Pharmacological Research Communications, Vol. 3. No. 2, 1971 139

THE EFFECT OFA 196 TETRAHYDROCANNABINOL ON BIOGENIC AMINES

AND THEIR AMINO ACID PRECURSORS IN THE RAT BRAIN

B. E. Leonard

Pharmacology Section, Imperial Chemical Industries Ltd.,

Pharmaceuticals Division, Alderley Park, Nr. Macclesfield, Cheshire.

Received 22 July 1971

SUMMARY A 1,6 T.H.C. caused excitement followed by pronounced

behavioural depression and catalepsy in rats when given at a dose

of 100 mg/kg i.p. (Lower doses were less effective in producing

these responses). Some 4 hr after administration, the animals

became aggressive when disturbed. Ten hr after injection catalepsy

was absent, the animals were slightly hyperactive -but no longer

aggressive. Despite the pronounced behavioural changes caused by

A 196 T.H.C.,the concentration and turnover of brain biogenic amines

remained unaffected. There was a slight decrease in brain and

blood tyrosine and in brain y-aminobutyric acid levels.

INTRODUCTION It is generally agreed that the tetrahydrocannabinols

are the active principles of marihuana or hashish although the

precise chemical nature of the active principle is uncertain.

Despite the widespread concern recently expressed in this country

and in the United States over the possible deleterious effects

of cannabis use, the literature seems deficient in any detailed

studies of the possible mechanisms of action of the tetrahydro-

cannabinolsonthebrain.Thisproblem has been reviewed by

Leonard (1969).

140 Pharmacological Research Communications, Vo/. 3, No. 2, 1971

The present study was therefore undertaken to see what effect

A 136 tetrahydrocannabinol (A 176 T.H.C.) had on the metabolism

and turnover of biogenic amines in the rat brain. Previous studies

of a number of structurally diverse hallucinogenic drugs had

shown that they all had a pronounced effect on these brain amines

and their precursors ( Leonard and Tonge, 1969; Tonge and

Leonard, 1969; Tonge and Leonard, 1970; Leonard and Shallice,

1971 1.

METHODS ,196 T.H.C. was dissolved in polyethylene glycol and

administered to groups of 5 albino rats (T; 90-110 g) of the

Alderley Park strain by the intraperitoneal route. The control

group was given an equivalent volume of the vehicle (0.2 ml/100 g

body weight). The gross behavioural effects produced were

observed periodically over 10 hr. At various times after

injection (shown in RESULTS) the animals were killed by

decapitation, and the brains minus the cerebella removed.

Noradrenaline dopamine, 5-hydroxytryptamine, 5-hydroxyindole

acetic acid and normetanephrine were determined on extracts

from the same brains. The brains were homogenized in 9 ml of

O.OlN HCl containing 0.2 ml 10% (w/v) ethylene diamine tetra-

acetic acid (as the disodium salt), centrifuged and 4.5 ml

of the supernatant fraction removed for the determination of

normetanephrine by the solvent extraction method of Anton

and Sayre (1966). 5-Hydroxytryptamine (GHT), s-hydroxyindole-

acetic acid (S-HlAA), noradrenaline and dopamine were determined

in the remaining portion of the brain extract by the solvent

extraction method of Welch and Welch (1969). Blood and brain

tyrosine and tryptophan, and brain y-amino butyric acid concentration

were determined by the spectrophoto-fluorimetric methods

outlined previously (Leonard and Shallice, 1971).

Pharmacological Research Communications, Vol. 3, No. 2, 1971 141

RESULTS AND DISCUSSION

Behavioural Effects. Some 15 min after injection of 100 mg/kg

i.p., the rats were excited. They became behaviourally depressed

approximately 15 min later but no ataxia could be detected.

About 1 hr after injection the animals lay prostrate on the cage

floor and were very depressed and slightly hypothermic. The

maximum decrease in the pharyngeal temperature throughout the

10 hr observation period was 2O. When disturbed the animals

squeaked frequently and became very agitated. Catalepsy was apparent

1$-2 hr after injection and persisted for a further 3-4 hrs.

During this period the rats became aggressive towards one

another when disturbed but were otherwise very depressed and

remained prostrate. Gradually the depressant effects wore off, and

7-10 hr after injection the rats became fairly hyperexcitable

and showed piloerection. Lower doses of A 1,6 T.H.C. (50 and

75 w/kg i.p.) had qualitatively similar effects but their

duration and intensity was less marked. No analgesia could

be detected.

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

T.H.C. No change could be detected in the concentrations of

noradrenaline, dopamine, 5-HT, 5-HlAA or normetanephrine at

these times. The concentrations of these amines and their

metabolites were:- noradrenaline 0.50 ? 0.06; dopamine 0.806 5

2 0.058; 5-HT 0.67 $ 0.04; 5-HlAA 0.288 + 0.03; normetanephrine

0.31 + 0.026. Each result represents the mean 2 s.e.m. of at

least five animals in pg/g fresh weight of brain.

