull kept level between lambda and bregma (27). Bilateral placement of 22 gauge cannulae were then targeted into the NA (1.7 mm anterior from bregma + 1.5 mm lateral from midline, and 6.8 mm ventral) and SN (5.3 mm posterior from bregma + 2.2 mm lateral from midline, and 8.2 mm ventral), respectively. Subjects were given at least 7 days of postsurgical recovery prior to the start of testing. ICV injections were performed by backloading the drug solution through a 28 gauge internal cannula via a length of PE20 tubing affixed to a 25 ~1 Hamilton microsyringe. The total injection volume was 5.0 ~1 administered at a rate of 1 &5 s. Bilateral NA and SN injections (0.5 ~1 unilaterally over a 2-min period) were delivered by a 25 ~1 Hamilton syringe mounted in a microinjection pump (CMA/lOO, Bioanalytical Systems Inc., West Lafayette, IN). All internal cannula extended 0.5 mm beyond the guide cannula. Drug injections were performed while gently holding the rat by hand. All microinjections were monitored by following an air bubble in the PE tubing, and the internal cannula remained in place for at least 30 s to allow complete drug delivery and pressure equalization. The internal injection cannula was inspected for positive fluid flow after each central injection. (-)Cathinone hydrochloride (NIDA) was dissolved in sterile 0.9% saline. Saline served as the ICV vehicle (VEH) control injection. In Experiment 1, four doses of CATH (4.0, 8.0, 16, and 64 &rat) were tested, whereas in Experiments 2 and 3, a single dose of CATH (20 &rat) was used. All drugs were prepared fresh on the day of use and drug doses are expressed as the salt.
AND
SC‘HEC’H7F.K
Four pairs of infrared photosensors (Phillips ECG), affixed four per side in a 45.5 X 35.5 X 20.5 cm Plexiglas cage. were employed to measurement activity. The sensors were situated 5.5 cm above the floor and 9.5 cm apart along the wall of the longer side as described in more detail (3). Each photosensor interruption resulted in one activity count that was recorded by a computer in 5-min intervals throughout the 30- and 60-min testing sessions All subjects underwent thorough habituation to transport in that each rat was handled daily and allowed 3 days (4 h duration) of habitation in activity cages prior to the onset of data collection. This degree of habituation was necessary to allow for the establishment of stable baseline conditions. Rats underwent 3 days of activity measurements. Following a predrug day (no injection), 2 days of testing with drug or VEH were conducted. On drug day 1, rats were randomly assigned to one of two groups to conform to a drug/VEH injection design counterbalanced for dav of administration. Thus, one-half of the rats received an lCV_injection of one of four doses of CATH (4.0,8.0, 16. and 64 pg/rat); the remaining half received an ICV injection of VEH. On drug day 2. the rats that had received CATH on drug day 1 received VEH. and rats that received VEH on drug day 1 received one of four doses of CATH. Following the ICV injections, rats were immediately placed into an activity cage and 1 h of activity measurements were collected. Using a group of rats with NA or SN cannulae in Experiments 2 and 3, intracerebral (NA or SN) injection of CATH (20 pg/ rat) substituted for the ICV procedure: all other procedures were identical to Experiment 1. Following the completion of the intracerebral injection, rats were immediately placed into the activity cage and activity was measured for 30 min postinjection. This design allowed each of the subjects to act as its own control as well as to control for the possible effects of Day of testing. The design of Experiment I conformed to an independent groups analysis. Activity data were analyzed by one-factor analysis of variance (ANOVA) and, where appropriate, were followed by Dunnett’s test (3 1). Results from Experiments 2 and 3 were analyzed by paired t-tests.
