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Environmental stress as a factor in the response of rat brain catecholamine metabolism to Δ8-tetrahydrocannabinol
European Journal o f Pharmacology, 41 (1977) 171--182
171
© Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
E N V I R O N M E N T A L S T R E S S AS A F A C T O R I N T H E R E S P O N S E O F R A T B R A I N CATECHOLAMINE METABOLISM TO AS-TETRAHYDROCANNABINOL KEITH I. MACLEAN * and JOHN M. LITTLETON Department o f Pharmacology, King's College, Strand, London WC2 R 2 LS, England
Received 30 June 1976, revised MS received 1 October 1976, accepted 6 October 1976
K.I. MACLEAN and J.M. LITTLETON, Environmental stress as a factor in the response o f rat brain catecholamine metabolism to AS-tetrahydrocannabinol, European J. Pharmacot. 41 (1977) 171--182. In rats housed normally (aggregated, food and water ad lib) for fourteen days AS-tetrahydrocannabinol (THC) produced mild sedation and minimal hypothermia. An increase in noradrenaline synthesis was observed, but brain dopamine metabolism was unchanged. In rats removed from this 'normal' environment to conditions of isolation and food deprivation for 24 h THC produced immobility, marked hyper-reactivity, and hypothermia. Brain noradrenaline metabolism was unchanged by THC under these conditions, but significant changes in striatal dopamine metabolism were observed. These changes are consistent with increased dopamine reuptake in striatum produced by this combination of THC and novel environment. It is suggested that some of the behavioural effects of cannabis administered under stressful conditions may be related to alterations in striatal dopamine metabolism. Central cateeholamine metabolism Striatum Dopamine uptake
A s-Tetrahydroeannabinol
1. I n t r o d u c t i o n O n e o f t h e m a j o r difficulties in t h e s t u d y o f c a n n a b i s is t h e wide v a r i a t i o n in t h e reported behavioural and biochemical effects of t h e drug. T h u s , t h e b e h a v i o u r a l e f f e c t s o f cannabis in m a n h a v e b e e n r e p o r t e d t o range f r o m s e d a t i o n t o p s y c h o t i c o r p a r a n o i d reactions, and similar v a r i a t i o n s are o b s e r v e d in animals. T h e r e is e v i d e n c e t h a t i n t e r a c t i o n s b e t w e e n c a n n a b i s and e n v i r o n m e n t a l stress m a y be o f i m p o r t a n c e in d e t e r m i n i n g b e h a v i o u r a l res p o n s e s to t h e drug. F o r e x a m p l e , Carlini a n d G o n z a l e z ( 1 9 7 2 ) and Carlini et al. {1972) have d e m o n s t r a t e d t h a t t h e stresses o f hunger, cold, and m o r p h i n e w i t h d r a w a l can all mark e d l y alter t h e b e h a v i o u r a l r e s p o n s e o f rats to c a n n a b i s a n d t e t r a h y d r o c a n n a b i n o l s . In m a n , * Present address: Glaxo Research Ltd., Greenford, Middlesex UB60HE, England.
Cannabis
Environment
it has b e e n r e p o r t e d t h a t a greater f r e q u e n c y o f adverse r e a c t i o n s o c c u r s in c a n n a b i s users t a k i n g t h e d r u g u n d e r stressful c o n d i t i o n s ( T a l b o t t and Teague, 1969). T h e r e is theref o r e g o o d e v i d e n c e t h a t v a r i a t i o n s in the rep o r t e d b e h a v i o u r a l e f f e c t s o f c a n n a b i s m a y be related t o the e n v i r o n m e n t u n d e r w h i c h t h e d r u g is t a k e n o r a d m i n i s t e r e d . As y e t little is k n o w n o f t h e basis f o r t h e s e differences, since d i r e c t e v i d e n c e t h a t envir o n m e n t a l c o n d i t i o n s can change t h e bioc h e m i c a l e f f e c t s o f c a n n a b i s is scarce. T h e add i t i o n a l p r o b l e m has b e e n t h a t , until r e c e n t l y , t h e active c o n s t i t u e n t s o f c a n n a b i s have b e e n u n k n o w n or unavailable in p u r e f o r m . T h e use of crude extracts of poorly defined strength for b i o c h e m i c a l a n d b e h a v i o u r a l e x p e r i m e n t s has c o m p l i c a t e d i n t e r p r e t a t i o n o f results. It is now known that the major constituents of c a n n a b i s r e s p o n s i b l e f o r its p s y c h o t r o p i c eff e c t are t h e t e t r a h y d r o c a n n a b i n o l s . T h e s e c o m p o u n d s are d e s i g n a t e d A 8- a n d A%tetra-
172 hydrocannabinol by virtue of the placement of the double bond in the terpenoid ring. Their structural similarity is such that since becoming available in synthetic form no qualitative differences in their biochemical or behavioural effects have been reported. It is now possible to use these compounds to explore the relationships between the effects of the tetrahydrocannabinols and environment. In this paper our results refer only to A S-tetrahydrocannabinol, but we have discussed evidence from studies on both Ag-tetrahydro cannabinol and /xS-tetrahydrocannabinol and have given equal weight to results obtained using either isomer. Throughout this paper we have referred to either tetrahydrocannabinol as THC, since we feel that any qualitative distinction between the two is, at present, unjustified. We have reported preliminary findings suggesting an environment-dependent change in dopamine metabolism produced by AS-tetra hydrocannabinol (THC) in rat brain (Littleton and MacLean, 1974; 1976). In addition, List et al. (1975) have reported that, in vitro, levels of adrenal steroids similar to those produced in stressful environments completely alter the effect of A9-THC on mouse brain homogenate respiration. Such results suggest that environmental conditions can alter the biochemical effects of TttCs, and that this could be the cause of the wide variations in resulting behavioural changes produced by cannabis. This paper explores further the relationships between THC, environment, and associated changes in central catecholamine metabolism. Many of the behavioural effects of cannabis, such as sedation, immobility, hyperreactivity, and hypothermia, could be mediated by brain catecholamines. Environmental stress is itself associated with changes in central catecholamine metabolism (Goldberg and Salama, 1972; Thierry, 1973). Clearly, therefore, the possibility exists that behavioural effects of cannabis are determined by interaction between the biochemical effects of the drug and the overall state of activation of the
K.I. MACLEAN, J.M. LITTLETON central catecholaminergic systems. Cannabis and THCs have been reported by m a n y authors to change catecholamine metabolism in animals (Holtzmann et al., 1969; Fuxe and Jonsson, 1971; Truitt and Anderson, 1971). ttowever, in several instances, the effects reported have been dissimilar and often contradictory (Maitre et al., 1972). Braes et al. (1975) have suggested that the hypothermia produced by THC is an important factor in these differences and report, that, when administered to animals in a thermoneutral environment, no changes in central catecholamine metabolism are produced by THC. Our work suggests that other environmental factors are also of importance. In addition, the methods used for assessing catecholamine metabolism frequently differ, making strict comparison of results impossible. In this paper, we have used several methods of assessing the changes in rat brain catecholamine metabolism to study the changes produced by the administration of TttC to rats housed under different environmental conditions. We have chosen to study two discrete areas of brain known for their high density of catecholamine containing neurones. Thus, to study dopaminergie mechanisms we have chosen the corpus striatum, while to study noradrenergie mechanisms we have chosen the hypothalamus. This choice may also be defended on functional grounds, since these areas are intimately concerned in extrapyramidal function and thermoregulation (e.g., Fuxe et al., 1970) and the behavioural effects of THC appear to involve alteration of both these functions. The hypothesis that behavioural effects of THC are determined by interaction between the biochemical effects of the drug and the state of activation of the central eatecholaminergie systems can be challenged by investigating the effects of the drug under environmental conditions known to alter central cateeholamine metabolism. Studies by Friedman et al. (1973) and earlier studies in our laboratory (Littleton and Walker, unpublish-
AS-THC, STRESS AND BRAIN CATECHOLAMINES
173
ed) indicated that food restriction was capable of altering dopamine metabolism in rat brain. In preliminary experiments we have previously reported that, in isolated, food deprived rats THC prevented the depletion of striatal dopamine after inhibition of catecholamine synthesis, whereas no effect was seen in normally housed (aggregated and fed) rats (Littleton and MacLean, 1974). In all subsequent experiments, therefore, we have compared the effects o f THC in rats housed under 'normal' environmental conditions with the effects of THC in isolated, food deprived rats.
immediately before use, and all injections t o o k place between 9.00--10.30 am.
2. Materials and methods 2.1. Materials 2.1.1. Animals Male, Wistar rats weighing 120--160 g (obtained from Animal Suppliers London Ltd., Welwyn Garden City, Hertfordshire) were acclimatised for 13 days in groups of 4 (cage dimensions 56 X 38 X 18 cm) with free access to food and water. Environmental temperature was 21°C and the lighting cycle was 12 h light {7.00 am--7.00 pm) and 12 h dark. For the next 24 h animals were either maintained under these conditions ('normal' environment) or isolated in smaller cages (33 X 15 X 13 cm) with access to water, but not to food ('stressful' environment). Preliminary experiments had shown that THC produced consistent behavioural and hypothermic effects at an i.p. dose of 10 mg/ kg, that the peak effects o f this dose occurred 2 h after injection, and that animals recovered after 5--6 h. In all experiments therefore, THC was suspended in Tween (4% in isotonic saline) to give a final THC concentration of 4 mg/ml and was injected i.p. in a dose volume o f 2.5 ml/kg after 22 h of the fourteenth day of acclimatisation. Control animals received Tween vehicle {2.5 ml/kg i.p.) alone and all animals were killed 2 h after injection. Fresh suspensions o f THC were made up
2.2. Methods 2.2.1. Fluorimetric measurement o f brain catecholamines Animals were killed by cervical fracture, decapitated, and whole heads immersed in liquid nitrogen for 30 sec. When sufficiently thawed, whole brains were removed and placed on solid carbon dioxide. Striata and hypothalami were dissected by the method of Glowinski and Iversen (1966) and pooled (4) striata or hypothalami were analysed for content of dopamine (DA) and noradrenaline (NA). After homogenisation (Turrax, 30 sec, 0°C) the catecholamines were extracted by the method of Shore and Olin (1958) and estimated fluorimetrically by the method of Laverty and Taylor (1968). Results are corrected for recovery and expressed as pg/g wet weight of tissue. Addition of THC to homogenates did not affect the recovery of catecholamines. 2.2.2. Inhibition o f catecholamine synthesis The rate of depletion of brain catecholamine after inhibition of synthesis reflects the extent to which synthesis contributes to the maintenance of steady state levels in catecholamine stores. The enzyme tyrosine hydroxylase, which catalyses the first step in the biosynthesis of catecholamines, may be inhibited by the soluble methyl ester of a-methyl-p-tyrosine (AMT) (And~n et al., 1966; Corrodi and Hanson, 1966). Preliminary studies showed that AMT injected i.p. at a dose of 400 mg/kg produced approximately a 50% depletion of striatal and hypothalamic catecholamines 2 h after injection. This was also the selected time course for THC, and in all subsequent experiments animals were killed and catecholamine depletion measured 2 h after the i.p. administration o f 400 mg/kg AMT. D,L-a -Methyl-p-tyrosine methyl ester
174 (AMT) (Sigma) was dissolved in isotonic saline and injected i.p. in a dose volume o f 2.5 ml/kg. Control animals received saline alone, and all animals were killed 2 h after injection. In o t h e r experiments, animals received AMT followed immediately by THC. Control animals received saline followed immediately by Tween vehicle, and all animals were killed 2 h after injection. Brains were dissected and catecholamines extracted and estimated as previously described.
