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BEHAVIORAL BIOLOGY 17, 313-332 (1976), Abstract No. 5276

S i m i l a r i t i e s b e t w e e n A 9 - T e t r a h y d r o c a n n a b i n o l

( A 9 -THC) a n d R e s e r p i n e - l i k e D r u g s I ,2

BROOKS CARDER 3 and STUART M. DEIKEL

Department of Psychology, 402 Hilgard Avenue,

University of California, Los Angeles, California 90024

Similarities between the behavioral and biochemical effects of Ag-THC and reserpine are reviewed. This interpretation is further supported by several experimental results. Pretreatment with a monoamine oxidase inhibitor (MAO-I) followed by 0.5 mg Ag-THC, produced straub-tail and excitatory effects on intracranial self-stimulation behavior. The identical dose of THC, without MAO-I pretreatment impaired self-stimulation. It was demonstrated that these effects were probably not due to nonspecific excitatory effects of the MAO-I. Further, rats made tolerant to A9-THC exhibited cross-tolerance to the hypothermic effects of the reserpine- congener tetrabenazene (TBZ). Postmortem examination suggested that these effects were probably not due to ineffective absorption of TBZ from the injection site. Finally, behavioral cross-tolerance between A9-THC and TBZ, in an unlearned swimming-escape task, was demonstrated. These data suggest further similarities between the effects of A 9-THC and reserpine. The possible role of monoamine disposition in the mechanism of cannabis action is discussed.

Similarities between selected behavioral and physiological effects of

Ag-Tetrahydrocannabinol (A9-THC) and other compounds, particularly

morphine and anticholinergics, have been noted by several investigators

(Lomax, 1971a, 1971b; Mechoulam, 1973 p. 258; Brown, 1971). While there

are indeed selected areas of comparison between cannabis and both morphine

and antimuscarinics, it is doubtful that marijuana shares a mechanism of

action common to either of these classes of compounds.

1This work was supported by Public Health Service Grant Number DA-00288 to Brooks Carder.

2The authors would like to thank Dr. Larry L. Butcher and Konrad Talbot for advice on the design of the experiments and preparation of the manuscript, and Michelle Black for assistance in carrying out the research.

3Address reprints to this author at Syanon Foundation, Marshall, California 94940.

313

Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

314 CARDER AND DEIKEL

The chemical property of cannabis which has been suggested to domin- ate its pharmacological actions (Paton et al., pp. 50-76, 1972) is its extreme lipophilicity; morphine, in contrast, is readily soluble in water. The pharmacological action of A 9-THC has also been described as far more specific than the general depressant effects which characterize the opiates (Loewe, 1945). While the addiction liability of morphine is well documented, no definitive evidence of physical dependence following the extended use of cannabis has yet been offered (Carlini et al., p. 154, 1972). Furthermore, while cross-tolerance between THC and morphine has been suggested (Kaymakcalan, p. 75, 1972), no data demonstrating that effect have yet been reported. Finally, cannabis is unaffected by opiate antagonists (McMillan et al., 1971).

It has recently been reported that THC may attenuate the symptoms of naloxone-precipitated morphine (Hine et al., 1975a) and methadone (Hine et al., 1975b) abstinence; on the basis of these data Hine et al. reassert the potential similarities between cannabis and opiate derivatives. However, these speculations should be regarded as tentative due to serious methodological limitations of the work (Carder, 1975; Deikel and Carder, 1975). Further- more, these data failed to represent a similarity between THC and either morphine or methadone since a variety of non-narcotic agents will suppress naloxone-precipitated indices of opiate abstinence (Kamei et al., 1973). Hirschborn and Rosecrans (1974) have demonstrated naloxone-induced abstinence following the chronic administration of Ag-THC. However, these investigators rightly conclude that naloxone-precipated abstinence may be produced following the chronic administration of a variety of substances.