All subjects underwent histological verification of cannula placements at the conclusion of activity testing. Following peripheral injection with sodium pentobarbital (200 mg/kg), rats were injected ICV with 4 ~1 of Staedtler (#C745) ink. NA and SN placements received a total of 1 cl1of ink bilaterally. Within 10 min after ink injection, each subject was perfused transcardially with physiological (0.9%) saline followed by a solution of buffered formalin (10%). The brains were rapidly removed from the cranial cavity and stored in formalin. After 24 h, a freezing microtome was used to prepare coronal sections (40 pm thick) for visual verification of placements for lateral ventricular, NA and SN targeted cannula. Positive cannula placement ICV was defined by the presence of ink throughout the ventricles. The following number of subjects were excluded from the analyses due to improper cannula placement, unverified cannula placement, or sickness: Experiment 1, three subjects; Experiments 2 and 3; two rats with NA targets, 1 rat with SN targets. Purging these subjects from the data base left 25 rats in Experiment 1; five rats with NA placements and six rats with SN placements for Experiments 2 and 3, respectively.
CENTRALLY
INJECTED
CATHINONE
845
AND ACTIVITY
RESULTS
The mean (plus the standard error of the mean) activity counts from rats receiving one of four ICV administered doses of CATH are shown in Fig. 1. A one-factor ANOVA revealed that there was no difference between the four ICV VEH control groups, F(3,24)= 0.82, p = 0.50. Therefore, the VEH dose groups were pooled for further comparison. A one-factor ANOVA also revealed that there were no differences between the pooled VEH groups and the 4 pg dose, F( 1, 3 1) = 0.04, p = 0.80. The 4 rg dose was then selected to act as the control group for a Dunnett’s comparison of CATH dose groups. A one-factor ANOVA revealed a significant difference for dose, F(3, 24) = 3.2, p < 0.05. Dunnett’s analysis revealed that the 16 and 64 pg doses were significantly different (&= 25, differences > 2.04; allp < 0.05) from the control group dose. These results demonstrate that CATH dose-dependently increased activity. Following the 16 and 64 pg/rats doses, these measurements increased 80- 120%, respectively, above control. Figure 2 depicts the mean (f SEM) of rats receiving bilaterally administered CATH (VEH and 20 rg/rat) into the NA or SN for Experiments 2 and 3. The results of paired t-tests demonstrate that CATH (20 pg/ rat) injection into the NA significantly increased, t(4) = 4.2, p < 0.05, activity @fivefold) over VEH administration. In contrast, CATH injection into the SN did not alter activity when compared to VEH, t(5) = 1.0, p = 0.35. DISCUSSION One objective of this series of experiments was to characterize the dose-related effects of centrally administered CATH upon spontaneous activity in rats. ICV administration of CATH (16 and 64 pg/rat) elevated activity (80 and 117%, respectively) in comparison to control. Activity counts following administration of the 4 and 8 rg doses of CATH were not significantly different from control. As the potency and time-course effects of CATH are nearly identical to AMPH in several operant behavioral measures (17) including drug discrimination (30), this finding is not unexpected.
Em l
E ? 9 e e $ 2 F B
3w
FIG. 2. Mean (SEM) activity counts in rats receiving bilaterally administered doses of CATH (VEHICLE and 20 &rat) into their nucleus accumbens or substantia nigral over a 0.5-h testing session. An asterisk indicates significant @ < 0.05) differences from baseline using paired ttests.
Similar to the results with ICV administration of CATH, bilateral microinjection of CATH (20 pg) into the NA resulted in a significant elevation (fivefold) of spontaneous activity. This finding is in agreement with other stbdies (1,2) reporting that microinjection of AMPH (lo-20 pg) into the NA significantly elevates activity several fold in rats. In vitro and in vivo analyses support the hypothesis that CATH effectively releases DA at this brain site (26,28). In contrast to results of NA-administered CATH, microinjections into the SN were without effect. As the SN is a site rich in DA cell bodies and believed to play a role in AMPH-induced activity [for review see (6)], it remains unknown why CATH failed to elevate activity. One clue for the lack of effect may reside in the observation that DA metabolism in the SN is insensitive to reserpine (32). This suggests that vesicular storage of DA at this site is low and, hence, with only small amounts of DA available for release, CATH’s effects might be minimal. Another possibilitv resides in the observation that CATH decreases DA release in this area by inhibiting the firing rate of cells in the SN (23). This inhibition is readily reversed by haloperidol (50 ccg/kg), further supporting the idea that CATH’s effects are dopaminergically mediated (23). In conclusion, these data demonstrate that both the ICV and NA administration of CATH produced a robust elevation of spontaneous activity in rats. In contrast, SN injections were without effect. The possibility remains that higher doses of cathinone might result in enhanced locomotor activity.