2.2.3. Incorporation of 3H-tyrosine into 3Hcatecholamines and 3H-O-methylated metabolites Over short time intervals, most of the labelled catecholamine formed from i.v. administered labelled precursor is retained and its tissue c o n t e n t reflects its rate o f synthesis (Sedvall et al., 1968). We have measured the rate o f f o r m a t i o n o f 3H-DA and 3H-NA as well as the rate o f f o r m a t i on o f their labelled Omethylated metabolites 3H-3-methoxytyramine (3H-MT) and 3H-normetanephrine (3HNM). This approach has allowed us to examine the effects o f THC n o t only on synthesis but also on the release and extraneuronal metabolism of DA and NA in striatum and hypothalamus. Preliminary studies showed that 0.75 h after i.v. administration of 3H-tyrosine there was a high degree o f 3H-incorporation into DA and NA stores, and t hat concentrations of 3H-MT and 3H-NM were sufficiently high to be reproducibly measurable. In all subsequent experiments, therefore, animals were killed and brain 3H-content measured 0.75 h after administration o f 3H-tyrosine. Animals received either THC (10 mg/kg i.p.) or Tween vehicle (2.5 ml/kg i.p.) and after 1.25 h 3H-l-tyrosine (2 mCi/kg) specific activity 53 Ci/mole (Radiochemical Centre, Amersham) was injected into the tail vein in a dose volume o f 2 ml/kg. After 0.75 h animals were killed and brains dissected as previously described. The separation o f the 3H-amines was by the m e t h o d s o f Persson and Waldeck (1968) and T a y l o r and Laverty (1969) in
K.I. MACLEAN, J.M. LITTLETON which amines are absorbed on to buffered (pH 6.0) DOWEX cation exchange columns and differentially eluted with increasing concentrations of hydrochloric acid. Tritium was determined by liquid scintillation spectromet ry in a 2 - e t h o x y e t h a n o l - - O m n i f l u o r - - t o l u ene scintillation m i xt ure and all results are expressed as cpm/g wet weight o f tissue.
2.2.4. Striatal uptake of 3H-DA Catecholamines can be accumulated in brain tissue by a neuronal membrane transport system which can be studied in vitro by the incubation o f labelled amines with synaptosomes (pinched-off nerve endings) (Coyle and Snyder, 1969). We have examined the effects o f stress alone, and a combination of THC and stress, on the uptake o f 3H-DA into striatal homogenates. Animals were injected with THC (10 mg/kg i.p.) or Tween vehicle and killed 2 h later. In experiments concerned with the effects of stress alone on DA uptake animals were killed at the end o f the f o u r t e e n t h day o f acclimatisation. Animals were killed by cervical fracture and whole brains were rapidly removed and chilled on ice. Striata were dissected as previously described and were pooled (2) for the preparation o f synaptosomes and the measurement o f DA uptake by the m e t h o d o f Palfreyman et al. (1974). Uptake was expressed as the Uptake Ratio calculated as cpm/g pellet to cpm/ml supernatant, corrected for uptake at 0 ° C.
2.2.5. Statistics In experiments designed to compare the effect o f THC with t h a t o f Tween vehicle, and to compare the effect of one environment with t hat o f a different environment, a twotailed t-test was used to determine probability values. The same test was used in the statistical analysis o f the effects o f THC on AMTinduced depletion in a named envi ronm ent and to compare the effects of different envir o n m e n t s on the depletions produced by a c o m b i n a t i o n of THC and AMT.
AS-THC, S T R E S S A N D B R A I N C A T E C H O L A M I N E S
175
3. Results
3.1. Behavioural change produced by THC
and
hypothermia
The administration of THC (10 mg/kg i.p.) to rats housed in a normal environment produced initial excitement followed after 5--10 min by signs of mild sedation. Many animals appeared drowsy b u t were easily roused. This effect lasted for a period of 5--6 h. Control injections o f Tween produced o n l y the initial excitement phase. The hypothermia produced by THC administered in a normal environment was not marked and reached a level of statistical significance (p ~ 0.05) only at the 3 h time interval (table 1). The administration of THC to rats isolated and food deprived 24 h before injection produced initial excitement followed by immobility and hyperreactivity. In this state rats stood immobile in the centre of the cage and
showed a marked startle response, even to mild external stimuli. Control injections in this environment produced only the shortlived phase of excitation. The hypothermia produced by THC administered in this environment was more marked than that produced in the normal environment but was still not great (table 1).
3.2. Catecholamine concentrations striatum and hypothalamus
in
rat
3.2.1. Effect o f environment The effect on endogenous catecholamine concentrations of housing rats under different environmental conditions is shown in table 2. No significant change in NA concentration in either brain region, nor in DA concentration in hypothalamus, was observed. A significant (p < 0.05) fall in DA concentration in the striatum was produced by isolation and food deprivation for 24 h.
TABLE 1 H y p o t h e r m i a p r o d u c e d b y ~ 8.THC. T h e rectal t e m p e r a t u r e s ( m e a n s +- S.E.) o f rats k e p t in c o n d i t i o n s o f aggregation a n d f o o d a n d w a t e r ad l i b i t u m ( " n o r m a l " e n v i r o n m e n t ) are c o m p a r e d w i t h t h o s e o f rats isolated a n d food d e p r i v e d ( " s t r e s s f u l " e n v i r o n m e n t ) . 4% T w e e n i.p. r e p r e s e n t s a c o n t r o l i n j e c t i o n o f t h e vehicle for AS-THC (10 m g / k g i.p.). N u m b e r s in p a r e n t h e s e s i n d i c a t e t h e n u m b e r o f a n i m a l s used. Environment
T i m e (h)
Rectal t e m p e r a t u r e (°C) 4% T w e e n i.p.
A 8-THC i.p.
"Normal"
0 1 2 3 4 6
38.37 38.20 37.90 38.17 37.93 37.93
± ± ± ± ± ±
0.11 0.24 0.19 0.16 0.15 0.22
(3) (3) (3) (3) (3) (3)
37.38 36.24 36.10 35.74 36.08 37.48
± 0.30 _+ 0.27 ± 0.38 ± 0.44 ± 0.57 ± 0.35
(4) (5) I (5) 2 (5) (5) (5)
"Stressful"
0 1 2 3 4 6
38.01 38.12 37.91 37.62 37.75 37.60
± ± ± ± ± ±
0.36 0.17 0.42 0.44 0.29 0.48
(3) (3) (3) (3) (3) (3)
37.92 35.32 34.80 34.96 35.60 36.47
_+ 0.22 ± 0.36 ± 0.31 _+ 0.58 ± 0.33 ± 0.51
(5) 2 (5) 1,2 (5) 2 (5) 2 (5) (5)
I S i g n i f i c a n t (p < 0 . 0 5 ) d i f f e r e n c e b e t w e e n e n v i r o n m e n t s . 2 Values w h e r e a c o m p a r i s o n i n d i c a t e s a statistically significant (p < 0 . 0 5 ) d i f f e r e n c e f r o m c o n t r o l .
176
K.I. MACLEAN, J.M. LITTLETON
TABLE 2 Endogenous concentrations of catecholamines in rat striatum and hypothalamus. The concentrations (means + S.E.) of catecholamines expressed as pg/g wet weight of brain tissue are compared in animals kept under different environmental conditions and given saline (2.5 ml/kg i.p.) 2 h before killing as a control injection. Environment
Treatment
Region
Dopamine (Ug/g)
"Normal"
Saline i.p.
"Stressful"
Saline i.p.
Stratum Hypothalamus Striatum Hypothalamus
3.82 0.41 2.94 0.41
± 0.37 + 0.07 ± 0.27 ± 0.09
Noradrenaline (~g/g) 1 (3) (3) ~ (9) (5)
0.46 1.46 0.55 1.41
± 0.07 ± 0.10 -+ 0.03 ± 0.04
(3) (3) (9) (5)
1 Significant (p < 0.05) difference between environments. Numbers in parentheses indicate the number of observations.
3.2.2. Effect of THC T h e a d m i n i s t r a t i o n of THC to rats h o u s e d in n o r m a l c o n d i t i o n s p r o d u c e d no change in e n d o g e n o u s c a t e c h o l a m i n e c o n c e n t r a t i o n s in s t r i a t u m o r h y p o t h a l a m u s . S i m i l a r l y , i n isolated and food deprived animals THC prod u c e d no f u r t h e r change in c a t e c h o l a m i n e c o n c e n t r a t i o n s ( t a b l e 3). T h e s e r e s u l t s d e m o n s t r a t e t h a t THC has no significant effect o n the steady-state concentration of catechola m i n e s in s t r i a t u m or h y p o t h a l a m u s in either t h e a c c u s t o m e d ( ' n o r m a l ' ) e n v i r o n m e n t or in the novel ('stressful') environment.
3.3. Effect o f THC on AMT-induced depletion o f catecholamines The administration of AMT (400 mg/kg i.p., 2 h ) p r o d u c e d m a r k e d d e p l e t i o n o f cate c h o l a m i n e s i n b o t h b r a i n r e g i o n s { t a b l e 4). T h e p e r c e n t a g e r e d u c t i o n in c a t e c h o l a m i n e c o n c e n t r a t i o n d i d n o t d i f f e r s i g n i f i c a n t l y between environments. A d m i n i s t r a t i o n o f T H C a t t h e s a m e t i m e as AMT in a n o r m a l e n v i r o n m e n t had no effect on the percentage reduction of concentrations of D A or N A in s t r i a t u m or h y p o t h a l a m u s . In
TABLE 3 Effect of AS-THC on catecholamine concentrations in rat striatum and hypothatamus. Catecholamine concentrations (means ± S.E.) in striatum and hypothalamus, after administration of THC (10 mg/ kg i.p.) or Tween as a vehicle control, are expressed as percentages of the appropriate control values. Injections were made in a "normal" or "stressful" environment (see Materials and methods) 2 h before killing animals. Numbers in parentheses indicate the number of observations. Environment
Treatment
Region
Dopamine
"Normal"
Tween THC Tween THC Tween THC Tween THC
Striatum
100.0 ± 101.5± 100.0 ± 102.9 ± .100.0 ± 102.7 ± 100.0 ± 92.9 ±
"Stressful"
Hypothalamus Striatum Hypothalamus
4.6 4.4 25.8 30.1 7.0 5.3 19.7 25.7
Noradrenaline (8) (8) (8) (6) (4) (6) (6) (5)
100.0 4 8.0 (8) 9 8 . 9 t 7.5 (8) 100.0 +_ 8.1 (7) 96.8 _+ 8.3 (7) 100.0 ± 4.0 (6) 96.0 ± 2.0(6) 100.0 ± 1.5 (6) 95.4 +_ 3.9 (6)
AS-THC, STRESS AND BRAIN CATECHOLAMINES
177
TABLE 4 Effect o f AS-THC on AMT-induced depletion of catecholamines in rat striatum and hypothalamus. Catecholamine concentrations (means ± S.E.) after the administration of control injections (Tween or saline), AMT (400 mg/kg i.p.) or AMT + THC (10 mg/kg i.p.) 2 h before the animals were killed are expressed as percentages of the appropriate control values. No significant difference was observed between values obtained after different control procedures in the same environment. Numbers in parentheses indicate the number of observations. Environment
Treatment
Region
Dopamine
Noradrenaline
"Normal"
Saline AMT Tween + saline THC+AMT Saline AMT Tween + saline THC+AMT Saline AMT Tween + saline THC+AMT Saline AMT Tween + saline THC+AMT
Striatum
100.0 ± 51.2+_ 100.0 ± 48.5± 100.0 ± 61.2± 100.0 ± 96.0+_ 100.0 ± 57.6± 100.0 ± 48.2± 100.0 ± 62.2 ± 100.0 ± 55.0±
100.0 ± 9.3 (4) 60.7 ± 8.2 (4) 100.0 ± 4.7 (4) 61.1± 7.5 (4) 100.0 ± 5.5 (9) 76.4± 7.1 (9) 100.0 ± 3.4 (9) 76.3÷ 6.7 (9) 100.0 +_ 10.2 (4) 62.6± 3.2 (4) 100.0 ± 5.0 (4) 62.3± 3 . 6 ( 4 ) 100.0 ± 2.8 (5) 78.7 ± 3.6 (6) 100.0 ± 4.0 (6) 74.4± 5.4 (5)
"Stressful"
"Normal"
"Stressful"
Striatum
Hypothalamus
Hypothalamus
9.6 (4) 10.8 (4) 5.9 (4) 4.82 (4) 8.5 (9) 4.8 I (8) 8.6 (9) 3.6 1,2 (9) 26.2 (4) 27.0 (4) 29.8 (4) 23.1 (4) 19.5 (5) 20.6 (6) 20.0 (6) 16.1 (5)
1 Significant (p < 0.001) effect of AS.THC. 2 Significant (p < 0.001) difference between environments, based on measured concentrations of striatal dopamine and not on percentages of appropriate controls. These data were: Environment
Treatment
Striatal dopamine (pg/g)
"NormaL" "Stressful"
THC + AMT THC + AMT
1.85 -+ 0.08 (4) 2.58 -+ 0.10 (9)
the isolated and food deprived environment THC prevented the AMT-induced depletion of D A in t h e s t r i a t u m a n d t h i s r e s u l t w a s h i g h l y s i g n i f i c a n t ( p < 0 . 0 0 1 ) . D e p l e t i o n o f N A in t h e s t r i a t u m a n d c a t e c h o l a m i n e s in t h e h y p o t h a l a m u s w a s u n a f f e c t e d { t a b l e 4). These results indicate that THC has a marked influence on striatal DA metabolism only when administered under specific environmental conditions and that, under these conditions, THC may reduce DA release, reduce D A c a t a b o l i s m , o r i n c r e a s e D A r e u p t a k e in the striatum.