The behavioral and pharmacological effects of A 9-THC also differ from the antimuscarinics in several important respects. First cannabinols exhibit paradoxical effects on general activity, some of which characterize the anticholinergics (Brown, 1971), while other actions are similar to anti- cholinesterases (Brown, 1972). Furthermore, unlike Ag-THC or reserpine, the typical anticholinergics, atropine sulfate and scopolamine, are water soluble (Innes and Nickerson, 478, 1970). Hypothermic responses accompanying the administration of Ag-THC have been widely described (Abel, pp. 120-142, 1972); however, opposite effects are observed after treatment with Belladonna alkaloids (Innes and Nickerson, p. 533, 1970). Finally cannabis, unlike antimuscarinics, fails to disrupt passive avoidance learning in rats (Miller and Drew, 1974). A more comprehensive review by Miller and Drew (1974) clearly outlines further inconsistencies implicit in an anticholinergic model of A 9-THC action.

Various findings, both from our laboratory and elsewhere, emphasize the similarities between Ag-THC and reserpine (Sofia and Dixit, 1971; Englert et al., 1973; Carder and Deikel, 1975). Table 1 depicts a comparison of selected effects of Ag-THC, reserpine, antimuscarinic agents such as atropine and scopolamine, and morphine. There is a very close correspondence between the

THC AND RESERPINE-LIKE DRUGS

TABLE 1

Comparison for Selected Effects of A 9 -THC, Reserpine, Anticholinergics, and Morphine a

315

Atropine/ Effect Ag-THC Reserpine scopolamine Morphine References

Increased barbiturate + + -* + 46, 69, 83, 40 sleeping time Biphasic motor + + -* + 15, 18, 75, 52

Response Blood pressure + + + + 24, 4, 22, 5 decreased Decreased CER + + -* + 29, 18, 19, 58 Catalepsy + + -* + 15, 18, 39, 44 Increased excitability + + -* -** 73, 69, 39, 3

to External Stimuli Hypothermia + + -* + 49, 18, 39, 84 Decreased respiration + + -* + 45, 69, 40, 2

at high doses Increased respiration + + -* -** 6, 69, 40, 80

at low doses Decreased responding + + -* + 56, 18, 19, 20

for food Decreased responding + + -* + 81, 65, 19, 64

or increased thresh- old for self-stimulation

Spontaneous activity + + + - 14, 69, 39, 72 Reduced Spontaneous activity + + ? -** 73, 7, 40 Increased after MAO-I Lipophilic + + -* -** 61, 18, 39, 40 Straub-tail + + - + 10, 18, 53

aDifferenees are denoted between the effects of A 9-THC and atropine/scopolamine (*) or morphine (**).

e f fec ts o f cannab i s and reserp ine ; m u c h closer t h a n t h a t b e t w e e n A 9-THC and

e i the r m o r p h i n e or the ant ichol inerg ics . The dispar i ty b e t w e e n these l a t t e r

c o m p o u n d s a n d cannab i s w i t h regard to " I n c r e a s e d Exc i t ab i l i ty to Ex t e rna l

S t i m u l i " a n d " I n c r e a s e d R e s p i r a t i o n at L o w Doses" ref lec ts the dose-specif ic

s t i m u l a n t e f fec t s wh ich are ev iden t a f te r the a d m i n i s t r a t i o n of e i the r A 9-THC

or reserp ine ; these e x c i t a t o r y e f fec t s are n o t d e t e c t e d fo l lowing the admin-

i s t r a t ion o f e i t he r m o r p h i n e or an t imuscar in ics . Also, the increase in spon-

t a n e o u s ac t iv i ty n o t e d a f te r A9-THC or reserp ine admin i s t r a t i on , fo l lowing

p r e t r e a t m e n t w i th a m o n o a m i n e oxidase i n h i b i t o r (MAO-I) , f u r t h e r suggests

s imilar i t ies b e t w e e n the ac t ion o f cannab i s and the Rauwol f i a a lkaloid .