2m
ACKNOWLEDGEMENTS
FIG. 1.Mean (+SEM) activity counts during a I-h testing session in rats administered one of four doses of CATH (4.0, 8.0, 16, and 64 &rat, ICV). An asterisk indicates significant (p < 0.05) differences from control
We thank the National Institute of Drug Abuse for the supply of (-)cathinone, Dr. Richmond E. Johnson for his constructive comments on an early draft of this manuscript and Susanne M. Meehan and Timothy Gordon for their contribution regarding the statistical treatment of these data. This research was funded by National Institute of Drug Abuse grant No. 3591 to M.D.S., whereas D.J.C. was funded by the State of Ohio Academic Challenge Program. All subjects were handled in compliance with the Guide for the Care and Use of bboratov Animals, Department of Health, Education and Welfare Publication, 1985 and full in-house IACUC approval was obtained for conducting the present
using Dunnett’s test.
experiments.
,m
I CATHlNONE @@rat)
846
CALC‘AGNETTI
AND
SCHECHTER
REFERENCES Bakshi, V. P.; Kelley, A. E. Dopaminergic regulation of feeding behavior: II. Differential effects of amphetamine microinfusion into three striatal subregions. Psychobiology 19:233-242; I99 1. 2. Boss, R.; Cools. A. R.; Ogren, S. Differential effects of the selective D2-antagonist raclopride in the nucleus accumbens of the rat on spontaneous and d-amphetamine-induced activity. Psychopharmacology (Berlin) 95:447-45 I ; 1988. -3 Calcagnetti, D. J.; Schechter, M. D. Conditioned place aversion fol_ lowing the central administration of a novel dopamine release inhibitor. CGS 10746B. Pharmacol. Biochem. Behav. 40:255-259; 1991. 4. Calcagnetti, D. J.; Schechter, M. D. Place preference for cathinone is blocked by pretreatment with a dopamine release inhibitor. Prog. Neuropsychopharmacol. Biol. Psychiat. (in press). 5. Carboni, E.; Imperato, A.; Perezzani, L.; Di Chiara, G. Amphetamine, cocaine, phencyclidine and nomifensine increase extracellular dopamine concentrations preferentially in the nucleus accumbens of freely moving rats. Neuroscience 28:653-661: 1989. 6. Cole, SI 0. Brain mechanisms of amphetamine-induced anorexia, locomotion, and stereotypy: A review. Neurosci. Biobehav. Rev. 2: 89-100; 1978. 7. Einhorn, L. C.; Johansen, P. A.; White, F. J. Electrophysiological effects of cocaine in the mesoaccumbens dopamine system: Studies in the ventral tegmental area. J. Neurosci. 8: 100-I 12: 1988. 8. George, F. G.; Porrino, L. J.; Ritz, M. C.; Goldberg, S. R. Inbred rat strain comparisons indicate different sites of action for cocaine and amphetamine locomotor stimulant effects. Psychopharmacology (Berlin) 104:457-462; 1991. 9. Giannini, A. J.; Castellani, S. A manic-like psychosis due to khat (Cutha edulis Forsk.). J. Toxicol. Clin. Toxicol. 19:455-459: 1982. 10. Glennon, R.; Schowalter, D. The effect of cathinone and several related derivatives on locomotor activity. Res. Commun. Sub. Abuse 2:186-192; 1981. I I. Gugelmann, R.; Von Allman, M.; Brenneisen, R.; Porzig, H. Quantitative differences in the pharmacological effects of (+) and (-) cathinone. Experientia 41:1568-1571; 1985. 12. Heacock, R.; Forrest, J. Khat. Can. J. Pharm. Sci. 9:64-66; 1974. 13. Heikkila. R.: Orlanskv. H.: Mvtilineou. C.: Cohen. G. Amphetamine: Evaluation of d- and-l-isomers as releasing agents and uptake inhibitors for [3H] dopamine and [3H] norepinephrine in slices of rat neo-striatum and cerebral cortex. J. Pharmacol. Exp. Ther. 194:4756; 1975. 14. Hemby, S. E.; Jones, G. H.; Justice, J. B.; Neill, D. B. Neuropharmacological assessment of cocaine-induced conditioned place preference using intracranial microinjections. Sot. Neurosci. Abstr. 16: 584: 1990. 15. Jackson, D. M.; Anden, N.; Dahlstrom, A. A functional effects of dopamine in the nucleus accumbens and in some other dopaminerich parts of the rat brain. Psychopharmacology (Berlin) 45: I39149; 1975. 16. Kalix, P. Hypermotility of the amphetamine type induced by a constituent of Khat leaves. Br. J. Pharmacol. 68:l l-13; 1980. 17. Kalix, P. Pharmacological properties of the stimulant Khat. Pharmacol. Ther. 48:397-4 16; 1990. 18. Kalix. P. Cathinone. a natural amphetamine. Pharmacol. Toxicol. 70:77-86: 1992. I