3.4. Synthesis of catecholamines and formation of O-methylated metabolites from 3Htyrosine 3.4.1. Effect of environment The effect of changing the housing conditions of rats on the metabolism of 3H-tyrosine is s h o w n in t a b l e 5. U n d e r c o n d i t i o n s o f i s o l a tion and food deprivation there was a marked i n c r e a s e in 3 H - t y r o s i n e u p t a k e i n t o b r a i n . T h i s r e s u l t is s i g n i f i c a n t { p < 0 . 0 1 ) d e s p i t e the s m a l l n u m b e r o f o b s e r v a t i o n s (n = 3). T h e r e a s o n s f o r t h i s d i f f e r e n c e in t y r o s i n e u p t a k e
178
K.I. MACLEAN, J.M. LITTLETON
TABLE 5 Metabolism of 3H-tyrosine in rat striatum and hypothalamus when administered under different environmental conditions. The incorporation of 3H-tyrosine into catecholamines and catecholamine metabolites in rat striatum and hypothalamus is expressed as cpm/g wet weight of tissue. Animals were given i.p. Tween as a control injection and 1.25 h later 3H-tyrosine (2 mCi/kg) was injected i.v. Animals were killed 0.75 h after injection of 3H-tyrosine. Each value represents the mean ± S.E. of three experiments. S, striatum; H, hypotalamus; DA, dopamine; NA, noradrenaline ; MT, 3-methoxytyramine ; NM, normetanephrine. Environment
Treatment
Region
Total 3 H
"Normal"
Tween
S
Tween
H
Tween
S
Tween
H
182,595 ±13,910 185,521 +-11,267 335,621 + 20,050 328,368 +_26,788
"Stressful"
' I I 1
3H-DA
3H-NA
3H-MT
3H-NM
1402 ±632 540 ±110 1686 +-39 691 ~11
438 ~124 840 ±79 672 +_17 960 +60
1128 +_310 290 +71 761 +_99 359 +_114
1039 !150 676 ~74 1001 ~18 920 +135
i Significant (p < 0.01) difference between environments.
are n o t k n o w n , b u t m a y have implications for o t h e r aspects o f this w o r k (see Discussion). Associated with increased t y r o s i n e u p t a k e t h e r e was increased i n c o r p o r a t i o n o f t r i t i u m into c a t e c h o l a m i n e s in striatum and h y p o thalamus. Similarly, increased c o n c e n t r a t i o n s o f 3H-MT and 3H-NM were observed in h y p o thalamus. In the s t r i a t u m 3H-MT c o n c e n t r a tions were r e d u c e d , whereas 3H-NM c o n c e n t r a t i o n s were u n c h a n g e d . A l t h o u g h these changes did n o t reach a level o f statistical significance ( p > 0.05) their c o n s i s t e n c y and relevance to o t h e r aspects o f this p a p e r m a k e m e n t i o n o f t h e m necessary. These results suggest t h a t e x p o s u r e to a stressful e n v i r o n m e n t is associated with a small increase in the synthesis o f catecholamines and with an increase in their release and c a t a b o l i s m in the h y p o t h a l a m u s . In the s t r i a t u m there m a y be a r e d u c t i o n in the release and catabolism, o r an increase in the reu p t a k e , o f DA.
3.4.2. Effect o f THC When T H C was a d m i n i s t e r e d 1.25 h b e f o r e the a d m i n i s t r a t i o n o f 3H-tyrosine no change in the u p t a k e o f t y r o s i n e was observed. Simi-
larly, no alteration in the c o n c e n t r a t i o n s o f 3H-DA o r 3H-MT was n o t e d . The c o n c e n t r a t i o n o f 3H-NA and 3H-NM was increased in b o t h s t r i a t u m and h y p o t h a l a m u s (table 6). This e f f e c t was significant (p < 0.05) b u t was o n l y seen w h e n T H C was a d m i n i s t e r e d in a n o r m a l e n v i r o n m e n t . When a d m i n i s t e r e d in a stressful e n v i r o n m e n t THC p r o d u c e d n o change in the synthesis o f c a t e c h o l a m i n e s or in c o n c e n t r a t i o n s o f their O - m e t h y l a t e d metabolites. These results suggest t h a t THC p r o d u c e s no change in the synthesis o f D A and N A w h e n a d m i n i s t e r e d u n d e r c o n d i t i o n s o f isolation and f o o d deprivation. The a p p a r e n t lack o f a n y e f f e c t on D A synthesis, o r on D A catabolism after release, suggests t h a t some o t h e r f a c t o r in the m e t a b o l i s m o f D A is i m p o r t a n t in explaining the T H C - i n d u c e d change in striatal D A d e p l e t i o n after AMT. Such a c h a n g e m i g h t involve r e d u c e d i n t r a n e u r o n a l catabolism, o r increased r e u p t a k e , o f DA.
3.5. Striatal uptake o f 3H-DA U n t r e a t e d rats k e p t u n d e r c o n d i t i o n s o f isolation and f o o d d e p r i v a t i o n b e f o r e killing
AS-THC, S T R E S S A N D B R A I N C A T E C H O L A M I N E S
179
TABLE 6 E f f e c t o f A8.THC o n t h e m e t a b o l i s m o f 3 H-tyrosine in rat s t r i a t u m a n d h y p o t h a l a m u s . Values r e p r e s e n t t h e m e a n +- S.E. o f c p m / g wet weight o f tissue o b t a i n e d 0.75 h a f t e r t h e a d m i n i s t r a t i o n o f 3Ht y r o s i n e (2 m C i / k g i.v.) in a n o r m a l or stressful e n v i r o n m e n t . T h e effect o f AS-THC (10 m g / k g i.p.), a d m i n i s t e r e d 2 h b e f o r e a n i m a l s were killed, is expressed as a p e r c e n t a g e of t h e a p p r o p r i a t e c o n t r o l . A b s o l u t e values for controls are given in table 5. Each value r e p r e s e n t s the m e a n of t h r e e e x p e r i m e n t s . S, s t r i a t u m ; H, h y p o t h a l a m u s ; DA, d o p a m i n e ; NA, n o r a d r e n a l i n e ; MT, 3 - m e t h o x y t y r a m i n e ; NM, n o r m e t a n e p h r i n e . Environment
Treatment
Region
Total 3 H
3 H-DA
3 H-NA
3 H-MT
3 H-NM
"Normal"
Tween
S
100.0 +-4.1 101.8 +-5.2 100.0 53.0 99.9 +-2.9 100.0 54.9 93.3 +4.1 100.0 +-5.1 96.9 ±3.7
100.0 +-9.1 115.5 -+7.8 100.0 +-16.4 91.1 +-11.5 100.0 +-12.3 94.8 -+13.9 100.0 +-4.6 104.0 +-5.5
100.0 ±12.6 154.1 +-9.2 1,2 100.0 +-9.4 140.6 -+8.8 1,2 100.0 ±12.5 100.4 513.1 2 100.0 +-6.2 107.7 +-6.0 2
100.0 +-13.6 101.6 +-14.4 100.0 +-24.3 97.5 522.9 100.0 -~13.0 98.2 +-13.7 100.0 +-13.6 112.2 -+13.6
100.0 +-10.7 154.5 +-14.2 2,2 100.0 +-10.8 129.9 +-8.6 100.0 +-11.8 95.0 -+10.2 2 100.0 ±14.6 94.5 +9.4 2
THC Tween
H
THC "Stressful"
Tween
S
THC Tween THC
H
1 Significant (p < 0 . 0 5 ) e f f e c t o f AS-THC. 2 Significant (p < 0 . 0 5 ) d i f f e r e n c e b e t w e e n e n v i r o n m e n t s .
TABLE 7 E f f e c t o f AS-THC o n u p t a k e o f 3 H - d o p a m i n e i n t o striata f r o m rats h o u s e d u n d e r d i f f e r e n t e n v i r o n m e n t a l conditions. Values r e p r e s e n t t h e u p t a k e ratios (see Materials a n d m e t h o d s ) for a c c u m u l a t i o n of 3H_dopamine i n t o striatal h o m o g e n a t e s . H o m o g e n a t e s were o b t a i n e d f r o m rats h o u s e d in " n o r m a l " or " s t r e s s f u l " e n v i r o n m e n t s e i t h e r a f t e r n o t r e a t m e n t o r a f t e r t h e i n j e c t i o n o f T w e e n (as c o n t r o l ) or a f t e r t h e i n j e c t i o n o f AS-THC ( 1 0 m g / k g i.p.) 2 h b e f o r e killing. Each value r e p r e s e n t s t h e m e a n ± S.E. o f t h e n u m b e r o f o b s e r v a t i o n s in p a r e n t h e s e s . Environment
Treatment
Uptake ratio
"Normal" "Stressful" "Normal"
None None Tween THC Tween THC
26.99 29.93 25.40 24.51 27.30 40.73
"Stressful"
_+ 0.83 ± 1.20 +- 3.27 +- 3.04 ± 2.66 ± 3.66
% Control (4) (4) (4) (8) (4) (8)
100.0 110.9 100.0 96.5 100.0 149.1
_+ 2.6 ± 3.8 +- 8.9 ± 8.4 ± 9.7 ± 9.1
2 (4) 2 (4) (4) 3 (8) 1 (4) 1,3 (8)
I Significant (p < 0 . 0 2 ) e f f e c t o f AS-THC p r e t r e a t m e n t . Significant differences b e t w e e n e n v i r o n m e n t s are i n d i c a t e d b y 2 (p < 0 . 0 5 ) a n d 3 (p < 0.02).
