The Effects Of (-)-A9-Tetrahydrocannabinol On Reserpine-Induced Hypothermia In Rats

Thread starter #1
L. F. ENGLERT, B. T. HO AND DOROTHY TAYLOR
Texas Research Institute of Mental Sciences, Houston, Texas 77025, U.S.A.

Summary
1. An intravenous injection into rats of 1 mg/kg (-)-A9-tetrahydrocannabinol
A9-THC) had no effect on rectal temperature and produced in the subcellular
fractions of the brain a shift of 5-hydroxytryptamine (5-HT) from the
particulate or 'bound' 5-HT to the supernatant or 'free' fraction, whereas
the noradrenaline (NA) decreased in both fractions.
2. Pretreatment of rats by an intravenous injection of 1 mg/kg A9-THC
three times a week for four weeks, prevented the hypothermia and the reduction
in brain 5-HT produced by an intraperitoneal injection of 15 mg/kg reserpine
given 24 h after the last A9-THC injection.
3. Pretreatment of rats by a single intravenous injection of 1 mg/kg
A9-THC prevented the hypothermia and reduction in brain 5-HT produced
by an intraperitoneal injection of reserpine given 1 h before. The reduction
in brain NA was not prevented except at the 18 h time interval.
4. An injection of 1 mg/kg A9-THC intravenously into rats 3 h after an
intraperitoneal injection of reserpine accentuated the reserpine hypothermia
as well as the reduction of 5-HT but not of NA in the brain.
5. The reserpine hypothermia was not prevented by a single intravenous
injection of 1 mg/kg A9-THC when cinanserin, a 5-HT inhibitor, was injected
30 min before the reserpine.
Introduction
There have been many attempts to relate brain amine concentrations with
thermoregulation. Based on the results obtained on injection of noradrenaline
(NA) and 5-hydroxytryptamine (5-HT), into the cerebral ventricles, Feldberg &
Myers (1963, 1964, 1965) suggested the involvement of catecholamines and 5-HT
in the central mechanism of temperature control. The effects of brain amines
on body temperature vary among different species; in rats 5-HT and NA produce
hypothermia, but the effect of NA changes to hyperthermia as the concentration
increases (Feldberg & Lotti, 1967). More recent evidence also suggests a role
for dopamine in thermoregulation. The hypothermic response to 6-hydroxydopamine
in rats has been related to the release of dopamine (Simmonds &
Uretsky, 1970). Fuxe, Hokfelt & Ungerstedt (1970) related the hypothermia
observed after administration of apomorphine to activation of dopaminergic
receptors. In addition, the dopamine-,8-oxidase inhibitor, FLA-63 (bis-(4-methyll-
homopiperazinylthiocarbonyl)disulphide), has been shown to decrease the effect
of dopamine on body temperature in mice (Svensson & Waldeck, 1969).