19. Kalix, P.; Braenden, 0. Pharmacological aspects of the chewing of Khat leaves. Pharmacol. Rev. 37:149-164; 1985. 20. Kelley. A. E.; Gauthier, A. M.; Lang, C. G. Amphetamine microinjections into distinct striatal subregions cause dissociable effects on motor and ingestive behavior. Behav. Brain Res. 35:27-39; 1989. 21. Kelly, P. H.; Iversen, S. D. Selective 60HDA-induced destruction of mesolimbic dopamine neurons: abolition of psychostimulant-induced locomotor activity in rats. Eur. J. Pharmacol. 40:45-56; 1976. 22. Kuczenski, R.; Segal, D. S.; Aizenstein, M. L. Amphetamine, cocaine, and fencamfamine: Relationship between locomotor and stereotypy response profiles and caudate and accumbens dopamine dynamics. J. Neurosci. 11:2703-2712: 1991. 23. Mereu, G. P.; Pacitti, C.; Argiolas, A. Effect of(-) cathinone. a Khat leaf constituent. on dopaminergic firing and dopamine metabolism in the rat brain. Life Sci. 32: 1383-1389: 1983. 24. Nencini, P.: Ahmed, A. M. Khat consumption: A pharmacological review. Drug Alcohol Depend. 23: 19-29; 1989. 25. Nencini, P.; Grassi, M.; Boton. A. A.; Asseyr, A. F.; Paroli, E. Khat chewing spread to the Somali community in Rome. Drug Alcohol Depend. 23:255-3 13; 1989. 26. Nielsen. J.: Schechter, M. D. Behavioral and neurochemical effects of (m-) and (+) cathinone: dose-response and time-course. Prog. Neuropsychopharmacol. Biol. Psychiatry 9:739-743; 1985. 27. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates, 2nd ed. New York: Academic Press; 1986. 28. Pehek, E. A.; Schechter. M. D.; Yamamoto, B. K. Effects ofcathinone and amphetamine on the neurochemistry of dopamine in vivo. Neuropharmacology 29: I I7 1-I 176: 1990. 29. Ritz, M. C.: Lamb, R. J.; Goldberg, S. R.; Kuhar, M. J. Cocaine receptors on dopamine transporters are related to self-administration ofcocaine. Science 237:1219-1223; 1987. 30. Schechter, M. D. Temporal parameters of cathinone, amphetamine and cocaine. Pharmacol. Biochem. Behav. 34:289-292; 1989. 31. Tallarida, R. J.; Murray, R. B. Manual of pharmacologic calculations, 2nd ed. New York: Springer-Verlag; 1987. 32. Tonon, G. C.: Saiani, L.; Spano. P. F.; Tarbucchi. M. Differential effects of reserpine on dopaminergic receptor function in rat substantia nigra and caudate nucleus. Brain Res. 160:553-558; 1979. 33. Valterio. C.: Kalix, P. The effect of the alkaloid (-) cathinone on the motor activity of mice. Arch. Int. Pharmacodyn. Ther. 255: 196203: 1982. 34. Wagner. G.; Preston, K.; Ricaurte, G.; Schuster. C.: Seiden, L. Neurochemical similarities between d,l-cathinone and d-amphetamine. Drug Alcohol Depend. 9:279-284; 1982. 35. Yanagita. T. Studies on cathinones: Cardiovascular and behavioral effects in rats and self-administration experiments in monkeys. In: Problems of drug dependence. vol. Z’I.Washington, DC: NIDA Res. Monograph, U.S. Government Printing Office; 1979:326-327. 36. Yeh, S. Y.; Haertzen, C. A. Cocaine induced locomotor activity in rats. Pharmacol. Biochem. Behav. 39:723-727; 1991. 37. Zelger, J. L.; Carlini, E. A. Influence of cathinone (alpha-aminopropiophenone) and cathine (phenylpropanoamine) on circling behavior and on uptake and release of [‘HI dopamine in striatal slices of rats. Neuropsychopharmacology 20:839-843: 1981.