Hardman, Domino, and Seevers (197l) described, on the basis of their similar effects on cardiovascular responses, the marked consistencies between the actions of synthetic cannabinoids and reserpine. Indeed Sofia et al. (1971) reported that THC administration attenuated the subsequent action of reserpine and suggested that the two compounds may have a similar site of action.

Finally Englert et al. (1973) have demonstrated that the prior administration of either THC or reserpine attenuated the hypothermic effects of the other compound; these data further suggest that these agents may bear similarities in their respective mechanisms of action.

Biochemical investigations, especially with regard to the catecholamines, further support the similarities between cannabis and reserpine. Several researchers report that cannabinols increase turnover and decrease endogenous levels of central catecholamines (Holtzman, et al. 1969; Schildkraut and Efron, 1971; Maitre et al., 1970). Maitre et al. (1973) have reported that increased utilization of catecholamines is especially marked in the hypothalamus, a region prominently involved in the mediation of many cannabis effects (Paton and Pertwee, pp. 222-225, 1973). Howes and Osgood (1974) have reported that THC, like amphetamine, blocks uptake of dopamine into striatal synaptosomes. However, conflicting data have been obtained, as a number of investigators report varying effects of A9-THC on central catecholamine levels (Ho et al., 1972). The controversial nature of reports reflecting the biochemical effects of cannabinols in animals suggested to us that a somewhat different approach to the problem might prove useful. Thus, we began our investigation by examining the behavioral effects of A9-THC. With this perspective we have attempted to demonstrate further similarities between Ag-THC and other compounds known to facilitate the increased turnover of central neurotransmitter substances.

EXPERIMENT 1

The depressant behavioral effects accompanying the administration of compounds which facilitate the release of monoamines (e.g., Rauwolfia alkaloids and related benzoquinolizines) are markedly reversed when subjects are pretreated with a monoamine oxidase inhibitor (MAO-I)(Carlsson, 1966). Reserpine alone, for example, has marked depressant effects on self- stimulation (Olds and Travis, 1960). However, following the administration of an MAO-I, reserpine has prolonged excitatory effects (Carlsson, 1966). Sabelli et al. (1974), using relatively gross observational techniques, reported that pretreatment with Nialamide caused an increase in the motor activity and general excitability of rats administered A 9-THC.

Prior work on self-stimulation indicated that A9-THC is an effective

THC AND RESERPINE-LIKE DRUGS 317

inhibitor of this behavior (Wayner, 1974). These data are, of course, com- patible with a hypothesis that A9-THC interferes with the central catecholamines, the significant neural transmitters in the mediation of self- stimulation (German and Bowden, 1974). This disruption may reflect a facilitation of the utilization in CAs systems in a manner similar to reserpine; an hypothesis which would predict that pretreatment of rats with MAO-I should result in a stimulant action of THC, rather than the depressant effect typically observed. Our first studies then investigated the joint influence of THC and MAO-I on intracranial self-stimulation with a view toward further investigation of similarities between THC and reserpine.

Method

Subjects

Subjects were 20 male, Sprague-Dawley rats acquired from Simonsen Breeding Laboratories, Gilroy, California. All subjects were at least 90 days old and weighed between 300 and 350 g at the beginning of the experiment. Each subject was housed individually with food and water freely available at all times.

Five to nine days prior to the beginning of the experiment subjects were anesthetized with Pentobarbital Sodium (35 mg/kg) and implanted bilaterally with bi-polar, Teflon coated, stainless steel electrodes directed at the lateral hypothalamic region (DeGroot coordinates: anterior from interaural axis: 5.5; lateral: 1.6; vertical from dura: 7.5).

Apparatus

Training and testing for self-stimulation were conducted in two Gerbrands Operant Conditioning Chambers. Each bar press delivered a 500 msec train of 60 Hz current of adjustable intensity.

