The effect of Δ1,6 tetrahydrocannabinol on biogenic amines and their amino acid precursors in the rat brain

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<ul><li><p>Pharmacological Research Communications, Vol. 3. No. 2, 1971 139 </p><p>THE EFFECT OFA 196 TETRAHYDROCANNABINOL ON BIOGENIC AMINES </p><p>AND THEIR AMINO ACID PRECURSORS IN THE RAT BRAIN </p><p>B. E. Leonard </p><p>Pharmacology Section, Imperial Chemical Industries Ltd., </p><p>Pharmaceuticals Division, Alderley Park, Nr. Macclesfield, Cheshire. </p><p>Received 22 July 1971 </p><p>SUMMARY A 1,6 T.H.C. caused excitement followed by pronounced </p><p>behavioural depression and catalepsy in rats when given at a dose </p><p>of 100 mg/kg i.p. (Lower doses were less effective in producing </p><p>these responses). Some 4 hr after administration, the animals </p><p>became aggressive when disturbed. Ten hr after injection catalepsy </p><p>was absent, the animals were slightly hyperactive -but no longer </p><p>aggressive. Despite the pronounced behavioural changes caused by </p><p>A 196 T.H.C.,the concentration and turnover of brain biogenic amines </p><p>remained unaffected. There was a slight decrease in brain and </p><p>blood tyrosine and in brain y-aminobutyric acid levels. </p><p>INTRODUCTION It is generally agreed that the tetrahydrocannabinols </p><p>are the active principles of marihuana or hashish although the </p><p>precise chemical nature of the active principle is uncertain. </p><p>Despite the widespread concern recently expressed in this country </p><p>and in the United States over the possible deleterious effects </p><p>of cannabis use, the literature seems deficient in any detailed </p><p>studies of the possible mechanisms of action of the tetrahydro- </p><p>cannabinolsonthebrain.Thisproblem has been reviewed by </p><p>Leonard (1969). </p></li><li><p>140 Pharmacological Research Communications, Vo/. 3, No. 2, 1971 </p><p>The present study was therefore undertaken to see what effect </p><p>A 136 tetrahydrocannabinol (A 176 T.H.C.) had on the metabolism </p><p>and turnover of biogenic amines in the rat brain. Previous studies </p><p>of a number of structurally diverse hallucinogenic drugs had </p><p>shown that they all had a pronounced effect on these brain amines </p><p>and their precursors ( Leonard and Tonge, 1969; Tonge and </p><p>Leonard, 1969; Tonge and Leonard, 1970; Leonard and Shallice, </p><p>1971 1. </p><p>METHODS ,196 T.H.C. was dissolved in polyethylene glycol and </p><p>administered to groups of 5 albino rats (T; 90-110 g) of the </p><p>Alderley Park strain by the intraperitoneal route. The control </p><p>group was given an equivalent volume of the vehicle (0.2 ml/100 g </p><p>body weight). The gross behavioural effects produced were </p><p>observed periodically over 10 hr. At various times after </p><p>injection (shown in RESULTS) the animals were killed by </p><p>decapitation, and the brains minus the cerebella removed. </p><p>Noradrenaline dopamine, 5-hydroxytryptamine, 5-hydroxyindole </p><p>acetic acid and normetanephrine were determined on extracts </p><p>from the same brains. The brains were homogenized in 9 ml of </p><p>O.OlN HCl containing 0.2 ml 10% (w/v) ethylene diamine tetra- </p><p>acetic acid (as the disodium salt), centrifuged and 4.5 ml </p><p>of the supernatant fraction removed for the determination of </p><p>normetanephrine by the solvent extraction method of Anton </p><p>and Sayre (1966). 