The effect of hashish extract on the norepinephrine in rabbit brain


Materials and methods


Pages: 33 to 34
Creation Date: 1971/01/01

The effect of hashish extract on the norepinephrine in rabbit brain

Professor C. J MIRAS
Department of Biological Chemistry, School of MedicineUniversity of Athens, Greece

The pharmacological effects of hashish have been connected with the adrenergic mechanism ( [ 1] ). Holtzman et al. ( [ 2] ) found changes in the 5-hydroxytryptamine and the norepinephrine in the whole brain of the mouse. Recently L. Maître et al. ( [ 3] ) have found that the administration of hashish extracts had no effect on the endogenous content of noradrenaline and dopamine in the rat brain.

We have examined the possibility of norepinephrine change in the different parts of the rabbit brain after the administration of a certain amount of extract and sacrificing the animals at a fixed time.

The result was that the total norepinephrine practically did not change in the whole brain but changed considerably in the different parts of it.

Materials and methods

The hashish extract was prepared by extracting hashish for 24 hours with ether. After removing the ether, the resin was weighed, dissolved in ether and then an amount of olive oil was added so that the final product, after removing the ether, contained 0.5 g of resin in each ml of olive oil. The injected dose was 0,3 g/kg.

Rabbits of approximately 2 kg were sacrificed one and a half hours after the intraperitoneal injection of the hashish extract.

The rabbit brain was immediately removed. The different parts were dissected and then placed into beakers containing HClO4 0.4N. For each determination the brains of two rabbits were used. This procedure of receiving pure extracts is based on the method of A. Bertler et al. ( [ 4] ).

The tissue was extracted twice with 10 ml HClO4 0.4N each time.

After the neutralization of the extracts to pH: 5.5 by means of K2CO3 5N, they were transferred to the resin Dowex 50W-X4 (200-400 mesh), dimensions of the column 5 cm height, internal diameter 0.5 cm. The regeneration of the column was done according to Bertler et al. ( [ 5] ).

The elution was performed by 10 ml HCl 1M. Noradrenaline was eluted quantitatively in this fraction.

After neutralizing the eluates to pH: 6, 1 ml was taken from the fraction for the determination.

The noradrenaline was determined as follows:

To 0.5 ml of buffer phosphate solution 0,1M 1ml of the sample was added and then 0,1 ml K3(Fe(CN)6) 0.25% as oxidizing agent. After 5 minutes, 0,2 ml of solution (ascorbic acid/ethylenediamine/NaOH) (6), was added.

The fluorescence was measured after the activation of the samples to fluorescent light for 35 minutes.

All measurements were performed with Aminco-Bowman fluorimeter and were compared with standard curves of NE.


The values listed below are given for 8 measurements, each from two rabbits.


NE g/gr ± SE


Part of the brain



% of change NE

Hypothalamus Anterior and Striam
0.23 ± 0.03
0.18 ± 0.01
Quadrigeminal area and Hypothalamus Pos-tero-superior
0.10 ± 0.01
0.18 ± 0.02
Pons-Medulla oblongata
0.19 ± 0.02
0.24 ± 0.02
0.14 ± 0.01
0.063 ± 0.008
0.070± 0.009
0.07 ± 0.01


The connection of hashish with the adrenergic system has been suggested long ago ( [ 7] ). It is obvious however that the accidental changes in the specific concentration of the adrenergic mediators cannot be reflected in the total amount of norepinephrine in the whole brain. This is evidently the reason why no significant changes have been observed by L. Maître et al. ( [ 3] ). Miras and Constantinidis ( [ 8] ) using histochemical methods found also changes in the green fluorescence (NE) in locus coeruleus and hypothalamus following hashish and THC administration in rats. The importance of adrenergic mediators in psycho-effects, as seems to occur in hashish users and in addicts to other psychodrugs has been discussed long ago. Undoubtedly further research is needed in order to elucidate the exact mechanism and the extent to which hashish influences the metabolism of the catecholamines.


An extract of cannabis injected intraperitoneally to rabbits caused changes in several areas (increase and decrease) in the concentrations of the endogenous norepinephrine in the brain. But there were no significant changes in the total amount of norepinephrine in the whole brain.



C. J. Miras (1965), Hashish, Its chemistry and pharmacology, Ciba Foundation Study Group No. 21, Churchill, London (Eds. G.E.W. Wolstenholme and J. Knight).


D. Holtzman, R. A. Lovell, J. H. Jaffe, and D. X. Freedman (1969) Science, 163, 1464-1467.


L. Maître, M. Staehelin and H. J. Bein (1970) Agents and Actions, Vol. 1/3, 136-143.


A. Bertler, A. Carlsson and E. Rosengren (1958) Acta physiol. Scand. 44, 273-292.


A. Bertler and E. Rosengren (1959) Acta physiol. Scand. 47, 350-361.


A. S. Welch and B. L. Welch (1969) Analyti. Biochem. 30, 1611-79.


G. Joachimoglu, J. Kiburis and C. Miras (1967) Prakt. Akad. Athenon. 70, 161.


C. J. Miras and J. Constantinidis, in press.