The role of brain monoamines in reserpine-induced hypothermia continues to
be a controversial problem. In rats, imipramine-like drugs prevent the hypothermia
produced by reserpine (Costa, Garattini & Valzelli, 1960; Garattini, Giachetti,
Jori, Pieri & Valzelli, 1962; Garattini & Jori, 1967), but when given during the
reserpine hypothermia they increase body temperature (Askew, 1963; Jori &
Garattini, 1965). This hyperthermia may be due to potentiation of NA action
because the imipramine-like drugs prevent the NA re-uptake, thus making more
NA available to the receptor. An antagonism of the hypothermic effect of reserpine
in rats by a-methyl-m-tyrosine, the precursor of a false transmitter metaraminol,
has also been reported (Garattini & Valzelli, 1961). Recently, the effect of
A9-tetrahydrocannabinol (A9-THC) on 5-HT metabolism has been studied in rats
by Sofia, Dixit & Barry (1971). These authors found that the reduction of brain
5-HT induced by reserpine was retarded by pretreatment with A9-THC.
The present investigation deals with the effect of chronic and acute A9-THC
pretreatment on the hypothermia and reduction of the brain monoamines produced
by reserpine as observed on their subcellular distribution. We also examined
whether A9-THC had an effect when given after reserpine at a time when the
hypothermia and reduction of brain monoamines had already occurred, and whether
the effect of acute A9-THC pretreatment could be influenced by cinanserin, an
inhibitor of 5-HT.
Methods
Male Sprague-Dawley rats weighing 150 to 170 g were used. Their rectal
temperature was taken every 30 or 60 min by means of a rectally inserted probe
with a telethermometer (Yellow Spring Instrument Company) calibrated at 0-10 C
intervals. The rats were used in groups of six animals and for each time interval
their mean temperature was calculated and the S.E. was determined.
For the chronic A9-THC pretreatment groups of four rats were used. Each
rat received intravenous injections of 1 mg/kg A9-THC three times a week for
four weeks and at least 24 h after the last injection, reserpine (15 mg/kg) was
given intraperitoneally; 18 h later the rats were killed for determination of
brain 5-HT and NA.
For the acute A9-THC pretreatment three groups of four rats were used. Each
rat was given a single intravenous injection of 1 mg/kg A9-THC 1 h before the
intraperitoneal injection of 15 mg/kg reserpine, and one group was killed 3 h,
another 6 h, and the third 18 h later for determination of the brain 5-HT and
NA. The same number of reserpine-control animals were killed at each time
interval. Groups of rats receiving only the intravenous A9-THC injectioin were
killed 4, 7 and 19 h after the injection so that the time intervals coincided with
those of the rats given both compounds.
In the experiments in which the effect of A9-THC was studied after an intraperitoneal
injection of 15 mg/kg of reserpine, the intravenous injection of 1 mg/kg
A9-THC was given 3 h later. This time interval was chosen because 3-6 h
after the injection of reserpine the rate of fall in body temperature was found
to be greatest. To determine the 5-HT and NA in the brain the rats, again in
groups of four, were killed 3, 6 and 18 h after the reserpine injection.

A9-THC and reserpine hypothermia
Subcellular distribution of 5-hydroxytryptamine and noradrenaline in
brain homogenates
The rats were killed by decapitation under light ether anaesthesia and the
brains removed and frozen until assayed. They were homogenized in 2 parts
0-025 M sucrose containing 1.5 mM ethylenediamine tetraacetic acid and 2 mM
tranylcypromine. The homogenate was centrifuged at 100,000 g for 20 min as
described by Giarman, Freedman & Schanberg (1964).
The supernatant and particulate fractions were separated and the pH adjusted
to 2 with dilute HCI to give a total volume of 3 ml. Both 5-HT and NA were
first extracted into n-butanol and then returned to 1-5 ml of 0-1 N HCI containing
1% cysteine according to a modified procedure of Curzon & Green (1970).
Duplicate 0-5 ml aliquots were taken and assayed separately for 5-HT and NA.
Internal standards of 5-HT and NA were added to the whole brain homogenates
and run through the extraction procedure.
5-HT was determined by the o-phthalaldehyde(OPT) reaction and the fluorescent
product measured on an Aminco-Bowman Spectrophotofluorometer (activation
X: 360 nm; fluorescence X: 470 nm). NA was oxidized by the procedure of
Laverty & Taylor (1968). This method utilizes oxidation with iodine, alkaline
rearrangement and subsequent measurement of the fluorescence at an acidic pH
(activation X: 380 nm; fluorescence X: 480 nm).
The concentration of the amines in the supernatant and particulate fractions
are expressed as ug/g (mean of 4 assays + S.E.M.). Total brain values were
estimated from the two fractions. Percentage values are the difference between
the A9-THC group and either the reserpine. or vehicle controls. The statistical
significance was calculated by Student's t test.
Drugs
A9-Tetrahydrocannabinol (A9-THC) was synthesized in our laboratories; it
contained less than 10% of unidentified impurities as determined by gas
chromatography (Idanpiian-Heikkila, Fritchie, Englert, Ho & McIsaac, 1969). As
its absorption on intraperitoneal injection is poor and erratic (Ho, Fritchie, Englert,
Mclsaac & Idanpaan-Heikkila, 1971) it was injected in a 4% Tween-80-0-9%
NaCl solution into the tail vein. A dose of 1 mg/kg in a volume of 0-1 ml
was injected each time. In the experiments in which the drug was injected for
four weeks the solutions were prepared fresh daily, and the dose was adjusted
for the increase in body weight. Reserpine (Regis Chemical Company) was
dissolved in 30% propylene glycol and a dose of 15 mg/kg was injected intraperitoneally
in a volume of 0-5 ml. Cinanserin (Nutritional Biochemical Corporation)
an inhibitor 5-HT was dissolved in 0-9% w/v NaCl and 10 mg/kg
was injected intraperitoneally in a volume of 0 5 ml.
Results
Figure 1 shows that an intravenous injection of 1 mg/kg A9-THC had little
or no effect on rectal temperature, but that an intravenous injection of 5 mg/kg
produced hypothermia which lasted for approximately 3 hours. The intravenous
injection of 1 mg/kg A9-THC affected the brain monoamines, 5-HT and NA,
differently. As shown in Table 1, the total level of brain 5-HT remained unchanged
but there was a shift of the 5-HT from the particulate or 'bound' fraction to
the supernatant or 'free ' fraction. On the other hand, the total level of brain
NA was reduced and both the particulate and supernatant fractions were lower
than the reserpine controls.
Since the intravenous injection of 1 mg/kg A9-THC caused very little change
in the rectal temperature, this dose was chosen in the following experiments for
studying the effect of A9-THC on the reserpine-induced hypothermia and reduction
of the brain monoamines in whole brain, as well as on its subcellular distribution.
Effect of chronic A9-tetra,hydrocannabinol pretreatment on the reserpine-induced
hypothermia and reduction of brain monoamines
The long-lasting hypothermia which developed a few hours after an intraperitoneal
injection of 15 mg/kg reserpine and was still evident 18 h later was
prevented by chronic A9-THC pretreatment. This is illustrated in Figure 2.