Because of the possibility that A 136 T.H.C. might cause

changes in the turnover of these amines which are ,not to be

reflected in changes in their absolute concentration, experiments

were carried out in which the effect of T.H.C. was studied on

the rate of depletion of brain noradrenaline and dopamine

D

142 Pharmacological Research Communications, Vol. 3, No. 2, 1971

following the administration of the tyrosine hydroxylase

inhibitor a-methyl-p-tyrosine (aMTP) and on the depletion

of brain 5-HT after inhibitionoftryptophan hydroxylase by

parachlorophenylalamine (PCPA).

For these experiments, 4 groups of 5 rats were used.

To determine the effect of A 1,6 T.H.C. on the depletion of

noradrenaline, 2 groups of rats were injected with 300 mg/kg

(i.p.) ofaMPT; one of these groups was injected simultaneously

with A 196 T.H.C. (100 mg/kg i.p.>. The third group of rats

was injected with A 136 T.H.C. alone and the fourth group

was given the vehicle (control group). The animals were killed

3 hr later and the catecholamines determined as described

under METHODS. For assessing the effect of A 176 T.H.C. on

the rate of depletion of brain 5-HT, PCPA (suspended in

Dispersol) was substituted for aMPT and given intraperitoneally

(300 mg/kg). The groups of rats were otherwise treated in the

same way as that described for the a.MPT experiment. The

animals were killed 10 hr later and the brain 5-HT concentration

determined by the method of Bogdanski and co-workers (1956).

Following the administration of u.MPT, the concentration

of brain noradrenaline was reduced by 40%; the concentration

of this amine in the control brain was 0.47 2 0.04 pg/g.

aMPT in combination with A 196 T.H.C. caused a reduction of

brain noradrenaline by 38%. After PCPA, the brain 5-HT

concentration was reduced by 35%; the control level was

0.64 f 0.05 Pg/g. PCPA in combination with A 196 T.H.C. caused

a decrease of 30%.

It is apparent from these experiments that A 196 T.H.C.

does not affect the rate of depletion of these amines

following inhibition of their synthesis.

Effect on some blood and brain amino acids. The effect of A1,6 T.H.C. on tyrosine, tryptophan and y-aminobutyric acid

Pharmacological Research Communications, Vol. 3, No. 2, 1971 143

was studied 3 and 10 hr after the administration of the drug.

The former time coincided with maximum behavioural depression

and catalepsy, while the latter coincided with maximum behavioural

excitation. A 1,6 T.H.C. was administered at 50 and 100 mg/kg.

The results are shown in Table 1; it is apparent that both

brain and blood tyrosine are reduced at both 3 and 10 hr

whereas the reduction in brain and blood tryptophan is negligible.

Brain y-aminobutyric acid levels were slightly reduced 3 and

10 hr after the drug had been administered.

The results of this investigation show that little neuro-

chemical change accompanies the behavioural effects produced

by A 196 T.H.C. This is somewhat surprising as all other

hallucinogenic drugs, of diverse chemical structure, which

have been studied cause marked changes in brain monoamines

(see references in INTRODUCTION). The changes in brain

tyrosine may indicate that A 196 T.H.C. affects the uptake

and/or utilization of the amino acid in the brain and that

the decrease in brain y-aminobutyric acid, which has been

implicated as an inhibitory transmitter substance in the

mammalian brain (Krnjevic and Schwartz, 1966), may be

correlated with the excitation seen 10 hr after the drug had

been administered.

Other investigators have studied the effect of crude cannabis

extracts on the gross behaviour of rodents and described some-

what similar effects to these reported here (Chopra and Chopra,

1939; Loewe, 1950; Schultz, Mohrmann and Haffner, 1959). Garatti-

ni (1965) carried out a detailed neuropharmacological investigation

of a crude cannabis extract but could find no changes which

could be specifically ascribed to the action of the drug. Further-

more, he did not find that the drug caused any change in the

levels of brain and heart noradrenaline or brain and intestinal

5-HT even when doses of up to 300 mg/kg (i.p.) of the extract

were given.

144 Pharmacological Research, Communications, Vql. 3, No. 2, 19il

0 v) 8

r3

Pharmacological Research Communications, Vol. 3, MO. 2, 1971 145

It can be concluded from this investigation that changes

in brain biogenic amines do not accompany the behavioural effects

of A1J6 T.H.C. There is clearly the need to extend this study

to include other putative transmitter substances which may be

primarily affected by the drug.

REFERENCES

Anton A.H. and Sayre D.F., 1966, J. Pharmac. exp. Therap. 153, 15-24.

Bogdanski D.F., Pletscher A., Brodie B.B. and Udenfried S. 1956, J. Pharmac. exp. Therap., 112_, 82.

Chopra R.N. and Chopra G.S., 1939, Indian J. Med. Res. Mem., 3l,l. Garattini S., 1965, In "Hashish: Its Chemistry and Pharmacology"

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.,

kJ6, 98. Tonge S.R. and Leonard B.E., 1969, Life Sci., ?3, 805-814.

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.