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THC aggravates rat muricide behavior induced by two levels of magnesium deficiency
Physiology & Behavior 77 (2002) 189 – 195
THC aggravates rat muricide behavior induced by two levels of magnesium deficiency Pierre Baca,*, Nicole Pagesa,b, Christine Herrenknechtc, Charlotte Duponta, Pierre Mauroisa, Joseph Vamecqa, Jean Durlachd a
Department of Neuropharmacology, Faculte´ de Pharmacie, 5 Rue J.B. Cle´ment, F-92296 Chaˆtenay Malabry Cedex, France b Department of Toxicology, Faculte´ de Pharmacie, Route du Rhin, F-67400 Illkirch Graffenstaden, France c Department of Analytical Chemistry, Faculte´ de Pharmacie, 5 Rue J.B. Cle´ment, F-92296 Chaˆtenay Malabry Cedex, France d SDRM, Hoˆpital Saint Vincent de Paul, Avenue Denfert Rochereau, F-75014 Paris, France Received 9 August 2001; received in revised form 15 April 2002; accepted 9 May 2002
Abstract A severe magnesium deprivation induces an interspecific aggressive behavior (muricidal behavior, MB) in different strains of rats. D9-tetrahydrocannabinol (THC) is also known to induce MB even after a single injection (11 mg/kg) in starving, isolated rats. In the present work, we investigated the MB behavior, for six successive assays 1 h delayed, of two groups of male Long – Evans rats fed 50- or 150-ppm Mg2 + -deficient diets, for 42 days after a single injection of THC at doses (2, 4 or 8 mg/kg) that did not induce aggressiveness in control rats. This treatment led to Mg2 + plasma levels of 5 ± 0.3 and 12.3 ± 0.9 mg/ml vs. 21 ± 1.5 mg/ml initially. In the 50-ppm Mg-deficient rat group, all the rats were muricidal but the MB pattern was severely aggravated by THC. In the 150-ppm Mg-deficient rat group, no rat was muricidal but all doses of THC induced a 100% MB. In addition, by quantifying the three phases of MB, we showed through six consecutive hourly muricidal assays, that the two first phases (attack latency and attack on the living mouse) decreased progressively, whereas the third phase (attack on the dead mouse) increased dramatically. This indicates firstly that Mg-deprivation decreases the responsiveness threshold of rats to THC. Secondly, these very low doses of THC induced an aggravation of MB and an acquired hyper-aggressiveness in both 50- and 150-ppm Mg-deficient rats, probably involving different neurotransmitters, mainly serotonin, which is decreased by both treatments. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Magnesium deficiency; Magnesium depletion; Cannabis; Rat; D9-Tetrahydrocannabinol (THC); Muricidal behavior; Aggressiveness
1. Introduction The predatory behavior of rats, which prompts them to prey on mice and sometimes to attack their congeners, may be the result of innate habits (natural killers) [1] or can be induced by long-term isolation [2,3], by chemical agents such as p-chlorophenylalanine or THC [4– 7], or by various regional brain lesions [8 – 12]. Deficiencies in nutrients such as thiamine [13 –16], tryptophan [17] or severe magnesium deficiency (50-ppm Mg-diet) [18] are also known to induce muricidal behavior (MB). Consequently, it may be assumed
* Corresponding author. Tel.: +33-1-4683-5551. E-mail address: [email protected] (P. Bac).
that any decrease in magnesium plasma levels may potentiate neurotoxicity of xenobiotics. It has been reported that acute or chronic administration of cannabis extracts or D9-tetrahydrocannabinol (THC) may alter aggressiveness in the rat [5– 7]. In agreement with Fujiwara and Ueki [7], we observed, 1 h after a single intraperitoneal injection (11 mg/kg b.w.) of THC, a high percentage (70%) MB among naive rats, in conditions of starvation and isolated housing. In addition, we showed that the muricidal pattern was significantly different from that of natural killer rats (NK) in the first assay. Indeed, 1 h after THC injection, the preying attack was disorganized and the duration of each phase increased notably, mainly the attack on the dead mouse which was far longer than in NK, indicating that THC induced an increased aggressiveness [19]. In addition, MB became more efficient through re-
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peated assays and resembled, on the sixth assay, that of NK, suggesting that the killer behavior was progressively acquired for the six assays whereas the aggressiveness disappeared with the elimination of THC. The aim of the present work was to study in rats presenting severe and moderate magnesium deficiencies (fed 50- and 150-ppm Mg-deficient diet, respectively, for 6 weeks) the influence of a single injection of THC at low dose (2, 4 or 8 mg/kg).
2.4. Statistical analysis Results were expressed as mean values ± S.E.M. The comparison between the different experimental groups was made by ANOVA. Significant differences ( P < .05) were estimated on the basis of Fischer – Scheffe´ and Dunnett’s tests.
3. Results 2. Materials and methods
3.1. Action of THC on rats fed a control diet (1700±100-ppm Mg)
2.1. Rats Male Long – Evans, 7-week-old rats (Janvier, France) were divided into three groups (n = 120/group). The first group was fed on a standard 1700-ppm Mg-diet. The two other groups were fed on UAR Mg-deficient diets, one group receiving 50 ppm and the other 150 ppm, for a period of 42 days. In order to assess the mineral intake more accurately, the rats drank only distilled water during this period. At the end of the magnesium deprivation, the rat body weight was 280 ± 22 g. Room temperature was maintained at 21 ± 1 °C with a dark – light cycle of 12:12 h. Care and treatment of animals was according to the guidelines for animal care. 2.2. Muricide assays after THC treatment Both rat groups were subjected to a 24-h period of starvation and a 48-h period of isolated housing. Three hours before treatment, they were fed ad libitum. They were then intraperitoneally injected a single dose, either 0-, 2-, 4- or 8-mg/kg THC (1% aqueous solution in Tween 80) under constant volume (2.5 ml/kg). All the rats were tested for muricidal activity 1, 2, 3, 4, 5 and 6 h after the injection. After each assay, 30 min after the mouse’s death, each rat was transferred with caution in a new clean cage and a new OF1 mouse (IFFA Credo, France) (body weight 22 – 26 g) was brought 30 min later. The occurrence and the muricidal pattern were then investigated. The pattern of MB has been reported previously [18,19]. Briefly, it presents three successive phases: (i) latency period before the preying attack on the mouse, (ii) attack on the living mouse (which is direct and efficient leading rapidly to the mouse’s death in natural killer rats), (iii) attack upon the dead mouse which generally stops in about 60 s. The duration of each phase was measured and expressed in seconds. 2.3. Plasma magnesium concentrations Magnesium concentrations were determined by atomic spectrophotometry in plasma and expressed in mg/ml according to Rousselet and Durlach [20].