0361-9230/92$5.00 + .W Copyright0 1992Pergamon Press Ltd.
Increases in the Locomotor Activity of Rats After Intracerebral Administration of Cathinone DANIEL J. CALCAGNETTI’
AND MARTIN D. SCHECHTER
Department of Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272-9989 Received 19 February 1992; Accepted 12 May 1992 CALCAGNETTI, D. J. AND M. D. SCHECHTER. Increases in the locomotoractivityof rats after intracerebraladministration of cathinone. BRAIN RES BULL 29(6) 843-846, 1992.-There is a widespread practice among people living in Eastern Africa and Southern Arabia of chewing the leaves of the Rhat shrub so as to produce pharmacological effects that are practically indistinguishable from those produced by amphetamine (AMPH). Cathinone (CATH) has been identified as the psychostimulant constituent of this plant and, although the locomotor elevating effects of centrally administered AMPH and cocaine (COC) in rats are well known, there is a paucity of data regarding CATH. Three experiments were, therefore, conducted to measure locomotor activity following central administeration of CATH in rats. The first experiment determined the dose-dependent effects of CATH on activity following intracerebroventricular (ICV) administration. As psychostimulant effects are believed, at least in part, to be mediated by dopaminergic systems, in Experiment 2 CATH was injected into the dopamine nerve terminals of the nucleus accumbens. Experiment 3 examined the effects of CATH injection into the dopamine cell body region of the substantia nigra, and activity was measured. Results of the ICV injection of CATH revealed a dose-dependent increase of activity. The highest dose tested (64 rg) yielded a 117% increase in activity when compared to baseline, whereas a 20 pg bilateral nucleus accumbens (NA) injection of CATH increased activity fivefold. These findings evidence the hypothesis that the effects of CATH are dopaminergically mediated. Substantia nigra (SN) injections of CATH were without effect. Dopamine
Cathinone
Activity
lntracerebroventricular
THERE is evidence that people have been cultivating the Khat shrub (Cutha edulis Forsk., family Celastraceae) for its psy-
chostimulant
Nucleus accumbens
Substantia nigra
Rats
( 11,16,35) have characterized peripherally administered doses of CATH in rats. Most recently, the locomotor elevating effects of the psychostimulants AMPH and cocaine (COC) have been shown following both central (1,2,14,20) and peripheral (8,22,36) administration in rats. However, there are no reports concerning the activity stimulating effects of CATH in rats following central administration. Therefore, Experiment 1 investigated the doseresponse effect on general activity in rats administered CATH by the intracerebroventricular (ICV) route. The doses of CATH for ICV injection were selected on the basis of data demonstrating that ICV CATH (16-32 pg) produces conditioned place preference (4). Support for the hypothesis that psychostimulants require an intact dopaminergic system to exert their effects has been evidenced by reports that dopaminergic antagonists (15), or pretreatment with the relatively DA-selective neurotoxin 6-hydroxydopamine, into mesolimbic (i.e., nucleus accumbens) pathways significantly attenuate stimulant-induced activity (2 1). CATH and AMPH are believed to exert their effects by penetrating intraneural sites (13,17,18) and, in turn, promoting the presynaptic release of neural DA (23,33). In contrast, COC is believed to exert its effects primarily by binding to a site in the DA reuptake transporter (29) that, subsequently, results in reduced DA reuptake from the extracellular space (5,7). Thus,
effects longer than they have grown coffee’(12).