180 showed a small but significant (p < 0.05) increase in striatal 3H-DA uptake when compared with rats housed under normal environmental conditions (table 7). Tween administration increased the variation in 3H-DA uptake rendering the effect o f e nvi r onm e nt nonsignificant. THC administration ( 1 0 m g / k g i.p.) 2 h before killing pr oduc e d a small nonsignificant reduction in 3H-DA uptake when administered to animals kept under normal environmental conditions. In isolated and f o o d deprived animals this t r e a t m e n t produced a marked and significant (p < 0.02) increase in 3H-DA uptake into striatal homogenates. These results suggest t hat THC produces an increase in striatal DA uptake only when administered to animals housed under certain environmental conditions. This effect could explain the observed change in AMT-induced depletion o f DA in the striatum. Increased neuronal recapture of released DA pr oduced by the c o m b i n a t i o n of environmental stress and THC could maintain striatal DA stores even in the presence o f inhibition o f catecholamine biosynthesis.
4. Discussion When administered in an environment to which rats were adapted, THC p r o d u c e d no observed effect on DA metabolism in corpus striatum or in hypothalamus. The only effect observed on NA metabolism was an increase in NA synthesis, and f or m at i on o f its extraneuronal metabolite, NM, bot h in striatum and in hypothalamus. These results are essentially in agreement with those o f Maitre et al. (1972). Removal o f rats from this 'normal' environm e n t to an en v ir o nm e nt in which t h e y were isolated and food deprived for 24 h resulted in subtle changes in central catecholamine metabolism. The endogenous striatal concentration o f DA was reduced, there was an apparent increase in capacity for tyrosine uptake into brain and increased synthesis of catechol-
K.I. MACLEAN, J.M. LITTLETON amines was noted. Striatal uptake o f DA was also increased. In support of these observations an increase in central catecholamine synthesis is a c o m m o n l y report ed response to environmental stress (Goldberg and Salama, 1972; Thierry, 1973). The change in striatal DA uptake under these environmental conditions is supported by the suggestions of Hutchins et al. (1975) based on indirect measurement. The administration of THC to rats housed in this stressful environment produced behavioural changes not observed in the normal environment. In particular, immobility or 'catalepsy' and hyper-reactivity were observed. In most respects no changes in catecholamine metabolism, o t h e r than those already associated with the stressful environment, were observed after administration o f THC. However, there were two marked differences in measured parameters relating to DA metabolism. First, there was a very marked red u c t i o n in AMT-induced depletion o f DA in the striatum. This result has been reported before (Fuxe and Jonsson, 1971) but the authors make no mention of the environmental conditions involved. Several o t h e r workers have not obtained this result (e.g., Bracs et al., 1975). Second, a large increase in the uptake o f DA into striatal synaptosomes was produced by THC p r e t r e a t m e n t of animals in the stressful environment. This result differs from those of previous experiments on the effect, in vitro, of THC on DA uptake (Howes and Osgood, 1974; Banerjee et al., 1975b). Differences in experimental procedure and in the uptake characteristics o f striata of rats from different environments may explain this anomaly. The inhibition o f AMT-induced depletion of DA by THC indicates that, after administration of THC, DA synthesis is of reduced i m port ance in maintaining the endogenous concentration o f stored DA. THC may reduce DA release, reduce DA catabolism, or increase DA reuptake by the dopaminergic neurone. The results obtained indicate t hat THC does n o t reduce DA release (sup-
AS-THC, STRESS AND BRAIN CATECHOLAMINES
181
ported b y Howes and Osgood, 1974) and does not reduce DA catabolism (supported by Osgood and Howes, 1974; Banerjee et al., 1975a). We do, however, report direct evidence that under stressful environmental conditions THC administration in vivo increases DA reuptake in rat striatum. We therefore suggest that this action may explain the effect on AMT-induced DA depletion and may contribute to the behavioural differences observed in response to THC in different environments. It is not certain whether these results relating DA metabolism and THC effects can be extrapolated to man. Available evidence (Messiha and Soskin, 1973} strongly suggests that smoking cannabis reduces urinary DA metabolites in man. The authors suggest that an increase in neuronal binding of DA may account for their findings. Our work, suggesting increased neuronal uptake of DA, provides some supportive evidence for this concept. Furthermore, we cannot speculate as to w h y environmental change should have so profound an effect on the behavioural and biochemical response to THC. Presumably, the change in activity of the central catecholaminergic systems induced by environmental change plays a part. The work of List et al. (1975) strongly implicates interaction with adrenal steroid hormones as a major determinant in the alteration of the biochemical effects o f THC by environment. There is little doubt, however, that our results further confirm the importance o f changes in the uptake characteristics of catecholaminergic neurones in the functional regulation of catechoamine metabolism. Adaptive changes in NA uptake (Henley et a1.,1973) and DA uptake (Hutchins et al., 1975) have both been reported in response to environmental change associated with fighting in mice. One of the most impressive alterations in behavioural responses to cannabis by environment is the bizarre fighting behaviour produced by chronic cannabis administration to rats stressed by food deprivation (Carlini et al., 1972).
In conclusion, we report that changing the environmental conditions under which A s, tetrahydrocannabinol is administered to rats changes both the behavioural and biochemical response to the drug. The main change in central catecholamine metabolism which we have observed is an increase in dopamine uptake in the corpus striatum. We suggest that this change may mediate some o f the behavioural effects o f cannabis which are observed specifically when the drug is administered under conditions o f stress. Acknowledgements We wish to thank Beecham Research Laboratories for gifts of AS-THC and radiochemicals. In particular, thanks are due to Dr. M.S.G. Clark and Dr. M.G. Palfreyman for help and advice. This study was supported in part by a CAPS award to K.I.M. from the SRC and Beecham Research Laboratories.
References Anddn, N.E., H. Corrodi, A. Dahlstr6m, K. Fuxe and T. H6kfelt, 1966, Effects of tyrosine hydroxylase inhibition on the amine levels of central monoamine neurons, Life Sci. 5,561. Banerjee, A., M.K. Poddar, S. Saha and J.J. Ghosh, 1975a, Effect of A9-tetrahydrocannabinol on monoamine oxidase activity of rat tissues in vivo, Biochem. Pharmacol. 24, 1435. Banerjee, S.P., S.H. Snyder and R. Mechoulam, 1975b, Cannabinoids: influence on neurotransmitter uptake in rat brain synaptosomes, J. Pharmacol. Exptl. Therap. 194, 74. Bracs, P., D.M. Jackson and G.B. Chesher, 1975, The effect of Ag-tetrahydrocannabinol on brain amine concentration and turnover in whole rat brain and in various regions of the brain, J. Pharm. Pharmacol. 27,713. Carlini, E.A. and C. Gonzales, 1972, Aggressive behaviour induced by marihuana compounds and amphetamine in rats previously made dependent on morphine, Experientia 28, 542. Carlini, E.A., A. Hamoui and R.M.W. M~rtz, 1972, Factors influencing the agressiveness elicited by marihuana in food deprived rats, Brit. J. Pharmacol. 44, 794. Corrodi, H. and L.C.F. Hanson, 1966, Central effects of an inhibitor of tyrosine hydroxylation, Psychopharmacologia 10, 116.
182 Coyle, J.T. and S.H. Snyder, 1969, Catecholamine uptake by synaptosomes in homogenates of rat brain: stereospecificity in different areas, J. Pharmacol. Exptl. Therap. 170, 221. Friedman, E., N. Starr and S. Gershon, 1973, Catecholamine synthesis and regulation of food intake in the rat, Life Sci. 12, 317. Fuxe, K. and G. Jonsson, 1971, The effect of tetrahydrocannabinols on central monoamine neurons, Acta Pharm. Suec. 8 , 6 9 5 . Fuxe, K., T. H6kfelt and U. Ungerstedt, 1970, Central monoaminergic tracts, in: Principles of Psychopharmacology, eds. W.G. Clark and J. Del Giudice (Academic Press, New York) p. 87. Glowinski, J. and L.L. Iversen, 1966, Regional studies of catecholamines in the rat brain. The disposition of (3-H)-norepinephrine, (3-H)-dopamine and (3-H)-DOPA in various regions of the brain, J. Neurochem. 13, 655. Goldberg, M.E. and A.I. Salama, 1972, Tolerance to drum stress and its relationship to dopamine turnover, European J. Pharmacol. 17,202. Henley, E.D., B. Moisset and B.L. Welch, 1973, Catecholamine uptake in cerebral cortex: adaptive change induced by fighting, Science 180, 1050. Holtzmann, D., R.A. Lovell, J.H. Jaffe and D.X. Freedman, 1969, 1-A9-tetrahydrocannabinol: neurochemical and behavioural effects in the mouse, Science 163, 1464. Howes, J. and P. Osgood, 1974, The effect of Ag-te trahydrocannabinol on the uptake and release of 14C-dopamine from crude striatal synaptosomal preparations, Neuropharmacology 13, 1109. Hutchins, D.A., J.D.M. Pearson and D.F. Sharman, 1975, Striatal metabolism of dopamine in mice made aggressive by isolation, J. Neurochem. 24, 1151. Laverty, R. and K.M. Taylor, 1968, The fluorometric assay of catecholamines and related compounds, Anal. Biochem. 22,269. List, A.F., S.F. Bartram, B.L. Nazar and J. Harclerode, 1975, Interactions of A9-tetrahydrocan nabinol, adrenal steroids and ethangl, J. Pharm. Pharmacol. 27,606. Littleton, J.M. and K.I. MacLean, 1974, The effect of AS-tetrahydrocannabinol (As-THC) on dopamine metabolism in the rat corpus striatum: the influence of environment, Brit. J. Pharmacol. 51, 117P.
K.I. MACLEAN, J.M. LITTLETON Littleton, J.M. and K.I. MacLean, 1976, Alterations in dopamine uptake in rat corpus striatum induced by combinations of stress and AS-tetrahydro cannabinol (AS-THC), Brit. J. Pharmacol. 56, 370P. Maftre, L., P.A. Baumann and A. Delini-Stula, 1972, Neurochemical tolerance to cannabinols, in: Cannabis and its Derivatives, eds. W.D.M. Paton and J. Crown (Oxford University Press, London) p. 101. Messiha, F.S. and R.A. Soskin, 1973, Effect of cannabis sativa administered by smoking on biogenic amine secretion in man, Res. Commun. Chem. Pathol. Pharmacol. 6,325. Osgood, P. and J. Howes, 1974, Cannabinoid-induced changes in mouse striatal homovanillic acid and dihydroxyphenylacetic acid: the effects of amphetamine and L-DOPA, Res. Commun. Chem. Pathol. Pharmacol. 9, 621. Palfreyman, M.G., E.S. Palfreyman and M.S.G. Clark, 1974, Effects of benapryzine, a new antiparkinson drug, on dopamine uptake into corpus striatum, European J. Pharmacol. 28,379. Persson, T. and B. Waldeck, 1968, The use of 3HDOPA for studying cerebral catecholamine metabolism, Acta Pharmacol. Toxicol. 26, 363. Sedvall, C.G., V.K. Weise and I.J. Kopin, 1968, The rate of norepinephrine synthesis measured in vivo during short intervals: influence of adrenergic nerve impulse activity, J. Pharmacol. Exptl. Therap. 159, 274. Shore, P.A. and J.S. Olin, 1958, Identification and chemical assay of norepinephrine in brain and other tissues, J. Pharmacol. Exptl. Therap. 122, 295. Talbott, J.A. and J.W. Teague, 1969, Marihuana psychosis, J. Amer. Med. Assoc. 210,299. Taylor, K.M. and R. Laverty, 1969, The metabolism of tritiated dopamine in regions of the rat brain in vivo, J. Neurochem. 16, 1361. Thierry, A.M., 1973, Effects of stress on various characteristics of noradrenaline metabolism in central noradrenaline neurones, Advan. Exptl. Med. Biol. 33,501. Truitt, E.B., Jr. and S.M. Anderson, 1971, Biogenic amine alterations produced in the brain by tetrahydrocannabinols aad their metabolites, Ann. N.Y. Acad. Sci. 191, 68.
