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

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  • 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