Local inhabitants of Eastern Africa and the Southern Arabian Peninsula (where the plant grows indigenously) gather in social settings to chew Kbat leaves. Consuming the juice from the
young leaves produces effects that are practically indistinguishable from amphetamine (AMPH); these include euphoria, allayed fatigue, insomnia, and excitation [for review see (18,24,25)]. The spread of Khat use was once limited because its psychoactive ingredients degrade rapidly once the leaves are picked (19). With the advent of modem rapid transportation, the growth of Khat use and abuse now seems to be spreading, as evidenced by its recent popularity in the Somalian community of Rome, Italy (25). Customs authorities in Great Britain, France, and the United States have also observed Khat shipments (19), and a toxic reaction to Khat chewing has been reported to have occurred in a patient living less than 35 miles from the site of the present studies (9). (-)Cathinone (CATH) is the major psychoactive alkaloid product of the Khat shrub (19). Several reports, using mice as subjects, have characterized the locomotor increases (fivefold above baseline) following peripherally administered doses (0.345 mg/kg) of CATH (10,33,37). In addition, three reports
’ Requests for reprints should be addressed to Daniel J. Calcagnetti, Ph.D., Department of Pharmacology, Northeastern Ohio Universities College of Medicine, P.O. Box 95, Rootstown, OH 44272-9989.
843
844
C/ZL~CAGNETTI
following administration of CATH. AMPH, or COC. the common end result is an increase in extracellular DA levels despite differing sites/mechanisms of action. As psychostimulants exert their effects via neural dopaminergic pathways, it was of interest to determine whether site-specific delivery of CATH would also result in elevated activity. Therefore, Experiment 2 examined the activity elevating effects of a single dose of CATH (20 pg) administered into the dopaminergic nerve terminal site of the nucleus accumbens (NA), and Experiment 3 determined the effects of CATH injected into the substantia nigra (SN), a site plentiful in dopamine cell bodies. METHOD
Male rats (purchased from Zivic-Miller Laboratories Inc., Allison Park, PA) of Sprague-Dawley descent served as subjects. Rats were individually housed in stainless steel hanging cages and allowed ad lib access to food (Purina 5008) and water. Housing took place in a colony room maintained at a constant temperature and humidity on a 12L: 12D h cycle (dark onset at 1800 h). Activity measurements took place in a room separate from the colony room during the latter half of the light phase (I 100-1600 h). A total of 28, 7, and 7 subjects were prepared for Experiments 1, 2, and 3. respectively. Surgcq~, Injection, and Drug
Rats (426-582 g) were anesthetized using 100 mg/kg of ketamine hydrochloride plus a 0.15 ml injection of xylazine ( 10 mg/ml, Sigma Chemical Co., St Louis, MO). For ICV implants, a single stainless steel outer guide cannula (22 gauge; Plastics One, Roanoke, VA) was stereotaxically implanted into the right lateral ventricle using the coordinates: 0.5 mm posterior to bregma, 1.5 mm lateral to midline, and 3.2 mm ventral to the surface of the dura, with the skull kept level between lambda and bregma (27). Bilateral placement of 22 gauge cannulae were then targeted into the NA (1.7 mm anterior from bregma + 1.5 mm lateral from midline, and 6.8 mm ventral) and SN (5.3 mm posterior from bregma + 2.2 mm lateral from midline, and 8.2 mm ventral), respectively. Subjects were given at least 7 days of postsurgical recovery prior to the start of testing. ICV injections were performed by backloading the drug solution through a 28 gauge internal cannula via a length of PE20 tubing affixed to a 25 ~1 Hamilton microsyringe. The total injection volume was 5.0 ~1 administered at a rate of 1 &5 s. Bilateral NA and SN injections (0.5 ~1 unilaterally over a 2-min period) were delivered by a 25 ~1 Hamilton syringe mounted in a microinjection pump (CMA/lOO, Bioanalytical Systems Inc., West Lafayette, IN). All internal cannula extended 0.5 mm beyond the guide cannula. Drug injections were performed while gently holding the rat by hand. All microinjections were monitored by following an air bubble in the PE tubing, and the internal cannula remained in place for at least 30 s to allow complete drug delivery and pressure equalization. The internal injection cannula was inspected for positive fluid flow after each central injection. (-)Cathinone hydrochloride (NIDA) was dissolved in sterile 0.9% saline. Saline served as the ICV vehicle (VEH) control injection. In Experiment 1, four doses of CATH (4.0, 8.0, 16, and 64 &rat) were tested, whereas in Experiments 2 and 3, a single dose of CATH (20 &rat) was used. All drugs were prepared fresh on the day of use and drug doses are expressed as the salt.