171
© Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
E N V I R O N M E N T A L S T R E S S AS A F A C T O R I N T H E R E S P O N S E O F R A T B R A I N CATECHOLAMINE METABOLISM TO AS-TETRAHYDROCANNABINOL KEITH I. MACLEAN * and JOHN M. LITTLETON Department o f Pharmacology, King's College, Strand, London WC2 R 2 LS, England
Received 30 June 1976, revised MS received 1 October 1976, accepted 6 October 1976
K.I. MACLEAN and J.M. LITTLETON, Environmental stress as a factor in the response o f rat brain catecholamine metabolism to AS-tetrahydrocannabinol, European J. Pharmacot. 41 (1977) 171--182. In rats housed normally (aggregated, food and water ad lib) for fourteen days AS-tetrahydrocannabinol (THC) produced mild sedation and minimal hypothermia. An increase in noradrenaline synthesis was observed, but brain dopamine metabolism was unchanged. In rats removed from this 'normal' environment to conditions of isolation and food deprivation for 24 h THC produced immobility, marked hyper-reactivity, and hypothermia. Brain noradrenaline metabolism was unchanged by THC under these conditions, but significant changes in striatal dopamine metabolism were observed. These changes are consistent with increased dopamine reuptake in striatum produced by this combination of THC and novel environment. It is suggested that some of the behavioural effects of cannabis administered under stressful conditions may be related to alterations in striatal dopamine metabolism. Central cateeholamine metabolism Striatum Dopamine uptake
A s-Tetrahydroeannabinol
1. I n t r o d u c t i o n O n e o f t h e m a j o r difficulties in t h e s t u d y o f c a n n a b i s is t h e wide v a r i a t i o n in t h e reported behavioural and biochemical effects of t h e drug. T h u s , t h e b e h a v i o u r a l e f f e c t s o f cannabis in m a n h a v e b e e n r e p o r t e d t o range f r o m s e d a t i o n t o p s y c h o t i c o r p a r a n o i d reactions, and similar v a r i a t i o n s are o b s e r v e d in animals. T h e r e is e v i d e n c e t h a t i n t e r a c t i o n s b e t w e e n c a n n a b i s and e n v i r o n m e n t a l stress m a y be o f i m p o r t a n c e in d e t e r m i n i n g b e h a v i o u r a l res p o n s e s to t h e drug. F o r e x a m p l e , Carlini a n d G o n z a l e z ( 1 9 7 2 ) and Carlini et al. {1972) have d e m o n s t r a t e d t h a t t h e stresses o f hunger, cold, and m o r p h i n e w i t h d r a w a l can all mark e d l y alter t h e b e h a v i o u r a l r e s p o n s e o f rats to c a n n a b i s a n d t e t r a h y d r o c a n n a b i n o l s . In m a n , * Present address: Glaxo Research Ltd., Greenford, Middlesex UB60HE, England.
Cannabis
Environment
it has b e e n r e p o r t e d t h a t a greater f r e q u e n c y o f adverse r e a c t i o n s o c c u r s in c a n n a b i s users t a k i n g t h e d r u g u n d e r stressful c o n d i t i o n s ( T a l b o t t and Teague, 1969). T h e r e is theref o r e g o o d e v i d e n c e t h a t v a r i a t i o n s in the rep o r t e d b e h a v i o u r a l e f f e c t s o f c a n n a b i s m a y be related t o the e n v i r o n m e n t u n d e r w h i c h t h e d r u g is t a k e n o r a d m i n i s t e r e d . As y e t little is k n o w n o f t h e basis f o r t h e s e differences, since d i r e c t e v i d e n c e t h a t envir o n m e n t a l c o n d i t i o n s can change t h e bioc h e m i c a l e f f e c t s o f c a n n a b i s is scarce. T h e add i t i o n a l p r o b l e m has b e e n t h a t , until r e c e n t l y , t h e active c o n s t i t u e n t s o f c a n n a b i s have b e e n u n k n o w n or unavailable in p u r e f o r m . T h e use of crude extracts of poorly defined strength for b i o c h e m i c a l a n d b e h a v i o u r a l e x p e r i m e n t s has c o m p l i c a t e d i n t e r p r e t a t i o n o f results. It is now known that the major constituents of c a n n a b i s r e s p o n s i b l e f o r its p s y c h o t r o p i c eff e c t are t h e t e t r a h y d r o c a n n a b i n o l s . T h e s e c o m p o u n d s are d e s i g n a t e d A 8- a n d A%tetra-
172 hydrocannabinol by virtue of the placement of the double bond in the terpenoid ring. Their structural similarity is such that since becoming available in synthetic form no qualitative differences in their biochemical or behavioural effects have been reported. It is now possible to use these compounds to explore the relationships between the effects of the tetrahydrocannabinols and environment. In this paper our results refer only to A S-tetrahydrocannabinol, but we have discussed evidence from studies on both Ag-tetrahydro cannabinol and /xS-tetrahydrocannabinol and have given equal weight to results obtained using either isomer. Throughout this paper we have referred to either tetrahydrocannabinol as THC, since we feel that any qualitative distinction between the two is, at present, unjustified. We have reported preliminary findings suggesting an environment-dependent change in dopamine metabolism produced by AS-tetra hydrocannabinol (THC) in rat brain (Littleton and MacLean, 1974; 1976). In addition, List et al. (1975) have reported that, in vitro, levels of adrenal steroids similar to those produced in stressful environments completely alter the effect of A9-THC on mouse brain homogenate respiration. Such results suggest that environmental conditions can alter the biochemical effects of TttCs, and that this could be the cause of the wide variations in resulting behavioural changes produced by cannabis. This paper explores further the relationships between THC, environment, and associated changes in central catecholamine metabolism. Many of the behavioural effects of cannabis, such as sedation, immobility, hyperreactivity, and hypothermia, could be mediated by brain catecholamines. Environmental stress is itself associated with changes in central catecholamine metabolism (Goldberg and Salama, 1972; Thierry, 1973). Clearly, therefore, the possibility exists that behavioural effects of cannabis are determined by interaction between the biochemical effects of the drug and the overall state of activation of the
K.I. MACLEAN, J.M. LITTLETON central catecholaminergic systems. Cannabis and THCs have been reported by m a n y authors to change catecholamine metabolism in animals (Holtzmann et al., 1969; Fuxe and Jonsson, 1971; Truitt and Anderson, 1971). ttowever, in several instances, the effects reported have been dissimilar and often contradictory (Maitre et al., 1972). Braes et al. (1975) have suggested that the hypothermia produced by THC is an important factor in these differences and report, that, when administered to animals in a thermoneutral environment, no changes in central catecholamine metabolism are produced by THC. Our work suggests that other environmental factors are also of importance. In addition, the methods used for assessing catecholamine metabolism frequently differ, making strict comparison of results impossible. In this paper, we have used several methods of assessing the changes in rat brain catecholamine metabolism to study the changes produced by the administration of TttC to rats housed under different environmental conditions. We have chosen to study two discrete areas of brain known for their high density of catecholamine containing neurones. Thus, to study dopaminergie mechanisms we have chosen the corpus striatum, while to study noradrenergie mechanisms we have chosen the hypothalamus. This choice may also be defended on functional grounds, since these areas are intimately concerned in extrapyramidal function and thermoregulation (e.g., Fuxe et al., 1970) and the behavioural effects of THC appear to involve alteration of both these functions. The hypothesis that behavioural effects of THC are determined by interaction between the biochemical effects of the drug and the state of activation of the central eatecholaminergie systems can be challenged by investigating the effects of the drug under environmental conditions known to alter central cateeholamine metabolism. Studies by Friedman et al. (1973) and earlier studies in our laboratory (Littleton and Walker, unpublish-
AS-THC, STRESS AND BRAIN CATECHOLAMINES
173
ed) indicated that food restriction was capable of altering dopamine metabolism in rat brain. In preliminary experiments we have previously reported that, in isolated, food deprived rats THC prevented the depletion of striatal dopamine after inhibition of catecholamine synthesis, whereas no effect was seen in normally housed (aggregated and fed) rats (Littleton and MacLean, 1974). In all subsequent experiments, therefore, we have compared the effects o f THC in rats housed under 'normal' environmental conditions with the effects of THC in isolated, food deprived rats.
immediately before use, and all injections t o o k place between 9.00--10.30 am.
2. Materials and methods 2.1. Materials 2.1.1. Animals Male, Wistar rats weighing 120--160 g (obtained from Animal Suppliers London Ltd., Welwyn Garden City, Hertfordshire) were acclimatised for 13 days in groups of 4 (cage dimensions 56 X 38 X 18 cm) with free access to food and water. Environmental temperature was 21°C and the lighting cycle was 12 h light {7.00 am--7.00 pm) and 12 h dark. For the next 24 h animals were either maintained under these conditions ('normal' environment) or isolated in smaller cages (33 X 15 X 13 cm) with access to water, but not to food ('stressful' environment). Preliminary experiments had shown that THC produced consistent behavioural and hypothermic effects at an i.p. dose of 10 mg/ kg, that the peak effects o f this dose occurred 2 h after injection, and that animals recovered after 5--6 h. In all experiments therefore, THC was suspended in Tween (4% in isotonic saline) to give a final THC concentration of 4 mg/ml and was injected i.p. in a dose volume o f 2.5 ml/kg after 22 h of the fourteenth day of acclimatisation. Control animals received Tween vehicle {2.5 ml/kg i.p.) alone and all animals were killed 2 h after injection. Fresh suspensions o f THC were made up
2.2. Methods 2.2.1. Fluorimetric measurement o f brain catecholamines Animals were killed by cervical fracture, decapitated, and whole heads immersed in liquid nitrogen for 30 sec. When sufficiently thawed, whole brains were removed and placed on solid carbon dioxide. Striata and hypothalami were dissected by the method of Glowinski and Iversen (1966) and pooled (4) striata or hypothalami were analysed for content of dopamine (DA) and noradrenaline (NA). After homogenisation (Turrax, 30 sec, 0°C) the catecholamines were extracted by the method of Shore and Olin (1958) and estimated fluorimetrically by the method of Laverty and Taylor (1968). Results are corrected for recovery and expressed as pg/g wet weight of tissue. Addition of THC to homogenates did not affect the recovery of catecholamines. 2.2.2. Inhibition o f catecholamine synthesis The rate of depletion of brain catecholamine after inhibition of synthesis reflects the extent to which synthesis contributes to the maintenance of steady state levels in catecholamine stores. The enzyme tyrosine hydroxylase, which catalyses the first step in the biosynthesis of catecholamines, may be inhibited by the soluble methyl ester of a-methyl-p-tyrosine (AMT) (And~n et al., 1966; Corrodi and Hanson, 1966). Preliminary studies showed that AMT injected i.p. at a dose of 400 mg/kg produced approximately a 50% depletion of striatal and hypothalamic catecholamines 2 h after injection. This was also the selected time course for THC, and in all subsequent experiments animals were killed and catecholamine depletion measured 2 h after the i.p. administration o f 400 mg/kg AMT. D,L-a -Methyl-p-tyrosine methyl ester
174 (AMT) (Sigma) was dissolved in isotonic saline and injected i.p. in a dose volume o f 2.5 ml/kg. Control animals received saline alone, and all animals were killed 2 h after injection. In o t h e r experiments, animals received AMT followed immediately by THC. Control animals received saline followed immediately by Tween vehicle, and all animals were killed 2 h after injection. Brains were dissected and catecholamines extracted and estimated as previously described.
