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
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,
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
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
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
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
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