A single injection of 2, 4 or 8 mg/kg in starving, isolated rats was without aggressive effect upon MB. 3.2. Action of THC on 50-ppm Mg-deficient (severe magnesium deficiency) At the end of the magnesium-deprivation, all the untreated 50-ppm Mg-deficient rats were muricidal and they were used as controls to compare the MB changes induced by injection of THC. Their magnesium plasma levels were 5.0 ± 0.3 vs. 21.0 ± 1.5 mg/ml at the beginning of the experiment. The MB pattern—i.e., the duration of each phase—was constant throughout the six successive assays: 14.1 ± 0.7 s for attack latency, 54.0 ± 1.5 s for attack on the living mouse and 68.0 ± 1.2 s for attack on the dead mouse (Fig. 1a – c). THC treatment resulted in a significant decrease ( P < .01) in the attack latency duration vs. controls from the first assay in the three treated groups (7.2 ± 0.6, 4.2 ± 0.8, 3.4 ± 0.3 s for 2-, 4- and 8-mg/kg THC, respectively; controls 14.7 ± 0.6 s) and reached a constant low level from the third to the sixth assay. The duration of the five last assays in the three THC-treated groups was 3.5 ± 0.5 s (the attack latency was reduced about four-fold as compared to controls) (Fig. 1a). The variations in the duration of the attack on the living mouse were more complex when compared to controls (Fig. 1b). THC 2 mg/kg induced a decrease in this duration, significant from the second assay (THC-treated 40.3 ± 5.6 s, controls 52.7 ± 0.9 s) and remained constant from the fourth to the sixth assay (9.4 ± 0.4 s). Higher doses of THC (4 and 8 mg/kg) induced a diphasic variation of this parameter: increase for the first (THC-treated 100.6 ± 12.8 and 150.6 ± 20.4 s, respectively; controls 55.3 ± 1.2 s) and the second assay, then decrease in the other ones (9.4 ± 2.5 s). The rats killed their prey progressively faster. It can be seen from Fig. 1b that from the fourth assay, the rats killed the mouse about six-fold faster than the controls, whatever the THC doses. Finally, the attack on the dead mouse (Fig. 1c) also varied differently according to the number of assays. In the first assay, the attack decreased in the rats treated with the two higher doses. In contrast, at the dose of 2-mg/kg THC, the rats began to attack the dead mouse. When repeating the
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Fig. 1. Effect of 2-, 4- or 8-mg/kg THC in 50 ppm Mg-deficient rat groups for six successive assays on the three phases of MB: (a) attack latency, (b) attack on the living mouse, (c) attack on the dead mouse. Control values are those of the 50-ppm Mg-deficient rat group untreated with THC. * P < .05, * * P < .01 vs. untreated 50-ppm Mg-deficient rats.
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Fig. 2. Effect of 2-, 4- or 8-mg/kg THC in 150 ppm Mg-deficient rat groups for six successive assays on the three phases of MB: (a) attack latency, (b) attack on the living mouse, (c) attack on the dead mouse. Control values are those of the 50-ppm Mg-deficient rat group untreated with THC since 150-ppm Mg-deficient rats did not exhibit MB. * P < .05, * * P < .01 vs. untreated 50-ppm Mg-deficient rats.
P. Bac et al. / Physiology & Behavior 77 (2002) 189–195
muricide assays, the duration of the attack on the dead mouse increased dramatically from the second assay to the sixth assay whatever the THC dose (8-mg/kg THC-treated 550.9 ± 69.5 s, controls 66.9 ± 1.2 s). The attack was all the more marked as the THC doses were higher. The increases induced by 2-, 4- and 8-mg/kg THC were, respectively, 337%, 523% and 823% vs. controls, in the sixth assay (Fig. 1c). 3.3. Action of THC on 150-ppm Mg-deficient group (moderate deficiency) The magnesium plasma concentration was decreased twice at the end of the magnesium-deficient feeding period (12.3 ± 0.9 vs. 21.0 ± 1.5 mg/ml initially). In this 150-ppm Mg-deficient group, the untreated rats were not muricidal. Consequently, we used the control rats of the preceding experiment (with a severe magnesium deficiency and no THC treatment) as controls to compare the MB pattern in rats suffering a moderate magnesium deficiency and injected a single low dose of THC. Indeed, all doses of THC induced a 100% MB in this group fed 150-ppm Mg-diet. The same pattern was observed as previously but the variations were less severe than in the 50-ppm Mg-deficient rat group (Fig. 2a– c). The attack latency duration (Fig. 2a) decreased significantly ( P < .01) from the first (6.5 ± 0.3 and 5.2 ± 0.5 s, respectively) to the sixth assay with the two higher doses of THC as compared to controls. However, this phase did not decrease as much as in the 50-ppm Mg-deficient rats treated with equivalent doses of THC, for the first to the fourth assay, but became equivalent in the two last ones (3.3 ± 0.3 s). For 2-mg/kg THC, the decrease was significant only for the fifth and the sixth assays (9.2 ± 0.7 s) (Fig. 2a). The same dose induced also a decrease in the duration of the attack on the living mouse (Fig. 2b) from the fourth to the sixth assay (45.7 ± 1.1, 20.8 ± 0.6, 18.2 ± 0.3 s, respectively; controls 56.3 ± 1 s). Higher doses of THC induced a biphasic variation: increase for the first, second and third assays for the 4-mg THC, for the first and the second assay for the 8-mg/kg THC, then decrease for the following ones (20.6 ± 0.6 and 19.6 ± 1.2 s, respectively, in the sixth assay). Finally, the duration of the attack on the dead mouse (Fig. 2c) increased significantly from the second assay for the higher dose (8 mg/kg) and from the fourth to the sixth assay whatever the THC doses. In the sixth assay, the attacks lasted 120.6 ± 6.7, 135.6 ± 10.8 and 175.9 ± 19.3 s with 2-, 4and 8-mg/kg THC, respectively.