The effect of chronic A9-THC pretreatment on the reduction of the brain
monoamines which occurred after the reserpine injection differed for 5-HT and
NA. The 5-HT reduction was blocked, whereas the NA reduction was not
affected. These results are summarized in Table 2.
Without the A9-THC pretreatment, both the 5-HT and NA levels in the brain
were greatly reduced 18 h after the intraperitoneal reserpine injection. Since the
reduction was more pronounced in the particulate than in the supernatant fraction,
the levels of both 5-HT and NA became approximately equal in the two fractions.
The reversal of the 5-HT reduction produced by the A9-THC pretreatment was
found in both fractions, but more significantly in the particulate fraction where
it resulted in an increase of 70% over the reserpine control. It is possible that
this reversal of the 5-HT reduction might account for the prevention of the reserpine
hypothermia by the chronic A9-THC pretreatment as illustrated in Figure 2.
A9-THC and reserpine hypothermia
Effect of acute A9-tetrahydrocannabinol pretreatment on the reserpine-induced
hypothermia and reduction of brain monoamines
An intravenous injection of 1 mg/kg A9-THC given 1 h before an intraperitoneal
injection of 15 mg/kg reserpine prevented the reserpine hypothermia.
The effect, as shown in Fig. 3 was significant from 5 h onwards during the 18 h
time interval. However, this prevention of the reserpine hypothermia by A9-THC
did not occur when 10 mg/kg of cinanserin was injected 30 min before the
reserpine. This is illustrated in Figure 4.
The effect of acute A9-THC pretreatment on the reduction of the brain monoamines
which occurred after the reserpine injection again differed for 5-HT and
NA. The reversal of the 5-HT reduction was even more pronounced than with
the chronic pretreatment while the NA reduction by reserpine was not consistently
affected until 18 hours. These results are summarized in Table 3.

Without the A9-THC pretreatment, both the 5-HT and NA levels were rapidly
reduced after the injection of 15 mg/kg of reserpine, and their levels were approximately
equal in both fractions. The depletion was produced as early as 3 h
after the injection, however, at 18 h a slight increase was obtained in both the
particulate and supernatant fractions. This could be due to the high dose of
reserpine used which caused the maximum depletion to occur before 18 hours.
The reversal of the 5-HT reduction produced by acute A9-THC pretreatment
was found in both fractions and was highly significant at all of the time intervals
studied, but was most pronounced at 6 hours. This coincides with the prevention
of the reserpine hypothermia, which was significant as early as 5 h as illustrated
in Figure 3.