AND
SC‘HEC’H7F.K
Four pairs of infrared photosensors (Phillips ECG), affixed four per side in a 45.5 X 35.5 X 20.5 cm Plexiglas cage. were employed to measurement activity. The sensors were situated 5.5 cm above the floor and 9.5 cm apart along the wall of the longer side as described in more detail (3). Each photosensor interruption resulted in one activity count that was recorded by a computer in 5-min intervals throughout the 30- and 60-min testing sessions All subjects underwent thorough habituation to transport in that each rat was handled daily and allowed 3 days (4 h duration) of habitation in activity cages prior to the onset of data collection. This degree of habituation was necessary to allow for the establishment of stable baseline conditions. Rats underwent 3 days of activity measurements. Following a predrug day (no injection), 2 days of testing with drug or VEH were conducted. On drug day 1, rats were randomly assigned to one of two groups to conform to a drug/VEH injection design counterbalanced for dav of administration. Thus, one-half of the rats received an lCV_injection of one of four doses of CATH (4.0,8.0, 16. and 64 pg/rat); the remaining half received an ICV injection of VEH. On drug day 2. the rats that had received CATH on drug day 1 received VEH. and rats that received VEH on drug day 1 received one of four doses of CATH. Following the ICV injections, rats were immediately placed into an activity cage and 1 h of activity measurements were collected. Using a group of rats with NA or SN cannulae in Experiments 2 and 3, intracerebral (NA or SN) injection of CATH (20 pg/ rat) substituted for the ICV procedure: all other procedures were identical to Experiment 1. Following the completion of the intracerebral injection, rats were immediately placed into the activity cage and activity was measured for 30 min postinjection. This design allowed each of the subjects to act as its own control as well as to control for the possible effects of Day of testing. The design of Experiment I conformed to an independent groups analysis. Activity data were analyzed by one-factor analysis of variance (ANOVA) and, where appropriate, were followed by Dunnett’s test (3 1). Results from Experiments 2 and 3 were analyzed by paired t-tests.
All subjects underwent histological verification of cannula placements at the conclusion of activity testing. Following peripheral injection with sodium pentobarbital (200 mg/kg), rats were injected ICV with 4 ~1 of Staedtler (#C745) ink. NA and SN placements received a total of 1 cl1of ink bilaterally. Within 10 min after ink injection, each subject was perfused transcardially with physiological (0.9%) saline followed by a solution of buffered formalin (10%). The brains were rapidly removed from the cranial cavity and stored in formalin. After 24 h, a freezing microtome was used to prepare coronal sections (40 pm thick) for visual verification of placements for lateral ventricular, NA and SN targeted cannula. Positive cannula placement ICV was defined by the presence of ink throughout the ventricles. The following number of subjects were excluded from the analyses due to improper cannula placement, unverified cannula placement, or sickness: Experiment 1, three subjects; Experiments 2 and 3; two rats with NA targets, 1 rat with SN targets. Purging these subjects from the data base left 25 rats in Experiment 1; five rats with NA placements and six rats with SN placements for Experiments 2 and 3, respectively.