4. Discussion MB was reported to be induced by a single dose of THC (11 mg/kg) in rats, in conditions of starvation and isolated housing [7]. Similarly, we showed in the same conditions (i.e., 11-mg/kg THC) that 70% of naive rats fed a normal 1700 ± 100-ppm Mg-containing diet became muricidal [19].
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Their muricidal pattern showed from the first assay an increased aggressiveness as compared to NK rats, with an important increase in the two first phases and a very prolonged attack on the dead mouse (the durations of the three respective phases in NK rats were 13.8 ± 0.6, 27.6 ± 0.7 and 69.2 ± 1 s, respectively). However, we observed previously while repeating the assays (six successive assays 1 h delayed) in rats having a physiological magnesium status that progressively the durations of the three phases decreased to levels similar to those of NK rats. This suggests that the killing behavior was progressively acquired through the six successive assays under the influence of 11-mg/kg THC [19]. In contrast, lower doses (2, 4 or 8 mg/kg) assayed on the same rats failed to induce MB. In the present work, we showed that MB appeared even at these low doses of THC (2, 4 or 8 mg/kg), in rats suffering a severe but also a moderate magnesium deficiency. The greater was the magnesium deficiency, the greater the MB in the rats. A severe magnesium deficiency (50-ppm diet) may induce MB by itself [18]. In the present work, we observed that 50-ppm Mg-deficient diet was responsible for 100% MB, whereas the 150-ppm one failed to induce MB. The corresponding Mg2 + plasma concentrations were four-fold and two-fold decreased at the end of the deprivation period. Injection of a dose as low as 2-mg/kg THC to 50-ppm Mgdeficient rats resulted in an increased aggressiveness represented by a progressive significant decrease in the two first phases and an increased attack on the dead mouse (which was not linked to hunger [19]). By repeating the muricidal assays, we observed that this aggressiveness increased dramatically on each successive assay. With higher doses of THC, the previous tendencies were greatly enhanced, mainly the attack on the dead mouse (nine-fold increased in the 8-mg/kg THC-treated group on the sixth assay vs. controls), but also the first two phases which reached a very low level identical whatever the THC doses (about five-fold decreased vs. controls). The apparent distortion observed in the two groups treated with 4 and 8 mg/kg, when the two first assays gave an opposite effect was probably attributable to an inadequate localization and holding of the prey linked to THC-induced motor incoordination in the two first hours following injection. Finally, it appeared that the rats attacked their prey faster and faster as a result of a true killing training and then tore the mouse to pieces more and more furiously. The more magnesium deficient were the rats, the more was their MB even when the single THC dose injected was as low as 2 mg/kg. A moderate Mg-deficient diet did not induce MB by itself. In contrast, when injected with 2, 4 and 8 mg/kg, those rats developed a 100% MB whatever the dose. All the previously reported alterations were observed but were less severe than in the 50-ppm Mg-deficient rats. By repeating the assays, a true killing training appeared as in the 50-ppm Mg-deficient rats and mainly for the two higher THC doses. The pattern included a decrease in the first two phases and
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an increase in the third phase (for example, at a dose of 8 mg/kg, the increase was three-fold higher than in controls but three-fold lower than in 50-ppm Mg-deficient rats injected with the corresponding doses). This increase cannot be attributed to an excited state generated by remaining blood or bones since the rats were transferred after each assay in a new clean cage. Although the neuroanatomical and neurotransmitter systems involved in MB are not yet fully known, it has been suggested that cerebral serotonin may have an inhibitory role in killing behavior [21,22]. Treatments, which decrease the serotoninergic function such as the injection of a tryptophan hydroxylase inhibitor ( p-chlorophenylalanine) [4,23], lesions of the dorsal and median raphe nuclei [8 – 12] and central injections of indoleamine neurotoxins (5,6and 5,7-dihydroxytryptamine) have been shown to facilitate MB in killer rats, or to induce killing in non-killer rats. Conversely, serotonin intraventricular injections [16] or systemic injections of the serotonin precursor, 5-hydroxytryptophan [14], have been shown to inhibit MB in killer rats. So, the MB observed in the 50-ppm Mg-deficient rat group, in the present experiment, was not surprising since magnesium is a cofactor of tryptophan hydroxylase, the limiting enzyme in the serotoninergic pathway, and is necessary in serotonin-receptor binding [21 – 27]. THC was also reported to induce a depressive effect on the serotoninergic turnover [28,29]. Consequently, the potentiation of the 50-ppm magnesium deficiency by low doses of THC was not surprising. In contrast, no MB appeared in the 150-ppm Mg-deficient rat group indicating that this diet induced probably a severe imbalance of the serotoninergic neurotransmission, which was not severe enough to induce alone MB. With injection of a single low dose of THC, the serotoninergic imbalance would become sufficient to provoke MB. THC would have acted as a trigger to induce aggressiveness in rats and reciprocally, magnesium deficiency revealed the potential neurotoxicity of a low dose of THC. The aggravation of such a behavior throughout the repetition of the assays led (i) to limit values in the duration of the first two phases of MB, which were in any case lower than those of NK rats; (ii) to a constant increase in the third phase, which was related both to the degree of Mg-deficiency and the dose of THC. Since the MB pattern remained identical for the six assays in the 50ppm Mg-deficient rats not treated with THC, whereas it was profoundly changed in both Mg-deficient THCinjected rats, it may be assumed that besides the potentialisation of the serotoninergic alterations [21,22,24], a lot of other mechanisms of neural increased excitability may also be involved [25 – 27]. These include dopaminergic neurons activation, which may be mediated through various systems, including cannabinoid and opioid receptors [28]. This hypothesis is in agreement with studies showing that drugs decreasing 5-HT turnover and/or increasing catecholamine levels potentiate cannabis-induced aggressiveness [29,30].
In conclusion, the present work showed that, in moderate or severe magnesium deficiency in rats, low doses of THC were responsible both for an aggravation of MB and for an increased aggressiveness. The social stress, represented by the mouse, released in the rats a violent reaction resulting with the repetition of the assays in attacking and killing the mouse progressively faster, but also in tearing it violently into pieces, this last phase being considerably increased as compared to controls. Finally, the results obtained showed (i) that in magnesium-deficient rats, even a single low dose of THC, lower than those reported in the literature, is sufficient to induce an increased aggressive state; (ii) that this aggressiveness is all the more severe as the rats are more magnesium-deficient; (iii) that serotonin is probably involved by both magnesium deficiency and THC but the implication of other neurotransmitters cannot be excluded.