Effect of A9-tetrahydrocannabinol after the hypothermia and reduction of brain
monoamines produced by reserpine
When A9-THC was injected 3 h after reserpine, the hypothermia was potentiated
as illustrated in Figure 5. The effect was significant as early as 1 h after the A9-THC
and was still evident after 8 hours.
After the reduction of brain amines by reserpine, A9-THC caused an enhancement
in the depletion of 5-HT whereas NA reduction was essentially not affected.
These results are summarized in Table 4.
Without the injection of A9-THC the 5-HT and NA levels were reduced in both
the particulate and supernatant fractions as early as 3 h after the reserpine. The
injection of A9-THC further depleted 5-HT in both fractions to approximately the
same extent from 6 to 18 hours. These results were directly opposite to those
obtained with either A9-THC chronic or acute pretreatment. A possible correlation
could exist between the further reduction of 5-HT in the brain and the potentiation
of the reserpine-induced hypothermia by A9-THC.
Discussion
Reserpine-induced hypothermia is prevented by both chronic and acute A9-THC
pre-treatment, and there is a concomitant blockade of the release of 5-HT in the
' free ' and ' bound ' forms in the rat brain. The reversal of reserpine action on
total brain 5-HT by A9-THC has also been demonstrated by Sofia et al. (1971).
When A9-THC is injected after reserpine the potentiation of the hypothermia
appears to coincide with an enhancement in the depletion of brain 5-HT. If the
initial rate of release of 5-HT from the storage vesicles is responsible for the fall
in rectal temperature as proposed by Brodie, Comer, Costa & Dlabac (1966), this
could explain why the hypothermia is further augmented in reserpine-treated
animals. The 5-HT, which has been released from the storage vesicles, stimulates
the cold receptors and the temperature falls.
In the presence of cinanserin, a 5-HT inhibitor, A9-THC pretreatment is no
longer effective in blocking the reserpine hypothermia. This antagonism by
cinanserin may be due to the loss of accessibility of 5-HT to the cold receptors.
A similar effect of cinanserin on the hyperthermia produced by lysergic acid
diethylamide in reserpine-treated animals has been reported by Rubin, Piala,
Burke & Craver (1964).
Although the results of the present study do not preclude that the effects of
A9-THC on reserpine hypothermia could also be of peripheral origin there appears
to be a correlation between the fall in rectal temperature and the release of brain
5-HT. There is, however, no obvious relation with brain NA. The involvement
of central 5-HT neurones in thermo-regulation in rats has been proposed by
Simmonds (1970) and 5-HT has been considered to be the more significant factor
responsible for the sedative and hypothermic effects of reserpine (Brodie et al.,
1966).
We wish to thank Mrs. Ann Rougeaux, Mrs. Sara Reese and Mrs. Sherrel Jochen for their
technical assistance.