5-Hydroxytryptamine (GHT), s-hydroxyindole- </p><p>acetic acid (S-HlAA), noradrenaline and dopamine were determined </p><p>in the remaining portion of the brain extract by the solvent </p><p>extraction method of Welch and Welch (1969). Blood and brain </p><p>tyrosine and tryptophan, and brain y-amino butyric acid concentration </p><p>were determined by the spectrophoto-fluorimetric methods </p><p>outlined previously (Leonard and Shallice, 1971). </p></li><li><p>Pharmacological Research Communications, Vol. 3, No. 2, 1971 141 </p><p>RESULTS AND DISCUSSION </p><p>Behavioural Effects. Some 15 min after injection of 100 mg/kg </p><p>i.p., the rats were excited. They became behaviourally depressed </p><p>approximately 15 min later but no ataxia could be detected. </p><p>About 1 hr after injection the animals lay prostrate on the cage </p><p>floor and were very depressed and slightly hypothermic. The </p><p>maximum decrease in the pharyngeal temperature throughout the </p><p>10 hr observation period was 2O. When disturbed the animals </p><p>squeaked frequently and became very agitated. Catalepsy was apparent </p><p>1$-2 hr after injection and persisted for a further 3-4 hrs. </p><p>During this period the rats became aggressive towards one </p><p>another when disturbed but were otherwise very depressed and </p><p>remained prostrate. Gradually the depressant effects wore off, and </p><p>7-10 hr after injection the rats became fairly hyperexcitable </p><p>and showed piloerection. Lower doses of A 1,6 T.H.C. (50 and </p><p>75 w/kg i.p.) had qualitatively similar effects but their </p><p>duration and intensity was less marked. No analgesia could </p><p>be detected. </p><p>Brain Amines. Groups of rats were killed at 0 (control), 1, 2, 3, 5, 7 and 10 hr after the administration of 100 mg/kg A 1,6 </p><p>T.H.C. No change could be detected in the concentrations of </p><p>noradrenaline, dopamine, 5-HT, 5-HlAA or normetanephrine at </p><p>these times. The concentrations of these amines and their </p><p>metabolites were:- noradrenaline 0.50 ? 0.06; dopamine 0.806 5 </p><p>2 0.058; 5-HT 0.67 $ 0.04; 5-HlAA 0.288 + 0.03; normetanephrine </p><p>0.31 + 0.026. Each result represents the mean 2 s.e.m. of at </p><p>least five animals in pg/g fresh weight of brain. </p><p>Because of the possibility that A 136 T.H.C. might cause </p><p>changes in the turnover of these amines which are ,not to be </p><p>reflected in changes in their absolute concentration, experiments </p><p>were carried out in which the effect of T.H.C. was studied on </p><p>the rate of depletion of brain noradrenaline and dopamine </p><p>D </p></li><li><p>142 Pharmacological Research Communications, Vol. 3, No. 2, 1971 </p><p>following the administration of the tyrosine hydroxylase </p><p>inhibitor a-methyl-p-tyrosine (aMTP) and on the depletion </p><p>of brain 5-HT after inhibitionoftryptophan hydroxylase by </p><p>parachlorophenylalamine (PCPA). </p><p>For these experiments, 4 groups of 5 rats were used. </p><p>To determine the effect of A 1,6 T.H.C. on the depletion of </p><p>noradrenaline, 2 groups of rats were injected with 300 mg/kg </p><p>(i.p.) ofaMPT; one of these groups was injected simultaneously </p><p>with A 196 T.H.C. (100 mg/kg i.p.&gt;. The third group of rats </p><p>was injected with A 136 T.H.C. alone and the fourth group </p><p>was given the vehicle (control group). The animals were killed </p><p>3 hr later and the catecholamines determined as described </p><p>under METHODS. For assessing the effect of A 176 T.H.C. on </p><p>the rate of depletion of brain 5-HT, PCPA (suspended in </p><p>Dispersol) was substituted for aMPT and given intraperitoneally </p><p>(300 mg/kg). The groups of rats were otherwise treated in the </p><p>same way as that described for the a.MPT experiment. The </p><p>animals were killed 10 hr later and the brain 5-HT concentration </p><p>determined by the method of Bogdanski and co-workers (1956). </p><p>Following the administration of u.MPT, the concentration </p><p>of brain noradrenaline was reduced by 40%; the concentration </p><p>of this amine in the control brain was 0.47 2 0.04 pg/g. </p><p>aMPT in combination with A 196 T.H.C. caused a reduction of </p><p>brain noradrenaline by 38%. After PCPA, the brain 5-HT </p><p>concentration was reduced by 35%; the control level was </p><p>0.64 f 0.05 Pg/g. PCPA in combination with A 196 T.H.C. caused </p><p>a decrease of 30%. </p><p>It is apparent from these