References [1] Karli P. The Norway rat’s killing response to the white mouse: an experimental analysis. Behavior 1956;10:81 – 103. [2] Gomita Y, Ueki S. Effects of long term isolation on aggressive behavior and excitability of the rat with olfactory bulbectomy. Life Sci 1979;24:2455 – 60. [3] Valzelli L, Garattini S. Biochemical and behavioral changes induced by isolation in rats. Neuropharmacology 1972;11:17 – 22. [4] Vergnes M. Induction du comportement d’agression Rat Souris par la p-chlorophenylalanine: roˆle de l’amygdale. Physiol Behav 1980;25: 353 – 6. [5] Ueki S, Fujiwara M, Ogawa N. Mouse-killing behavior (muricide) induced by delta-9-tetrahydrocannabinol in the rat. Physiol Behav 1972;9:585 – 7. [6] Miczeck KA. Does THC induce aggression? Suppression and induction of aggressive reactions by chronic and acute D9-THC treatments in laboratory rats. In: Braude MC, Szara S, editors. Pharmacology of marihuana, vol. 2. New York: Raven Press, 1976. p. 499 – 514. [7] Fujiwara M, Ueki S. The course of aggressive behavior induced by a single injection of D9-tetrahydrocannabinol and its characteristics. Physiol Behav 1979;22:535 – 9. [8] Albert DJ, Walsh ML, Ryan J, Siemens Y. Mouse killing in rats: a comparison of spontaneous killers and rats with lesions of the medial hypothalamus or the medial accumbens nucleus. Physiol Behav 1982; 29:989 – 94. [9] Grant LD, Cosuna DV, Grossman SP, Freedman DX. Muricide after serotonin depleting lesions of midbrain raphe nuclei. Pharmacol, Biochem Behav 1977;1:105 – 10. [10] Shibata S, Yamamoto T, Ueki S. Differential effects of medial, central and basolateral amygdaloid lesions on four models of experimentally induced aggression in rats. Physiol Behav 1982;28:289 – 94. [11] Ueki S, Nurimoto S, Ogawa N. Characteristics in emotional behavior of the rat with bilateral olfactory bulb ablations. Folia Psychiatr Neurol Jpn 1972;26:227 – 37. [12] Yamamoto T, Ueki S. Characteristics in aggressive behavior induced by midbrain raphe lesions in rats. Physiol Behav 1977;19:105 – 10. [13] Abe Y, Tadano T, Yonezawa A, Kisara K. Suppressive effect of intraventricular injected dopamine and nomifensine on muricide induced by thiamine deficiency. Pharmacol, Biochem Behav 1987;26: 77 – 82. [14] Onodera K, Kisara K, Ogura Y. Effect of 5-hydroxytryptophan on muricide response induced by thiamin deficiency. Arch Int Pharmacodyn 1979;240:220 – 7.
P. Bac et al. / Physiology & Behavior 77 (2002) 189–195 [15] Vorhees CV. Thiamin deficiency induced muricide behavior in rats. Physiol Behav 1979;23:211 – 4. [16] Onodera K, Ogura K, Kisara K. Characteristics of muricidal induced by thiamin deficiency and its suppression by antidepressants or intraventricular serotonin in the rat. Brain Res 1981;169:139 – 53. [17] Gibbons JL, Barr GA, Bridger WH, Leibowitz JF. Manipulations of dietary tryptophan: effects on mouse killing and brain serotonin in the rat. Brain Res 1979;169:139 – 53. [18] Bac P, Pages N, Herrenknecht C, Teste JF. Inhibition of mouse-killing behaviour in magnesium-deficient rats: effect of pharmacological doses of magnesium pidolate, magnesium aspartate, magnesium lactate, magnesium gluconate and magnesium chloride. Magnesium Res 1995;8:37 – 45. [19] Bac P, Pages N, Herrenknecht C, Paris M. Measurement of the three phases of muricide behavior induced by D9-tetrahydrocannabinol in isolated, fasting rats. Physiol Behav 1998;63:815 – 20. [20] Rousselet F, Durlach J. Techniques analytiques et explorations pratiques du me´tabolisme magne´sique en clinique humaine. In: Durlach J, editor. 1er Symposium International sur le De´ficit Magne´sique en Pathologie Humaine. I. Vol. des Rapports, SGEMV ed. Vittel, 1971, p. 65 – 90. [21] Barr GA, Gibbons J, Bridger WH. Neuropharmacological regulation of mouse killing by rat. Behav Biol 1976;17:143 – 59. [22] Segawa T, Bando S, Hosakawa M. Brain serotonin metabolism and
[23]
[24] [25]
[26] [27] [28]
[29]
[30]
195
D9-tetrahydrocannabinol-induced muricidal behavior in rats. Jpn J Pharmacol 1977;27:581 – 2. Bac P, Pages N, Herrenknecht C, Dewulf C, Binet P, Durlach J. Effect of various serotoninergically induced manipulations on audiogenic seizures in magnesium deficient mice. Magnesium Res 1994;7: 107 – 15. Valzelli L. Serotoninergic inhibitory control of experimental aggression. Pharmacol Res Commun 1982;14:1 – 13. Durlach J, Bac P, Bara M, Guiet-Bara A. Physiopathology of symptomatic and latent forms of central nervous hyperexcitability: a current general scheme. Magnesium Res 2000;13:293 – 303. Durlach J. Magnesium in clinical practice. London: John Libbey, 1988. Durlach J, Bara M. Le Magne´sium en Biologie et en Me´decine. Cachan, Ed. Me´d. Inter., 2000. Melis M, Gessa GL, Diana M. Different mechanisms for dopaminergic excitation induced by opiates and cannabinoids in rat midbrain. Prog Neuropsychopharmacol Biol Psychiatry 2000;24:993 – 1006. Palermo Neto J, Carlini EA. Aggressive behaviour elicited in rats by cannabis sativa: effects of p-chlorophenylalanine and DOPA. Eur J Pharmacol 1972;17:215 – 20. Fujiwara M, Kataoka Y, Yori H, Ueki S. Irritable aggression induced by delta-9 tetrahydrocannabinol in rats pretreated with 6-hydroxydopamine. Pharmacol, Biochem Behav 1984;20:457 – 62.