CENTRALLY
INJECTED
CATHINONE
845
AND ACTIVITY
RESULTS
The mean (plus the standard error of the mean) activity counts from rats receiving one of four ICV administered doses of CATH are shown in Fig. 1. A one-factor ANOVA revealed that there was no difference between the four ICV VEH control groups, F(3,24)= 0.82, p = 0.50. Therefore, the VEH dose groups were pooled for further comparison. A one-factor ANOVA also revealed that there were no differences between the pooled VEH groups and the 4 pg dose, F( 1, 3 1) = 0.04, p = 0.80. The 4 rg dose was then selected to act as the control group for a Dunnett’s comparison of CATH dose groups. A one-factor ANOVA revealed a significant difference for dose, F(3, 24) = 3.2, p < 0.05. Dunnett’s analysis revealed that the 16 and 64 pg doses were significantly different (&= 25, differences > 2.04; allp < 0.05) from the control group dose. These results demonstrate that CATH dose-dependently increased activity. Following the 16 and 64 pg/rats doses, these measurements increased 80- 120%, respectively, above control. Figure 2 depicts the mean (f SEM) of rats receiving bilaterally administered CATH (VEH and 20 rg/rat) into the NA or SN for Experiments 2 and 3. The results of paired t-tests demonstrate that CATH (20 pg/ rat) injection into the NA significantly increased, t(4) = 4.2, p < 0.05, activity @fivefold) over VEH administration. In contrast, CATH injection into the SN did not alter activity when compared to VEH, t(5) = 1.0, p = 0.35. DISCUSSION One objective of this series of experiments was to characterize the dose-related effects of centrally administered CATH upon spontaneous activity in rats. ICV administration of CATH (16 and 64 pg/rat) elevated activity (80 and 117%, respectively) in comparison to control. Activity counts following administration of the 4 and 8 rg doses of CATH were not significantly different from control. As the potency and time-course effects of CATH are nearly identical to AMPH in several operant behavioral measures (17) including drug discrimination (30), this finding is not unexpected.
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FIG. 2. Mean (SEM) activity counts in rats receiving bilaterally administered doses of CATH (VEHICLE and 20 &rat) into their nucleus accumbens or substantia nigral over a 0.5-h testing session. An asterisk indicates significant @ < 0.05) differences from baseline using paired ttests.
Similar to the results with ICV administration of CATH, bilateral microinjection of CATH (20 pg) into the NA resulted in a significant elevation (fivefold) of spontaneous activity. This finding is in agreement with other stbdies (1,2) reporting that microinjection of AMPH (lo-20 pg) into the NA significantly elevates activity several fold in rats. In vitro and in vivo analyses support the hypothesis that CATH effectively releases DA at this brain site (26,28). In contrast to results of NA-administered CATH, microinjections into the SN were without effect. As the SN is a site rich in DA cell bodies and believed to play a role in AMPH-induced activity [for review see (6)], it remains unknown why CATH failed to elevate activity. One clue for the lack of effect may reside in the observation that DA metabolism in the SN is insensitive to reserpine (32). This suggests that vesicular storage of DA at this site is low and, hence, with only small amounts of DA available for release, CATH’s effects might be minimal. Another possibilitv resides in the observation that CATH decreases DA release in this area by inhibiting the firing rate of cells in the SN (23). This inhibition is readily reversed by haloperidol (50 ccg/kg), further supporting the idea that CATH’s effects are dopaminergically mediated (23). In conclusion, these data demonstrate that both the ICV and NA administration of CATH produced a robust elevation of spontaneous activity in rats. In contrast, SN injections were without effect. The possibility remains that higher doses of cathinone might result in enhanced locomotor activity.
2m
ACKNOWLEDGEMENTS
FIG. 1.Mean (+SEM) activity counts during a I-h testing session in rats administered one of four doses of CATH (4.0, 8.0, 16, and 64 &rat, ICV). An asterisk indicates significant (p < 0.05) differences from control
We thank the National Institute of Drug Abuse for the supply of (-)cathinone, Dr. Richmond E. Johnson for his constructive comments on an early draft of this manuscript and Susanne M. Meehan and Timothy Gordon for their contribution regarding the statistical treatment of these data. This research was funded by National Institute of Drug Abuse grant No. 3591 to M.D.S., whereas D.J.C. was funded by the State of Ohio Academic Challenge Program. All subjects were handled in compliance with the Guide for the Care and Use of bboratov Animals, Department of Health, Education and Welfare Publication, 1985 and full in-house IACUC approval was obtained for conducting the present
using Dunnett’s test.
experiments.
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I CATHlNONE @@rat)
846
CALC‘AGNETTI
AND
SCHECHTER
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