experiments that A 196 T.H.C. </p><p>does not affect the rate of depletion of these amines </p><p>following inhibition of their synthesis. </p><p>Effect on some blood and brain amino acids. The effect of A1,6 T.H.C. on tyrosine, tryptophan and y-aminobutyric acid </p></li><li><p>Pharmacological Research Communications, Vol. 3, No. 2, 1971 143 </p><p>was studied 3 and 10 hr after the administration of the drug. </p><p>The former time coincided with maximum behavioural depression </p><p>and catalepsy, while the latter coincided with maximum behavioural </p><p>excitation. A 1,6 T.H.C. was administered at 50 and 100 mg/kg. </p><p>The results are shown in Table 1; it is apparent that both </p><p>brain and blood tyrosine are reduced at both 3 and 10 hr </p><p>whereas the reduction in brain and blood tryptophan is negligible. </p><p>Brain y-aminobutyric acid levels were slightly reduced 3 and </p><p>10 hr after the drug had been administered. </p><p>The results of this investigation show that little neuro- </p><p>chemical change accompanies the behavioural effects produced </p><p>by A 196 T.H.C. This is somewhat surprising as all other </p><p>hallucinogenic drugs, of diverse chemical structure, which </p><p>have been studied cause marked changes in brain monoamines </p><p>(see references in INTRODUCTION). The changes in brain </p><p>tyrosine may indicate that A 196 T.H.C. affects the uptake </p><p>and/or utilization of the amino acid in the brain and that </p><p>the decrease in brain y-aminobutyric acid, which has been </p><p>implicated as an inhibitory transmitter substance in the </p><p>mammalian brain (Krnjevic and Schwartz, 1966), may be </p><p>correlated with the excitation seen 10 hr after the drug had </p><p>been administered. </p><p>Other investigators have studied the effect of crude cannabis </p><p>extracts on the gross behaviour of rodents and described some- </p><p>what similar effects to these reported here (Chopra and Chopra, </p><p>1939; Loewe, 1950; Schultz, Mohrmann and Haffner, 1959). Garatti- </p><p>ni (1965) carried out a detailed neuropharmacological investigation </p><p>of a crude cannabis extract but could find no changes which </p><p>could be specifically ascribed to the action of the drug. Further- </p><p>more, he did not find that the drug caused any change in the </p><p>levels of brain and heart noradrenaline or brain and intestinal </p><p>5-HT even when doses of up to 300 mg/kg (i.p.) of the extract </p><p>were given. </p></li><li><p>144 Pharmacological Research, Communications, Vql. 3, No. 2, 19il </p><p>0 v) 8 </p><p>r3 </p></li><li><p>Pharmacological Research Communications, Vol. 3, MO. 2, 1971 145 </p><p>It can be concluded from this investigation that changes </p><p>in brain biogenic amines do not accompany the behavioural effects </p><p>of A1J6 T.H.C. There is clearly the need to extend this study </p><p>to include other putative transmitter substances which may be </p><p>primarily affected by the drug. </p><p>REFERENCES </p><p>Anton A.H. and Sayre D.F., 1966, J. Pharmac. exp. Therap. 153, 15-24. </p><p>Bogdanski D.F., Pletscher A., Brodie B.B. and Udenfried S. 1956, J. Pharmac. exp. Therap., 112_, 82. </p><p>Chopra R.N. and Chopra G.S., 1939, Indian J. Med. Res. Mem., 3l,l. Garattini S., 1965, In "Hashish: Its Chemistry and Pharmacology" </p><p>Ed. Wolstenholme G.E.W. and Knight J. Pub. Churchill.p.70-78 Krnjevic K. and Schwartz S., 1966, Exp. Brain Res.,3,320-336. Leonard B.E., 1969, Br. J. Addict., 64, 121-130. Leonard B.E. and Shallice S.A., 1971, Br. J. Pharmac. 41, 198-212,. Leonard B.E. and Tonge S.R., 1969, Life Sci., g, 815-825. Loewe S., 1950, Arch.exp. Path. Pharmak., 211, 175. Schultz D.E., Mohrmann H.L. and Haffner G., 1959, Z. Naturforsch., </p><p>kJ6, 98. Tonge S.R. and Leonard B.E., 1969, Life Sci., ?3, 805-814. </p><p>Tonge S.R. and Leonard B.E., 1970, Life Sci., 2, 1327-133s. Welch A.S. and Welch B.L., 1969, Anal. Biochem., 2, 161-179. </p></li></ul>