2.2.3. Incorporation of 3H-tyrosine into 3Hcatecholamines and 3H-O-methylated metabolites Over short time intervals, most of the labelled catecholamine formed from i.v. administered labelled precursor is retained and its tissue c o n t e n t reflects its rate o f synthesis (Sedvall et al., 1968). We have measured the rate o f f o r m a t i o n o f 3H-DA and 3H-NA as well as the rate o f f o r m a t i on o f their labelled Omethylated metabolites 3H-3-methoxytyramine (3H-MT) and 3H-normetanephrine (3HNM). This approach has allowed us to examine the effects o f THC n o t only on synthesis but also on the release and extraneuronal metabolism of DA and NA in striatum and hypothalamus. Preliminary studies showed that 0.75 h after i.v. administration of 3H-tyrosine there was a high degree o f 3H-incorporation into DA and NA stores, and t hat concentrations of 3H-MT and 3H-NM were sufficiently high to be reproducibly measurable. In all subsequent experiments, therefore, animals were killed and brain 3H-content measured 0.75 h after administration o f 3H-tyrosine. Animals received either THC (10 mg/kg i.p.) or Tween vehicle (2.5 ml/kg i.p.) and after 1.25 h 3H-l-tyrosine (2 mCi/kg) specific activity 53 Ci/mole (Radiochemical Centre, Amersham) was injected into the tail vein in a dose volume o f 2 ml/kg. After 0.75 h animals were killed and brains dissected as previously described. The separation o f the 3H-amines was by the m e t h o d s o f Persson and Waldeck (1968) and T a y l o r and Laverty (1969) in
K.I. MACLEAN, J.M. LITTLETON which amines are absorbed on to buffered (pH 6.0) DOWEX cation exchange columns and differentially eluted with increasing concentrations of hydrochloric acid. Tritium was determined by liquid scintillation spectromet ry in a 2 - e t h o x y e t h a n o l - - O m n i f l u o r - - t o l u ene scintillation m i xt ure and all results are expressed as cpm/g wet weight o f tissue.
2.2.4. Striatal uptake of 3H-DA Catecholamines can be accumulated in brain tissue by a neuronal membrane transport system which can be studied in vitro by the incubation o f labelled amines with synaptosomes (pinched-off nerve endings) (Coyle and Snyder, 1969). We have examined the effects o f stress alone, and a combination of THC and stress, on the uptake o f 3H-DA into striatal homogenates. Animals were injected with THC (10 mg/kg i.p.) or Tween vehicle and killed 2 h later. In experiments concerned with the effects of stress alone on DA uptake animals were killed at the end o f the f o u r t e e n t h day o f acclimatisation. Animals were killed by cervical fracture and whole brains were rapidly removed and chilled on ice. Striata were dissected as previously described and were pooled (2) for the preparation o f synaptosomes and the measurement o f DA uptake by the m e t h o d o f Palfreyman et al. (1974). Uptake was expressed as the Uptake Ratio calculated as cpm/g pellet to cpm/ml supernatant, corrected for uptake at 0 ° C.
2.2.5. Statistics In experiments designed to compare the effect o f THC with t h a t o f Tween vehicle, and to compare the effect of one environment with t hat o f a different environment, a twotailed t-test was used to determine probability values. The same test was used in the statistical analysis o f the effects o f THC on AMTinduced depletion in a named envi ronm ent and to compare the effects of different envir o n m e n t s on the depletions produced by a c o m b i n a t i o n of THC and AMT.
AS-THC, S T R E S S A N D B R A I N C A T E C H O L A M I N E S
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3. Results
3.1. Behavioural change produced by THC
and
hypothermia
The administration of THC (10 mg/kg i.p.) to rats housed in a normal environment produced initial excitement followed after 5--10 min by signs of mild sedation. Many animals appeared drowsy b u t were easily roused. This effect lasted for a period of 5--6 h. Control injections o f Tween produced o n l y the initial excitement phase. The hypothermia produced by THC administered in a normal environment was not marked and reached a level of statistical significance (p ~ 0.05) only at the 3 h time interval (table 1). The administration of THC to rats isolated and food deprived 24 h before injection produced initial excitement followed by immobility and hyperreactivity. In this state rats stood immobile in the centre of the cage and
showed a marked startle response, even to mild external stimuli. Control injections in this environment produced only the shortlived phase of excitation. The hypothermia produced by THC administered in this environment was more marked than that produced in the normal environment but was still not great (table 1).
3.2. Catecholamine concentrations striatum and hypothalamus
in
rat
3.2.1. Effect o f environment The effect on endogenous catecholamine concentrations of housing rats under different environmental conditions is shown in table 2. No significant change in NA concentration in either brain region, nor in DA concentration in hypothalamus, was observed. A significant (p < 0.05) fall in DA concentration in the striatum was produced by isolation and food deprivation for 24 h.
TABLE 1 H y p o t h e r m i a p r o d u c e d b y ~ 8.THC. T h e rectal t e m p e r a t u r e s ( m e a n s +- S.E.) o f rats k e p t in c o n d i t i o n s o f aggregation a n d f o o d a n d w a t e r ad l i b i t u m ( " n o r m a l " e n v i r o n m e n t ) are c o m p a r e d w i t h t h o s e o f rats isolated a n d food d e p r i v e d ( " s t r e s s f u l " e n v i r o n m e n t ) . 4% T w e e n i.p. r e p r e s e n t s a c o n t r o l i n j e c t i o n o f t h e vehicle for AS-THC (10 m g / k g i.p.). N u m b e r s in p a r e n t h e s e s i n d i c a t e t h e n u m b e r o f a n i m a l s used. Environment
T i m e (h)
Rectal t e m p e r a t u r e (°C) 4% T w e e n i.p.
A 8-THC i.p.
"Normal"
0 1 2 3 4 6
38.37 38.20 37.90 38.17 37.93 37.93
± ± ± ± ± ±
0.11 0.24 0.19 0.16 0.15 0.22
(3) (3) (3) (3) (3) (3)
37.38 36.24 36.10 35.74 36.08 37.48
± 0.30 _+ 0.27 ± 0.38 ± 0.44 ± 0.57 ± 0.35
(4) (5) I (5) 2 (5) (5) (5)
"Stressful"
0 1 2 3 4 6
38.01 38.12 37.91 37.62 37.75 37.60
± ± ± ± ± ±
0.36 0.17 0.42 0.44 0.29 0.48
(3) (3) (3) (3) (3) (3)
37.92 35.32 34.80 34.96 35.60 36.47
_+ 0.22 ± 0.36 ± 0.31 _+ 0.58 ± 0.33 ± 0.51
(5) 2 (5) 1,2 (5) 2 (5) 2 (5) (5)
I S i g n i f i c a n t (p < 0 . 0 5 ) d i f f e r e n c e b e t w e e n e n v i r o n m e n t s . 2 Values w h e r e a c o m p a r i s o n i n d i c a t e s a statistically significant (p < 0 . 0 5 ) d i f f e r e n c e f r o m c o n t r o l .
176
K.I. MACLEAN, J.M. LITTLETON
TABLE 2 Endogenous concentrations of catecholamines in rat striatum and hypothalamus. The concentrations (means + S.E.) of catecholamines expressed as pg/g wet weight of brain tissue are compared in animals kept under different environmental conditions and given saline (2.5 ml/kg i.p.) 2 h before killing as a control injection. Environment
Treatment
Region
Dopamine (Ug/g)
"Normal"
Saline i.p.
"Stressful"
Saline i.p.
Stratum Hypothalamus Striatum Hypothalamus
3.82 0.41 2.94 0.41
± 0.37 + 0.07 ± 0.27 ± 0.09
Noradrenaline (~g/g) 1 (3) (3) ~ (9) (5)
0.46 1.46 0.55 1.41
± 0.07 ± 0.10 -+ 0.03 ± 0.04
(3) (3) (9) (5)
1 Significant (p < 0.05) difference between environments. Numbers in parentheses indicate the number of observations.
3.2.2. Effect of THC T h e a d m i n i s t r a t i o n of THC to rats h o u s e d in n o r m a l c o n d i t i o n s p r o d u c e d no change in e n d o g e n o u s c a t e c h o l a m i n e c o n c e n t r a t i o n s in s t r i a t u m o r h y p o t h a l a m u s . S i m i l a r l y , i n isolated and food deprived animals THC prod u c e d no f u r t h e r change in c a t e c h o l a m i n e c o n c e n t r a t i o n s ( t a b l e 3). T h e s e r e s u l t s d e m o n s t r a t e t h a t THC has no significant effect o n the steady-state concentration of catechola m i n e s in s t r i a t u m or h y p o t h a l a m u s in either t h e a c c u s t o m e d ( ' n o r m a l ' ) e n v i r o n m e n t or in the novel ('stressful') environment.
3.3. Effect o f THC on AMT-induced depletion o f catecholamines The administration of AMT (400 mg/kg i.p., 2 h ) p r o d u c e d m a r k e d d e p l e t i o n o f cate c h o l a m i n e s i n b o t h b r a i n r e g i o n s { t a b l e 4). T h e p e r c e n t a g e r e d u c t i o n in c a t e c h o l a m i n e c o n c e n t r a t i o n d i d n o t d i f f e r s i g n i f i c a n t l y between environments. A d m i n i s t r a t i o n o f T H C a t t h e s a m e t i m e as AMT in a n o r m a l e n v i r o n m e n t had no effect on the percentage reduction of concentrations of D A or N A in s t r i a t u m or h y p o t h a l a m u s . In
TABLE 3 Effect of AS-THC on catecholamine concentrations in rat striatum and hypothatamus. Catecholamine concentrations (means ± S.E.) in striatum and hypothalamus, after administration of THC (10 mg/ kg i.p.) or Tween as a vehicle control, are expressed as percentages of the appropriate control values. Injections were made in a "normal" or "stressful" environment (see Materials and methods) 2 h before killing animals. Numbers in parentheses indicate the number of observations. Environment
Treatment
Region
Dopamine
"Normal"
Tween THC Tween THC Tween THC Tween THC
Striatum
100.0 ± 101.5± 100.0 ± 102.9 ± .100.0 ± 102.7 ± 100.0 ± 92.9 ±
"Stressful"
Hypothalamus Striatum Hypothalamus
4.6 4.4 25.8 30.1 7.0 5.3 19.7 25.7
Noradrenaline (8) (8) (8) (6) (4) (6) (6) (5)
100.0 4 8.0 (8) 9 8 . 9 t 7.5 (8) 100.0 +_ 8.1 (7) 96.8 _+ 8.3 (7) 100.0 ± 4.0 (6) 96.0 ± 2.0(6) 100.0 ± 1.5 (6) 95.4 +_ 3.9 (6)
AS-THC, STRESS AND BRAIN CATECHOLAMINES
177
TABLE 4 Effect o f AS-THC on AMT-induced depletion of catecholamines in rat striatum and hypothalamus. Catecholamine concentrations (means ± S.E.) after the administration of control injections (Tween or saline), AMT (400 mg/kg i.p.) or AMT + THC (10 mg/kg i.p.) 2 h before the animals were killed are expressed as percentages of the appropriate control values. No significant difference was observed between values obtained after different control procedures in the same environment. Numbers in parentheses indicate the number of observations. Environment
Treatment
Region
Dopamine
Noradrenaline
"Normal"
Saline AMT Tween + saline THC+AMT Saline AMT Tween + saline THC+AMT Saline AMT Tween + saline THC+AMT Saline AMT Tween + saline THC+AMT
Striatum
100.0 ± 51.2+_ 100.0 ± 48.5± 100.0 ± 61.2± 100.0 ± 96.0+_ 100.0 ± 57.6± 100.0 ± 48.2± 100.0 ± 62.2 ± 100.0 ± 55.0±
100.0 ± 9.3 (4) 60.7 ± 8.2 (4) 100.0 ± 4.7 (4) 61.1± 7.5 (4) 100.0 ± 5.5 (9) 76.4± 7.1 (9) 100.0 ± 3.4 (9) 76.3÷ 6.7 (9) 100.0 +_ 10.2 (4) 62.6± 3.2 (4) 100.0 ± 5.0 (4) 62.3± 3 . 6 ( 4 ) 100.0 ± 2.8 (5) 78.7 ± 3.6 (6) 100.0 ± 4.0 (6) 74.4± 5.4 (5)
"Stressful"
"Normal"
"Stressful"
Striatum
Hypothalamus
Hypothalamus
9.6 (4) 10.8 (4) 5.9 (4) 4.82 (4) 8.5 (9) 4.8 I (8) 8.6 (9) 3.6 1,2 (9) 26.2 (4) 27.0 (4) 29.8 (4) 23.1 (4) 19.5 (5) 20.6 (6) 20.0 (6) 16.1 (5)
1 Significant (p < 0.001) effect of AS.THC. 2 Significant (p < 0.001) difference between environments, based on measured concentrations of striatal dopamine and not on percentages of appropriate controls. These data were: Environment
Treatment
Striatal dopamine (pg/g)
"NormaL" "Stressful"
THC + AMT THC + AMT
1.85 -+ 0.08 (4) 2.58 -+ 0.10 (9)
the isolated and food deprived environment THC prevented the AMT-induced depletion of D A in t h e s t r i a t u m a n d t h i s r e s u l t w a s h i g h l y s i g n i f i c a n t ( p < 0 . 0 0 1 ) . D e p l e t i o n o f N A in t h e s t r i a t u m a n d c a t e c h o l a m i n e s in t h e h y p o t h a l a m u s w a s u n a f f e c t e d { t a b l e 4). These results indicate that THC has a marked influence on striatal DA metabolism only when administered under specific environmental conditions and that, under these conditions, THC may reduce DA release, reduce D A c a t a b o l i s m , o r i n c r e a s e D A r e u p t a k e in the striatum.