REFERENCES
ASKEW, B. M. (1963). A simple screening procedure for imipramine-like antidepressant agents.
Life Sci., Oxford, 10, 725-730.
BRODIE, B. B., COMER, M. S., COSTA, E. & DLABAC, A. (1966). The role of brain serotonin in the
mechanism of the central action of reserpine. J. Pharmac. exp. Ther., 152, 340-349.
COSTA, E., GARA1-INI, S. & VALZELLI, L. (1960). Interaction between reserpine, imipramine and
chlorpromazine. Experientia, 16, 461-463.
CURZON, G. & GREEN, A. R. (1970). Rapid method for the determination of 5-hydroxytryptamine and
5-hydroxyindoleacetic acid in small regions of the rat brain. Br. J. Pharmac., 39, 653-655.
FELDBERG, W. & Lorri, V. J. (1967). Temperature changes produced in the unanaesthetized rat by
monoamines and tranylcypromine injected into the cerebral ventricles. J. Physiol., Lond., 191
35-36P.
FELDBERG, W. & MYERS, R. D. (1963). A new concept of temperature regulation by amines in the
hypothalamus. Nature, Lond., 200, 1325.
FELDBERG, W. & MYERs, R. D. (1964). Effects on temperature of amines injected into the cerebral
ventricles. A new concept of temperature regulation. J. Physiol., Lond., 173, 226-237.
FELDBERG, W. & MYERS, R. D. (1965). Changes in temperature produced by microinjections of
amines into the anterior hypbthalamus of cats. J. Physiol., Lond., 177, 239-245.
FUXE, K., HOKFELT, T. & UNGERSTEDT, U. (1970). Morphological and functional aspects of central
monoamine neurons. Int. Rev. Neurobiol., 13, 92-126.
GARATTINI, S., GiAcHETTI, A., JORI, A., PIERi, L. & VALZELLI, L. (1962). Effect of imipramine,
amitriptylene and their monomethylderivatives in reserpine activity. J. Pharm. Pharmac., 14,
509-514.
GARATI-NI, S. & JoRI, A. (1967). Interactions between imipramine-like drugs and reserpine on body
temperature. In: Anti-depressant drugs. Proceedings of the First International Symposium, ed.
Garattini, S. & Dukes, M. N. G., pp. 179-193. Amsterdam: Excerpta Medica Foundation.
GARATriNi, S., VALZELLI, L. (1961). Biochemistry and pharmacology of serotonin in the central
nervous system. In: Monoamines et systime nerveux central, ed. Ajuria-guerra, J. de, pp. 59-88.
Geneva: Symposium Bel-Air.
GIARMAN, N. J., FREEDMAN, D. X. & SCHANBERG, S. M. (1964). Drug-induced changes in the subcellular
distribution of serotonin in rat brain with special reference to the action of reserpine.
In: Progress in brain research-Brain amines, ed. Himmich, H. E. & Himmich, W. A., pp. 72-80.
New York: Elsevier Publishing Co.
Ho, B. T., FRITCHIE, G. E., ENGLERT, L. F., MCISAAC, W. M. & IDANPXXN-HEIKKILX, J. E. (1971).
Marijuana: importance of the route of administration. J. Pharm. Pharmac., 23, 309-310.
IDANPXAN-HEIKKILA, J., FRITCHIE, G. E., ENGLERT, L. F., Ho, B. T. & MCISAAC, W. M. (1969).
Placental transfer of tritiated-l-A'l-tetrahydrocannabinol. New England J. Med., 281, 330.
JORI, A. & GARATTINI, S. (1965). Interaction between imipramine-like agents and catecholamineinduced
hyperthermia. J. Pharm. Pharmac., 17, 480-488.
LAVERTY, R. & TAYLOR, K. M. (1968). The fluorometric assay of catecholamines and related compounds:
Improvements and extensions of the hydroxyindole technique. Anal. Biochem., 22,
269-279.
RUBIN, B., PIALA, J. J., BURKE, J. C. & CRAVER, B. N. (1964). A new, potent and specific serotonin
inhibitor 2'-(3'-dimethylaminopropylthio) cinnamanilide hydrochloride: Antiserotonin activity
on uterus and on gastrointestinal, vascular and respiratory systems of animals. Arch. int.
Pharmacodyn., 152, 132-143.
SIMMONDS, M. A. (1970). Effect of environmental temperature on the turnover of 5-hydroxytryptamine
in various areas of rat brain. J. Physiol., Lond., 211, 93-108.
SIMMONDs, M. A. & URETSKY, N. J. (1970). Central effects of 6-hydroxydopamine on the body temperature
of the rat. Br. J. Pharmac., 40, 630-638.
SOFA, R. D., DIxIT, B. N. & BARRY III, H. (1971). The effect of AO-tetrahydrocannabinol on serotonin
metabolism in the rat brain. Life Sci., Oxford, 10, (1), 425-436.
SVENSSON, T. H. & WALDECK, B. (1969). On the significance of central noradrenaline for motor
activity: Experiments with a new dopamine-f-hydroxylase inhibitor. Eur. J. Pharmac., 7, 278-282.


Source: The Effects Of (-)-A9-Tetrahydrocannabinol On Reserpine-Induced Hypothermia In Rats