THC aggravates rat muricide behavior induced by two levels of magnesium deficiency Pierre Baca,*, Nicole Pagesa,b, Christine Herrenknechtc, Charlotte Duponta, Pierre Mauroisa, Joseph Vamecqa, Jean Durlachd a
Department of Neuropharmacology, Faculte´ de Pharmacie, 5 Rue J.B. Cle´ment, F-92296 Chaˆtenay Malabry Cedex, France b Department of Toxicology, Faculte´ de Pharmacie, Route du Rhin, F-67400 Illkirch Graffenstaden, France c Department of Analytical Chemistry, Faculte´ de Pharmacie, 5 Rue J.B. Cle´ment, F-92296 Chaˆtenay Malabry Cedex, France d SDRM, Hoˆpital Saint Vincent de Paul, Avenue Denfert Rochereau, F-75014 Paris, France Received 9 August 2001; received in revised form 15 April 2002; accepted 9 May 2002
Abstract A severe magnesium deprivation induces an interspecific aggressive behavior (muricidal behavior, MB) in different strains of rats. D9-tetrahydrocannabinol (THC) is also known to induce MB even after a single injection (11 mg/kg) in starving, isolated rats. In the present work, we investigated the MB behavior, for six successive assays 1 h delayed, of two groups of male Long – Evans rats fed 50- or 150-ppm Mg2 + -deficient diets, for 42 days after a single injection of THC at doses (2, 4 or 8 mg/kg) that did not induce aggressiveness in control rats. This treatment led to Mg2 + plasma levels of 5 ± 0.3 and 12.3 ± 0.9 mg/ml vs. 21 ± 1.5 mg/ml initially. In the 50-ppm Mg-deficient rat group, all the rats were muricidal but the MB pattern was severely aggravated by THC. In the 150-ppm Mg-deficient rat group, no rat was muricidal but all doses of THC induced a 100% MB. In addition, by quantifying the three phases of MB, we showed through six consecutive hourly muricidal assays, that the two first phases (attack latency and attack on the living mouse) decreased progressively, whereas the third phase (attack on the dead mouse) increased dramatically. This indicates firstly that Mg-deprivation decreases the responsiveness threshold of rats to THC. Secondly, these very low doses of THC induced an aggravation of MB and an acquired hyper-aggressiveness in both 50- and 150-ppm Mg-deficient rats, probably involving different neurotransmitters, mainly serotonin, which is decreased by both treatments. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Magnesium deficiency; Magnesium depletion; Cannabis; Rat; D9-Tetrahydrocannabinol (THC); Muricidal behavior; Aggressiveness
1. Introduction The predatory behavior of rats, which prompts them to prey on mice and sometimes to attack their congeners, may be the result of innate habits (natural killers) [1] or can be induced by long-term isolation [2,3], by chemical agents such as p-chlorophenylalanine or THC [4– 7], or by various regional brain lesions [8 – 12]. Deficiencies in nutrients such as thiamine [13 –16], tryptophan [17] or severe magnesium deficiency (50-ppm Mg-diet) [18] are also known to induce muricidal behavior (MB). Consequently, it may be assumed
* Corresponding author. Tel.: +33-1-4683-5551. E-mail address: [email protected] (P. Bac).
that any decrease in magnesium plasma levels may potentiate neurotoxicity of xenobiotics. It has been reported that acute or chronic administration of cannabis extracts or D9-tetrahydrocannabinol (THC) may alter aggressiveness in the rat [5– 7]. In agreement with Fujiwara and Ueki [7], we observed, 1 h after a single intraperitoneal injection (11 mg/kg b.w.) of THC, a high percentage (70%) MB among naive rats, in conditions of starvation and isolated housing. In addition, we showed that the muricidal pattern was significantly different from that of natural killer rats (NK) in the first assay. Indeed, 1 h after THC injection, the preying attack was disorganized and the duration of each phase increased notably, mainly the attack on the dead mouse which was far longer than in NK, indicating that THC induced an increased aggressiveness [19]. In addition, MB became more efficient through re-
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peated assays and resembled, on the sixth assay, that of NK, suggesting that the killer behavior was progressively acquired for the six assays whereas the aggressiveness disappeared with the elimination of THC. The aim of the present work was to study in rats presenting severe and moderate magnesium deficiencies (fed 50- and 150-ppm Mg-deficient diet, respectively, for 6 weeks) the influence of a single injection of THC at low dose (2, 4 or 8 mg/kg).
2.4. Statistical analysis Results were expressed as mean values ± S.E.M. The comparison between the different experimental groups was made by ANOVA. Significant differences ( P < .05) were estimated on the basis of Fischer – Scheffe´ and Dunnett’s tests.
3. Results 2. Materials and methods
3.1. Action of THC on rats fed a control diet (1700±100-ppm Mg)
2.1. Rats Male Long – Evans, 7-week-old rats (Janvier, France) were divided into three groups (n = 120/group). The first group was fed on a standard 1700-ppm Mg-diet. The two other groups were fed on UAR Mg-deficient diets, one group receiving 50 ppm and the other 150 ppm, for a period of 42 days. In order to assess the mineral intake more accurately, the rats drank only distilled water during this period. At the end of the magnesium deprivation, the rat body weight was 280 ± 22 g. Room temperature was maintained at 21 ± 1 °C with a dark – light cycle of 12:12 h. Care and treatment of animals was according to the guidelines for animal care. 2.2. Muricide assays after THC treatment Both rat groups were subjected to a 24-h period of starvation and a 48-h period of isolated housing. Three hours before treatment, they were fed ad libitum. They were then intraperitoneally injected a single dose, either 0-, 2-, 4- or 8-mg/kg THC (1% aqueous solution in Tween 80) under constant volume (2.5 ml/kg). All the rats were tested for muricidal activity 1, 2, 3, 4, 5 and 6 h after the injection. After each assay, 30 min after the mouse’s death, each rat was transferred with caution in a new clean cage and a new OF1 mouse (IFFA Credo, France) (body weight 22 – 26 g) was brought 30 min later. The occurrence and the muricidal pattern were then investigated. The pattern of MB has been reported previously [18,19]. Briefly, it presents three successive phases: (i) latency period before the preying attack on the mouse, (ii) attack on the living mouse (which is direct and efficient leading rapidly to the mouse’s death in natural killer rats), (iii) attack upon the dead mouse which generally stops in about 60 s. The duration of each phase was measured and expressed in seconds. 2.3. Plasma magnesium concentrations Magnesium concentrations were determined by atomic spectrophotometry in plasma and expressed in mg/ml according to Rousselet and Durlach [20].