3.4. Synthesis of catecholamines and formation of O-methylated metabolites from 3Htyrosine 3.4.1. Effect of environment The effect of changing the housing conditions of rats on the metabolism of 3H-tyrosine is s h o w n in t a b l e 5. U n d e r c o n d i t i o n s o f i s o l a tion and food deprivation there was a marked i n c r e a s e in 3 H - t y r o s i n e u p t a k e i n t o b r a i n . T h i s r e s u l t is s i g n i f i c a n t { p < 0 . 0 1 ) d e s p i t e the s m a l l n u m b e r o f o b s e r v a t i o n s (n = 3). T h e r e a s o n s f o r t h i s d i f f e r e n c e in t y r o s i n e u p t a k e
178
K.I. MACLEAN, J.M. LITTLETON
TABLE 5 Metabolism of 3H-tyrosine in rat striatum and hypothalamus when administered under different environmental conditions. The incorporation of 3H-tyrosine into catecholamines and catecholamine metabolites in rat striatum and hypothalamus is expressed as cpm/g wet weight of tissue. Animals were given i.p. Tween as a control injection and 1.25 h later 3H-tyrosine (2 mCi/kg) was injected i.v. Animals were killed 0.75 h after injection of 3H-tyrosine. Each value represents the mean ± S.E. of three experiments. S, striatum; H, hypotalamus; DA, dopamine; NA, noradrenaline ; MT, 3-methoxytyramine ; NM, normetanephrine. Environment
Treatment
Region
Total 3 H
"Normal"
Tween
S
Tween
H
Tween
S
Tween
H
182,595 ±13,910 185,521 +-11,267 335,621 + 20,050 328,368 +_26,788
"Stressful"
' I I 1
3H-DA
3H-NA
3H-MT
3H-NM
1402 ±632 540 ±110 1686 +-39 691 ~11
438 ~124 840 ±79 672 +_17 960 +60
1128 +_310 290 +71 761 +_99 359 +_114
1039 !150 676 ~74 1001 ~18 920 +135
i Significant (p < 0.01) difference between environments.
are n o t k n o w n , b u t m a y have implications for o t h e r aspects o f this w o r k (see Discussion). Associated with increased t y r o s i n e u p t a k e t h e r e was increased i n c o r p o r a t i o n o f t r i t i u m into c a t e c h o l a m i n e s in striatum and h y p o thalamus. Similarly, increased c o n c e n t r a t i o n s o f 3H-MT and 3H-NM were observed in h y p o thalamus. In the s t r i a t u m 3H-MT c o n c e n t r a tions were r e d u c e d , whereas 3H-NM c o n c e n t r a t i o n s were u n c h a n g e d . A l t h o u g h these changes did n o t reach a level o f statistical significance ( p > 0.05) their c o n s i s t e n c y and relevance to o t h e r aspects o f this p a p e r m a k e m e n t i o n o f t h e m necessary. These results suggest t h a t e x p o s u r e to a stressful e n v i r o n m e n t is associated with a small increase in the synthesis o f catecholamines and with an increase in their release and c a t a b o l i s m in the h y p o t h a l a m u s . In the s t r i a t u m there m a y be a r e d u c t i o n in the release and catabolism, o r an increase in the reu p t a k e , o f DA.
3.4.2. Effect o f THC When T H C was a d m i n i s t e r e d 1.25 h b e f o r e the a d m i n i s t r a t i o n o f 3H-tyrosine no change in the u p t a k e o f t y r o s i n e was observed. Simi-
larly, no alteration in the c o n c e n t r a t i o n s o f 3H-DA o r 3H-MT was n o t e d . The c o n c e n t r a t i o n o f 3H-NA and 3H-NM was increased in b o t h s t r i a t u m and h y p o t h a l a m u s (table 6). This e f f e c t was significant (p < 0.05) b u t was o n l y seen w h e n T H C was a d m i n i s t e r e d in a n o r m a l e n v i r o n m e n t . When a d m i n i s t e r e d in a stressful e n v i r o n m e n t THC p r o d u c e d n o change in the synthesis o f c a t e c h o l a m i n e s or in c o n c e n t r a t i o n s o f their O - m e t h y l a t e d metabolites. These results suggest t h a t THC p r o d u c e s no change in the synthesis o f D A and N A w h e n a d m i n i s t e r e d u n d e r c o n d i t i o n s o f isolation and f o o d deprivation. The a p p a r e n t lack o f a n y e f f e c t on D A synthesis, o r on D A catabolism after release, suggests t h a t some o t h e r f a c t o r in the m e t a b o l i s m o f D A is i m p o r t a n t in explaining the T H C - i n d u c e d change in striatal D A d e p l e t i o n after AMT. Such a c h a n g e m i g h t involve r e d u c e d i n t r a n e u r o n a l catabolism, o r increased r e u p t a k e , o f DA.
3.5. Striatal uptake o f 3H-DA U n t r e a t e d rats k e p t u n d e r c o n d i t i o n s o f isolation and f o o d d e p r i v a t i o n b e f o r e killing
AS-THC, S T R E S S A N D B R A I N C A T E C H O L A M I N E S
179
TABLE 6 E f f e c t o f A8.THC o n t h e m e t a b o l i s m o f 3 H-tyrosine in rat s t r i a t u m a n d h y p o t h a l a m u s . Values r e p r e s e n t t h e m e a n +- S.E. o f c p m / g wet weight o f tissue o b t a i n e d 0.75 h a f t e r t h e a d m i n i s t r a t i o n o f 3Ht y r o s i n e (2 m C i / k g i.v.) in a n o r m a l or stressful e n v i r o n m e n t . T h e effect o f AS-THC (10 m g / k g i.p.), a d m i n i s t e r e d 2 h b e f o r e a n i m a l s were killed, is expressed as a p e r c e n t a g e of t h e a p p r o p r i a t e c o n t r o l . A b s o l u t e values for controls are given in table 5. Each value r e p r e s e n t s the m e a n of t h r e e e x p e r i m e n t s . S, s t r i a t u m ; H, h y p o t h a l a m u s ; DA, d o p a m i n e ; NA, n o r a d r e n a l i n e ; MT, 3 - m e t h o x y t y r a m i n e ; NM, n o r m e t a n e p h r i n e . Environment
Treatment
Region
Total 3 H
3 H-DA
3 H-NA
3 H-MT
3 H-NM
"Normal"
Tween
S
100.0 +-4.1 101.8 +-5.2 100.0 53.0 99.9 +-2.9 100.0 54.9 93.3 +4.1 100.0 +-5.1 96.9 ±3.7
100.0 +-9.1 115.5 -+7.8 100.0 +-16.4 91.1 +-11.5 100.0 +-12.3 94.8 -+13.9 100.0 +-4.6 104.0 +-5.5
100.0 ±12.6 154.1 +-9.2 1,2 100.0 +-9.4 140.6 -+8.8 1,2 100.0 ±12.5 100.4 513.1 2 100.0 +-6.2 107.7 +-6.0 2
100.0 +-13.6 101.6 +-14.4 100.0 +-24.3 97.5 522.9 100.0 -~13.0 98.2 +-13.7 100.0 +-13.6 112.2 -+13.6
100.0 +-10.7 154.5 +-14.2 2,2 100.0 +-10.8 129.9 +-8.6 100.0 +-11.8 95.0 -+10.2 2 100.0 ±14.6 94.5 +9.4 2
THC Tween
H
THC "Stressful"
Tween
S
THC Tween THC
H
1 Significant (p < 0 . 0 5 ) e f f e c t o f AS-THC. 2 Significant (p < 0 . 0 5 ) d i f f e r e n c e b e t w e e n e n v i r o n m e n t s .
TABLE 7 E f f e c t o f AS-THC o n u p t a k e o f 3 H - d o p a m i n e i n t o striata f r o m rats h o u s e d u n d e r d i f f e r e n t e n v i r o n m e n t a l conditions. Values r e p r e s e n t t h e u p t a k e ratios (see Materials a n d m e t h o d s ) for a c c u m u l a t i o n of 3H_dopamine i n t o striatal h o m o g e n a t e s . H o m o g e n a t e s were o b t a i n e d f r o m rats h o u s e d in " n o r m a l " or " s t r e s s f u l " e n v i r o n m e n t s e i t h e r a f t e r n o t r e a t m e n t o r a f t e r t h e i n j e c t i o n o f T w e e n (as c o n t r o l ) or a f t e r t h e i n j e c t i o n o f AS-THC ( 1 0 m g / k g i.p.) 2 h b e f o r e killing. Each value r e p r e s e n t s t h e m e a n ± S.E. o f t h e n u m b e r o f o b s e r v a t i o n s in p a r e n t h e s e s . Environment
Treatment
Uptake ratio
"Normal" "Stressful" "Normal"
None None Tween THC Tween THC
26.99 29.93 25.40 24.51 27.30 40.73
"Stressful"
_+ 0.83 ± 1.20 +- 3.27 +- 3.04 ± 2.66 ± 3.66
% Control (4) (4) (4) (8) (4) (8)
100.0 110.9 100.0 96.5 100.0 149.1
_+ 2.6 ± 3.8 +- 8.9 ± 8.4 ± 9.7 ± 9.1
2 (4) 2 (4) (4) 3 (8) 1 (4) 1,3 (8)
I Significant (p < 0 . 0 2 ) e f f e c t o f AS-THC p r e t r e a t m e n t . Significant differences b e t w e e n e n v i r o n m e n t s are i n d i c a t e d b y 2 (p < 0 . 0 5 ) a n d 3 (p < 0.02).