A single injection of 2, 4 or 8 mg/kg in starving, isolated rats was without aggressive effect upon MB. 3.2. Action of THC on 50-ppm Mg-deficient (severe magnesium deficiency) At the end of the magnesium-deprivation, all the untreated 50-ppm Mg-deficient rats were muricidal and they were used as controls to compare the MB changes induced by injection of THC. Their magnesium plasma levels were 5.0 ± 0.3 vs. 21.0 ± 1.5 mg/ml at the beginning of the experiment. The MB pattern—i.e., the duration of each phase—was constant throughout the six successive assays: 14.1 ± 0.7 s for attack latency, 54.0 ± 1.5 s for attack on the living mouse and 68.0 ± 1.2 s for attack on the dead mouse (Fig. 1a – c). THC treatment resulted in a significant decrease ( P < .01) in the attack latency duration vs. controls from the first assay in the three treated groups (7.2 ± 0.6, 4.2 ± 0.8, 3.4 ± 0.3 s for 2-, 4- and 8-mg/kg THC, respectively; controls 14.7 ± 0.6 s) and reached a constant low level from the third to the sixth assay. The duration of the five last assays in the three THC-treated groups was 3.5 ± 0.5 s (the attack latency was reduced about four-fold as compared to controls) (Fig. 1a). The variations in the duration of the attack on the living mouse were more complex when compared to controls (Fig. 1b). THC 2 mg/kg induced a decrease in this duration, significant from the second assay (THC-treated 40.3 ± 5.6 s, controls 52.7 ± 0.9 s) and remained constant from the fourth to the sixth assay (9.4 ± 0.4 s). Higher doses of THC (4 and 8 mg/kg) induced a diphasic variation of this parameter: increase for the first (THC-treated 100.6 ± 12.8 and 150.6 ± 20.4 s, respectively; controls 55.3 ± 1.2 s) and the second assay, then decrease in the other ones (9.4 ± 2.5 s). The rats killed their prey progressively faster. It can be seen from Fig. 1b that from the fourth assay, the rats killed the mouse about six-fold faster than the controls, whatever the THC doses. Finally, the attack on the dead mouse (Fig. 1c) also varied differently according to the number of assays. In the first assay, the attack decreased in the rats treated with the two higher doses. In contrast, at the dose of 2-mg/kg THC, the rats began to attack the dead mouse. When repeating the
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Fig. 1. Effect of 2-, 4- or 8-mg/kg THC in 50 ppm Mg-deficient rat groups for six successive assays on the three phases of MB: (a) attack latency, (b) attack on the living mouse, (c) attack on the dead mouse. Control values are those of the 50-ppm Mg-deficient rat group untreated with THC. * P < .05, * * P < .01 vs. untreated 50-ppm Mg-deficient rats.
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Fig. 2. Effect of 2-, 4- or 8-mg/kg THC in 150 ppm Mg-deficient rat groups for six successive assays on the three phases of MB: (a) attack latency, (b) attack on the living mouse, (c) attack on the dead mouse. Control values are those of the 50-ppm Mg-deficient rat group untreated with THC since 150-ppm Mg-deficient rats did not exhibit MB. * P < .05, * * P < .01 vs. untreated 50-ppm Mg-deficient rats.
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muricide assays, the duration of the attack on the dead mouse increased dramatically from the second assay to the sixth assay whatever the THC dose (8-mg/kg THC-treated 550.9 ± 69.5 s, controls 66.9 ± 1.2 s). The attack was all the more marked as the THC doses were higher. The increases induced by 2-, 4- and 8-mg/kg THC were, respectively, 337%, 523% and 823% vs. controls, in the sixth assay (Fig. 1c). 3.3. Action of THC on 150-ppm Mg-deficient group (moderate deficiency) The magnesium plasma concentration was decreased twice at the end of the magnesium-deficient feeding period (12.3 ± 0.9 vs. 21.0 ± 1.5 mg/ml initially). In this 150-ppm Mg-deficient group, the untreated rats were not muricidal. Consequently, we used the control rats of the preceding experiment (with a severe magnesium deficiency and no THC treatment) as controls to compare the MB pattern in rats suffering a moderate magnesium deficiency and injected a single low dose of THC. Indeed, all doses of THC induced a 100% MB in this group fed 150-ppm Mg-diet. The same pattern was observed as previously but the variations were less severe than in the 50-ppm Mg-deficient rat group (Fig. 2a– c). The attack latency duration (Fig. 2a) decreased significantly ( P < .01) from the first (6.5 ± 0.3 and 5.2 ± 0.5 s, respectively) to the sixth assay with the two higher doses of THC as compared to controls. However, this phase did not decrease as much as in the 50-ppm Mg-deficient rats treated with equivalent doses of THC, for the first to the fourth assay, but became equivalent in the two last ones (3.3 ± 0.3 s). For 2-mg/kg THC, the decrease was significant only for the fifth and the sixth assays (9.2 ± 0.7 s) (Fig. 2a). The same dose induced also a decrease in the duration of the attack on the living mouse (Fig. 2b) from the fourth to the sixth assay (45.7 ± 1.1, 20.8 ± 0.6, 18.2 ± 0.3 s, respectively; controls 56.3 ± 1 s). Higher doses of THC induced a biphasic variation: increase for the first, second and third assays for the 4-mg THC, for the first and the second assay for the 8-mg/kg THC, then decrease for the following ones (20.6 ± 0.6 and 19.6 ± 1.2 s, respectively, in the sixth assay). Finally, the duration of the attack on the dead mouse (Fig. 2c) increased significantly from the second assay for the higher dose (8 mg/kg) and from the fourth to the sixth assay whatever the THC doses. In the sixth assay, the attacks lasted 120.6 ± 6.7, 135.6 ± 10.8 and 175.9 ± 19.3 s with 2-, 4and 8-mg/kg THC, respectively.
4. Discussion MB was reported to be induced by a single dose of THC (11 mg/kg) in rats, in conditions of starvation and isolated housing [7]. Similarly, we showed in the same conditions (i.e., 11-mg/kg THC) that 70% of naive rats fed a normal 1700 ± 100-ppm Mg-containing diet became muricidal [19].
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Their muricidal pattern showed from the first assay an increased aggressiveness as compared to NK rats, with an important increase in the two first phases and a very prolonged attack on the dead mouse (the durations of the three respective phases in NK rats were 13.8 ± 0.6, 27.6 ± 0.7 and 69.2 ± 1 s, respectively). However, we observed previously while repeating the assays (six successive assays 1 h delayed) in rats having a physiological magnesium status that progressively the durations of the three phases decreased to levels similar to those of NK rats. This suggests that the killing behavior was progressively acquired through the six successive assays under the influence of 11-mg/kg THC [19]. In contrast, lower doses (2, 4 or 8 mg/kg) assayed on the same rats failed to induce MB. In the present work, we showed that MB appeared even at these low doses of THC (2, 4 or 8 mg/kg), in rats suffering a severe but also a moderate magnesium deficiency. The greater was the magnesium deficiency, the greater the MB in the rats. A severe magnesium deficiency (50-ppm diet) may induce MB by itself [18]. In the present work, we observed that 50-ppm Mg-deficient diet was responsible for 100% MB, whereas the 150-ppm one failed to induce MB. The corresponding Mg2 + plasma concentrations were four-fold and two-fold decreased at the end of the deprivation period. Injection of a dose as low as 2-mg/kg THC to 50-ppm Mgdeficient rats resulted in an increased aggressiveness represented by a progressive significant decrease in the two first phases and an increased attack on the dead mouse (which was not linked to hunger [19]). By repeating the muricidal assays, we observed that this aggressiveness increased dramatically on each successive assay. With higher doses of THC, the previous tendencies were greatly enhanced, mainly the attack on the dead mouse (nine-fold increased in the 8-mg/kg THC-treated group on the sixth assay vs. controls), but also the first two phases which reached a very low level identical whatever the THC doses (about five-fold decreased vs. controls). The apparent distortion observed in the two groups treated with 4 and 8 mg/kg, when the two first assays gave an opposite effect was probably attributable to an inadequate localization and holding of the prey linked to THC-induced motor incoordination in the two first hours following injection. Finally, it appeared that the rats attacked their prey faster and faster as a result of a true killing training and then tore the mouse to pieces more and more furiously. The more magnesium deficient were the rats, the more was their MB even when the single THC dose injected was as low as 2 mg/kg. A moderate Mg-deficient diet did not induce MB by itself. In contrast, when injected with 2, 4 and 8 mg/kg, those rats developed a 100% MB whatever the dose. All the previously reported alterations were observed but were less severe than in the 50-ppm Mg-deficient rats. By repeating the assays, a true killing training appeared as in the 50-ppm Mg-deficient rats and mainly for the two higher THC doses. The pattern included a decrease in the first two phases and
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an increase in the third phase (for example, at a dose of 8 mg/kg, the increase was three-fold higher than in controls but three-fold lower than in 50-ppm Mg-deficient rats injected with the corresponding doses). This increase cannot be attributed to an excited state generated by remaining blood or bones since the rats were transferred after each assay in a new clean cage. Although the neuroanatomical and neurotransmitter systems involved in MB are not yet fully known, it has been suggested that cerebral serotonin may have an inhibitory role in killing behavior [21,22]. Treatments, which decrease the serotoninergic function such as the injection of a tryptophan hydroxylase inhibitor ( p-chlorophenylalanine) [4,23], lesions of the dorsal and median raphe nuclei [8 – 12] and central injections of indoleamine neurotoxins (5,6and 5,7-dihydroxytryptamine) have been shown to facilitate MB in killer rats, or to induce killing in non-killer rats. Conversely, serotonin intraventricular injections [16] or systemic injections of the serotonin precursor, 5-hydroxytryptophan [14], have been shown to inhibit MB in killer rats. So, the MB observed in the 50-ppm Mg-deficient rat group, in the present experiment, was not surprising since magnesium is a cofactor of tryptophan hydroxylase, the limiting enzyme in the serotoninergic pathway, and is necessary in serotonin-receptor binding [21 – 27]. THC was also reported to induce a depressive effect on the serotoninergic turnover [28,29]. Consequently, the potentiation of the 50-ppm magnesium deficiency by low doses of THC was not surprising. In contrast, no MB appeared in the 150-ppm Mg-deficient rat group indicating that this diet induced probably a severe imbalance of the serotoninergic neurotransmission, which was not severe enough to induce alone MB. With injection of a single low dose of THC, the serotoninergic imbalance would become sufficient to provoke MB. THC would have acted as a trigger to induce aggressiveness in rats and reciprocally, magnesium deficiency revealed the potential neurotoxicity of a low dose of THC. The aggravation of such a behavior throughout the repetition of the assays led (i) to limit values in the duration of the first two phases of MB, which were in any case lower than those of NK rats; (ii) to a constant increase in the third phase, which was related both to the degree of Mg-deficiency and the dose of THC. Since the MB pattern remained identical for the six assays in the 50ppm Mg-deficient rats not treated with THC, whereas it was profoundly changed in both Mg-deficient THCinjected rats, it may be assumed that besides the potentialisation of the serotoninergic alterations [21,22,24], a lot of other mechanisms of neural increased excitability may also be involved [25 – 27]. These include dopaminergic neurons activation, which may be mediated through various systems, including cannabinoid and opioid receptors [28]. This hypothesis is in agreement with studies showing that drugs decreasing 5-HT turnover and/or increasing catecholamine levels potentiate cannabis-induced aggressiveness [29,30].
In conclusion, the present work showed that, in moderate or severe magnesium deficiency in rats, low doses of THC were responsible both for an aggravation of MB and for an increased aggressiveness. The social stress, represented by the mouse, released in the rats a violent reaction resulting with the repetition of the assays in attacking and killing the mouse progressively faster, but also in tearing it violently into pieces, this last phase being considerably increased as compared to controls. Finally, the results obtained showed (i) that in magnesium-deficient rats, even a single low dose of THC, lower than those reported in the literature, is sufficient to induce an increased aggressive state; (ii) that this aggressiveness is all the more severe as the rats are more magnesium-deficient; (iii) that serotonin is probably involved by both magnesium deficiency and THC but the implication of other neurotransmitters cannot be excluded.