180 showed a small but significant (p < 0.05) increase in striatal 3H-DA uptake when compared with rats housed under normal environmental conditions (table 7). Tween administration increased the variation in 3H-DA uptake rendering the effect o f e nvi r onm e nt nonsignificant. THC administration ( 1 0 m g / k g i.p.) 2 h before killing pr oduc e d a small nonsignificant reduction in 3H-DA uptake when administered to animals kept under normal environmental conditions. In isolated and f o o d deprived animals this t r e a t m e n t produced a marked and significant (p < 0.02) increase in 3H-DA uptake into striatal homogenates. These results suggest t hat THC produces an increase in striatal DA uptake only when administered to animals housed under certain environmental conditions. This effect could explain the observed change in AMT-induced depletion o f DA in the striatum. Increased neuronal recapture of released DA pr oduced by the c o m b i n a t i o n of environmental stress and THC could maintain striatal DA stores even in the presence o f inhibition o f catecholamine biosynthesis.
4. Discussion When administered in an environment to which rats were adapted, THC p r o d u c e d no observed effect on DA metabolism in corpus striatum or in hypothalamus. The only effect observed on NA metabolism was an increase in NA synthesis, and f or m at i on o f its extraneuronal metabolite, NM, bot h in striatum and in hypothalamus. These results are essentially in agreement with those o f Maitre et al. (1972). Removal o f rats from this 'normal' environm e n t to an en v ir o nm e nt in which t h e y were isolated and food deprived for 24 h resulted in subtle changes in central catecholamine metabolism. The endogenous striatal concentration o f DA was reduced, there was an apparent increase in capacity for tyrosine uptake into brain and increased synthesis of catechol-
K.I. MACLEAN, J.M. LITTLETON amines was noted. Striatal uptake o f DA was also increased. In support of these observations an increase in central catecholamine synthesis is a c o m m o n l y report ed response to environmental stress (Goldberg and Salama, 1972; Thierry, 1973). The change in striatal DA uptake under these environmental conditions is supported by the suggestions of Hutchins et al. (1975) based on indirect measurement. The administration of THC to rats housed in this stressful environment produced behavioural changes not observed in the normal environment. In particular, immobility or 'catalepsy' and hyper-reactivity were observed. In most respects no changes in catecholamine metabolism, o t h e r than those already associated with the stressful environment, were observed after administration o f THC. However, there were two marked differences in measured parameters relating to DA metabolism. First, there was a very marked red u c t i o n in AMT-induced depletion o f DA in the striatum. This result has been reported before (Fuxe and Jonsson, 1971) but the authors make no mention of the environmental conditions involved. Several o t h e r workers have not obtained this result (e.g., Bracs et al., 1975). Second, a large increase in the uptake o f DA into striatal synaptosomes was produced by THC p r e t r e a t m e n t of animals in the stressful environment. This result differs from those of previous experiments on the effect, in vitro, of THC on DA uptake (Howes and Osgood, 1974; Banerjee et al., 1975b). Differences in experimental procedure and in the uptake characteristics o f striata of rats from different environments may explain this anomaly. The inhibition o f AMT-induced depletion of DA by THC indicates that, after administration of THC, DA synthesis is of reduced i m port ance in maintaining the endogenous concentration o f stored DA. THC may reduce DA release, reduce DA catabolism, or increase DA reuptake by the dopaminergic neurone. The results obtained indicate t hat THC does n o t reduce DA release (sup-
AS-THC, STRESS AND BRAIN CATECHOLAMINES
181
ported b y Howes and Osgood, 1974) and does not reduce DA catabolism (supported by Osgood and Howes, 1974; Banerjee et al., 1975a). We do, however, report direct evidence that under stressful environmental conditions THC administration in vivo increases DA reuptake in rat striatum. We therefore suggest that this action may explain the effect on AMT-induced DA depletion and may contribute to the behavioural differences observed in response to THC in different environments. It is not certain whether these results relating DA metabolism and THC effects can be extrapolated to man. Available evidence (Messiha and Soskin, 1973} strongly suggests that smoking cannabis reduces urinary DA metabolites in man. The authors suggest that an increase in neuronal binding of DA may account for their findings. Our work, suggesting increased neuronal uptake of DA, provides some supportive evidence for this concept. Furthermore, we cannot speculate as to w h y environmental change should have so profound an effect on the behavioural and biochemical response to THC. Presumably, the change in activity of the central catecholaminergic systems induced by environmental change plays a part. The work of List et al. (1975) strongly implicates interaction with adrenal steroid hormones as a major determinant in the alteration of the biochemical effects o f THC by environment. There is little doubt, however, that our results further confirm the importance o f changes in the uptake characteristics of catecholaminergic neurones in the functional regulation of catechoamine metabolism. Adaptive changes in NA uptake (Henley et a1.,1973) and DA uptake (Hutchins et al., 1975) have both been reported in response to environmental change associated with fighting in mice. One of the most impressive alterations in behavioural responses to cannabis by environment is the bizarre fighting behaviour produced by chronic cannabis administration to rats stressed by food deprivation (Carlini et al., 1972).
In conclusion, we report that changing the environmental conditions under which A s, tetrahydrocannabinol is administered to rats changes both the behavioural and biochemical response to the drug. The main change in central catecholamine metabolism which we have observed is an increase in dopamine uptake in the corpus striatum. We suggest that this change may mediate some o f the behavioural effects o f cannabis which are observed specifically when the drug is administered under conditions o f stress. Acknowledgements We wish to thank Beecham Research Laboratories for gifts of AS-THC and radiochemicals. In particular, thanks are due to Dr. M.S.G. Clark and Dr. M.G. Palfreyman for help and advice. This study was supported in part by a CAPS award to K.I.M. from the SRC and Beecham Research Laboratories.
References Anddn, N.E., H. Corrodi, A. Dahlstr6m, K. Fuxe and T. H6kfelt, 1966, Effects of tyrosine hydroxylase inhibition on the amine levels of central monoamine neurons, Life Sci. 5,561. Banerjee, A., M.K. Poddar, S. Saha and J.J. Ghosh, 1975a, Effect of A9-tetrahydrocannabinol on monoamine oxidase activity of rat tissues in vivo, Biochem. Pharmacol. 24, 1435. Banerjee, S.P., S.H. Snyder and R. Mechoulam, 1975b, Cannabinoids: influence on neurotransmitter uptake in rat brain synaptosomes, J. Pharmacol. Exptl. Therap. 194, 74. Bracs, P., D.M. Jackson and G.B. Chesher, 1975, The effect of Ag-tetrahydrocannabinol on brain amine concentration and turnover in whole rat brain and in various regions of the brain, J. Pharm. Pharmacol. 27,713. Carlini, E.A. and C. Gonzales, 1972, Aggressive behaviour induced by marihuana compounds and amphetamine in rats previously made dependent on morphine, Experientia 28, 542. Carlini, E.A., A. Hamoui and R.M.W. M~rtz, 1972, Factors influencing the agressiveness elicited by marihuana in food deprived rats, Brit. J. Pharmacol. 44, 794. Corrodi, H. and L.C.F. Hanson, 1966, Central effects of an inhibitor of tyrosine hydroxylation, Psychopharmacologia 10, 116.
182 Coyle, J.T. and S.H. Snyder, 1969, Catecholamine uptake by synaptosomes in homogenates of rat brain: stereospecificity in different areas, J. Pharmacol. Exptl. Therap. 170, 221. Friedman, E., N. Starr and S. Gershon, 1973, Catecholamine synthesis and regulation of food intake in the rat, Life Sci. 12, 317. Fuxe, K. and G. Jonsson, 1971, The effect of tetrahydrocannabinols on central monoamine neurons, Acta Pharm. Suec. 8 , 6 9 5 . Fuxe, K., T. H6kfelt and U. Ungerstedt, 1970, Central monoaminergic tracts, in: Principles of Psychopharmacology, eds. W.G. Clark and J. Del Giudice (Academic Press, New York) p. 87. Glowinski, J. and L.L. Iversen, 1966, Regional studies of catecholamines in the rat brain. The disposition of (3-H)-norepinephrine, (3-H)-dopamine and (3-H)-DOPA in various regions of the brain, J. Neurochem. 13, 655. Goldberg, M.E. and A.I. Salama, 1972, Tolerance to drum stress and its relationship to dopamine turnover, European J. Pharmacol. 17,202. Henley, E.D., B. Moisset and B.L. Welch, 1973, Catecholamine uptake in cerebral cortex: adaptive change induced by fighting, Science 180, 1050. Holtzmann, D., R.A. Lovell, J.H. Jaffe and D.X. Freedman, 1969, 1-A9-tetrahydrocannabinol: neurochemical and behavioural effects in the mouse, Science 163, 1464. Howes, J. and P. Osgood, 1974, The effect of Ag-te trahydrocannabinol on the uptake and release of 14C-dopamine from crude striatal synaptosomal preparations, Neuropharmacology 13, 1109. Hutchins, D.A., J.D.M. Pearson and D.F. Sharman, 1975, Striatal metabolism of dopamine in mice made aggressive by isolation, J. Neurochem. 24, 1151. Laverty, R. and K.M. Taylor, 1968, The fluorometric assay of catecholamines and related compounds, Anal. Biochem. 22,269. List, A.F., S.F. Bartram, B.L. Nazar and J. Harclerode, 1975, Interactions of A9-tetrahydrocan nabinol, adrenal steroids and ethangl, J. Pharm. Pharmacol. 27,606. Littleton, J.M. and K.I. MacLean, 1974, The effect of AS-tetrahydrocannabinol (As-THC) on dopamine metabolism in the rat corpus striatum: the influence of environment, Brit. J. Pharmacol. 51, 117P.
K.I. MACLEAN, J.M. LITTLETON Littleton, J.M. and K.I. MacLean, 1976, Alterations in dopamine uptake in rat corpus striatum induced by combinations of stress and AS-tetrahydro cannabinol (AS-THC), Brit. J. Pharmacol. 56, 370P. Maftre, L., P.A. Baumann and A. Delini-Stula, 1972, Neurochemical tolerance to cannabinols, in: Cannabis and its Derivatives, eds. W.D.M. Paton and J. Crown (Oxford University Press, London) p. 101. Messiha, F.S. and R.A. Soskin, 1973, Effect of cannabis sativa administered by smoking on biogenic amine secretion in man, Res. Commun. Chem. Pathol. Pharmacol. 6,325. Osgood, P. and J. Howes, 1974, Cannabinoid-induced changes in mouse striatal homovanillic acid and dihydroxyphenylacetic acid: the effects of amphetamine and L-DOPA, Res. Commun. Chem. Pathol. Pharmacol. 9, 621. Palfreyman, M.G., E.S. Palfreyman and M.S.G. Clark, 1974, Effects of benapryzine, a new antiparkinson drug, on dopamine uptake into corpus striatum, European J. Pharmacol. 28,379. Persson, T. and B. Waldeck, 1968, The use of 3HDOPA for studying cerebral catecholamine metabolism, Acta Pharmacol. Toxicol. 26, 363. Sedvall, C.G., V.K. Weise and I.J. Kopin, 1968, The rate of norepinephrine synthesis measured in vivo during short intervals: influence of adrenergic nerve impulse activity, J. Pharmacol. Exptl. Therap. 159, 274. Shore, P.A. and J.S. Olin, 1958, Identification and chemical assay of norepinephrine in brain and other tissues, J. Pharmacol. Exptl. Therap. 122, 295. Talbott, J.A. and J.W. Teague, 1969, Marihuana psychosis, J. Amer. Med. Assoc. 210,299. Taylor, K.M. and R. Laverty, 1969, The metabolism of tritiated dopamine in regions of the rat brain in vivo, J. Neurochem. 16, 1361. Thierry, A.M., 1973, Effects of stress on various characteristics of noradrenaline metabolism in central noradrenaline neurones, Advan. Exptl. Med. Biol. 33,501. Truitt, E.B., Jr. and S.M. Anderson, 1971, Biogenic amine alterations produced in the brain by tetrahydrocannabinols aad their metabolites, Ann. N.Y. Acad. Sci. 191, 68.