References [1] Karli P. The Norway rat’s killing response to the white mouse: an experimental analysis. Behavior 1956;10:81 – 103. [2] Gomita Y, Ueki S. Effects of long term isolation on aggressive behavior and excitability of the rat with olfactory bulbectomy. Life Sci 1979;24:2455 – 60. [3] Valzelli L, Garattini S. Biochemical and behavioral changes induced by isolation in rats. Neuropharmacology 1972;11:17 – 22. [4] Vergnes M. Induction du comportement d’agression Rat Souris par la p-chlorophenylalanine: roˆle de l’amygdale. Physiol Behav 1980;25: 353 – 6. [5] Ueki S, Fujiwara M, Ogawa N. Mouse-killing behavior (muricide) induced by delta-9-tetrahydrocannabinol in the rat. Physiol Behav 1972;9:585 – 7. [6] Miczeck KA. Does THC induce aggression? Suppression and induction of aggressive reactions by chronic and acute D9-THC treatments in laboratory rats. In: Braude MC, Szara S, editors. Pharmacology of marihuana, vol. 2. New York: Raven Press, 1976. p. 499 – 514. [7] Fujiwara M, Ueki S. The course of aggressive behavior induced by a single injection of D9-tetrahydrocannabinol and its characteristics. Physiol Behav 1979;22:535 – 9. [8] Albert DJ, Walsh ML, Ryan J, Siemens Y. Mouse killing in rats: a comparison of spontaneous killers and rats with lesions of the medial hypothalamus or the medial accumbens nucleus. Physiol Behav 1982; 29:989 – 94. [9] Grant LD, Cosuna DV, Grossman SP, Freedman DX. Muricide after serotonin depleting lesions of midbrain raphe nuclei. Pharmacol, Biochem Behav 1977;1:105 – 10. [10] Shibata S, Yamamoto T, Ueki S. Differential effects of medial, central and basolateral amygdaloid lesions on four models of experimentally induced aggression in rats. Physiol Behav 1982;28:289 – 94. [11] Ueki S, Nurimoto S, Ogawa N. Characteristics in emotional behavior of the rat with bilateral olfactory bulb ablations. Folia Psychiatr Neurol Jpn 1972;26:227 – 37. [12] Yamamoto T, Ueki S. Characteristics in aggressive behavior induced by midbrain raphe lesions in rats. Physiol Behav 1977;19:105 – 10. [13] Abe Y, Tadano T, Yonezawa A, Kisara K. Suppressive effect of intraventricular injected dopamine and nomifensine on muricide induced by thiamine deficiency. Pharmacol, Biochem Behav 1987;26: 77 – 82. [14] Onodera K, Kisara K, Ogura Y. Effect of 5-hydroxytryptophan on muricide response induced by thiamin deficiency. Arch Int Pharmacodyn 1979;240:220 – 7.
P. Bac et al. / Physiology & Behavior 77 (2002) 189–195 [15] Vorhees CV. Thiamin deficiency induced muricide behavior in rats. Physiol Behav 1979;23:211 – 4. [16] Onodera K, Ogura K, Kisara K. Characteristics of muricidal induced by thiamin deficiency and its suppression by antidepressants or intraventricular serotonin in the rat. Brain Res 1981;169:139 – 53. [17] Gibbons JL, Barr GA, Bridger WH, Leibowitz JF. Manipulations of dietary tryptophan: effects on mouse killing and brain serotonin in the rat. Brain Res 1979;169:139 – 53. [18] Bac P, Pages N, Herrenknecht C, Teste JF. Inhibition of mouse-killing behaviour in magnesium-deficient rats: effect of pharmacological doses of magnesium pidolate, magnesium aspartate, magnesium lactate, magnesium gluconate and magnesium chloride. Magnesium Res 1995;8:37 – 45. [19] Bac P, Pages N, Herrenknecht C, Paris M. Measurement of the three phases of muricide behavior induced by D9-tetrahydrocannabinol in isolated, fasting rats. Physiol Behav 1998;63:815 – 20. [20] Rousselet F, Durlach J. Techniques analytiques et explorations pratiques du me´tabolisme magne´sique en clinique humaine. In: Durlach J, editor. 1er Symposium International sur le De´ficit Magne´sique en Pathologie Humaine. I. Vol. des Rapports, SGEMV ed. Vittel, 1971, p. 65 – 90. [21] Barr GA, Gibbons J, Bridger WH. Neuropharmacological regulation of mouse killing by rat. Behav Biol 1976;17:143 – 59. [22] Segawa T, Bando S, Hosakawa M. Brain serotonin metabolism and
[23]
[24] [25]
[26] [27] [28]
[29]
[30]
195
D9-tetrahydrocannabinol-induced muricidal behavior in rats. Jpn J Pharmacol 1977;27:581 – 2. Bac P, Pages N, Herrenknecht C, Dewulf C, Binet P, Durlach J. Effect of various serotoninergically induced manipulations on audiogenic seizures in magnesium deficient mice. Magnesium Res 1994;7: 107 – 15. Valzelli L. Serotoninergic inhibitory control of experimental aggression. Pharmacol Res Commun 1982;14:1 – 13. Durlach J, Bac P, Bara M, Guiet-Bara A. Physiopathology of symptomatic and latent forms of central nervous hyperexcitability: a current general scheme. Magnesium Res 2000;13:293 – 303. Durlach J. Magnesium in clinical practice. London: John Libbey, 1988. Durlach J, Bara M. Le Magne´sium en Biologie et en Me´decine. Cachan, Ed. Me´d. Inter., 2000. Melis M, Gessa GL, Diana M. Different mechanisms for dopaminergic excitation induced by opiates and cannabinoids in rat midbrain. Prog Neuropsychopharmacol Biol Psychiatry 2000;24:993 – 1006. Palermo Neto J, Carlini EA. Aggressive behaviour elicited in rats by cannabis sativa: effects of p-chlorophenylalanine and DOPA. Eur J Pharmacol 1972;17:215 – 20. Fujiwara M, Kataoka Y, Yori H, Ueki S. Irritable aggression induced by delta-9 tetrahydrocannabinol in rats pretreated with 6-hydroxydopamine. Pharmacol, Biochem Behav 1984;20:457 – 62.