Procedure

Following recuperation from surgery subjects were tested for self- stimulation. Current intensities were adjusted to yield response rates of 1500 to 3600 responses per hour and remained constant for each subject throughout the experiment. Intensities ranged from 130 to 220/~A RMS. Each subject was allowed 4 hr of practice prior to the initiation of the experiment. Subjects were allowed 2 days of training during which they were each allowed to respond for l hr. Beginning on the evening of the second day, and continuing on throughout the experiment, 12 subjects were administered 100rag Nialamide/kg, while the remaining animals were administered 1 ml saline/kg. During the drug test session, conducted on the fifth day of the experiment,


subjects were allowed to respond for 40 min after which they were administered either 0.5 mg Ag-THC/kg, 24 mg Nembutal/kg, or 1 ml propylene glycol/kg.

At the beginning of each session on Days 1-4 subjects were placed in the chamber and allowed to respond for 1 hr; response totals were recorded at 20 rain intervals. During the drug test session, conducted on the fifth day of the experiment, after 40 min of responding subjects were dosed with the appropriate drug and immediately returned to the experimental chamber. Response totals were recorded for the following 2 hr at 20 rain intervals.

A9-THC was supplied by NIMH as a solution in dehydrated alcohol with 1000 rag/5.3 ml. This was diluted in propylene glycol to yield a solution of 0.5 mg Ag-THC/ml. Nialamide was supplied by Charles Pfizer, Incorporated. This compound was dissolved in 1 M HC1 and diluted in distilled water to yield a concentration of 80 mg/ml. Sodium bicarbonate was used to adjust the solution to a pH of approximately 4. Nembutal was used in a concentration of 50 mg/ml. Propylene glycol and physiological saline were prepared in sealed and sterile injection vials. All drug solutions were prepared prior to the beginning of the experiment and refrigerated throughout. All drugs were administered by intraperitoneal injection.

Following the completion of the experiment all subjects were sacrificed with Pentobarbital-sodium, perfused with 10% formalin, and frozen sections prepared to locate the electrode tips. Placements were located in the lateral regions of the lateral hypothalamic area at the approximate level of the ventromedial nucleus.

Results

Table 2 presents the mean response rate per hour for the entire session before and after each drug treatment. The data indicate that pretreatment with saline followed by Ag-THC administration induced a suppression of responding. However, animals pretreated with Nialamide prior to the administration of THC exhibited a significant increase in response rate IF(5, 3) = 9.33, P < 0.05]. Subjects treated with Nialamide and then dosed with Nembutal (25 mg/kg) also demonstrated a marked reduction in responding, much greater than that exhibited by subjects administered saline prior to Nembutal injection [F(5, 6)=5.81, P < 0 . 0 3 ] . The administration of Nialamide followed by propylene glycol injection had a slight excitatory effect; however, this failed to reach statistical significance (F3.21, P < 0.08).

These effects are most apparent in Fig. 1 which displays cumulative records of individual response rates over time, following various pretreatments and drug administrations. It is evident from Record A that saline pretreatment followed by THC administration had a marked depressant effect on the

THC AND RESERPINE-LIKE DRUGS

TABLE 2

Mean Self-Stimulation, per Hour, among Rats Administered Various Pretreatment and Test Drugs

319

Mean number of responses per hour

Pretreatment Baseline Pretreatment drug Test drug

Test

(Days 1-2) (Days 3-4) (Day 5)

Nialamide THC 2392 (± 36.21) 3312 (± 28.05) Saline THC 2197 (± 28.77) 2857 (± 29.72) Nialamide Nembutal 1380 (5 29.86) 1513 (5 29.63) Saline Nembutal 2726 (5 32.65) 3819 (5 33.09) Nialamide Propylene-

glycol 2767 (5 24.36) 2829 (± 27.14)

4569 (+- 28.38) 1425 (+- 32.61)

43 (± 30.89) 768 (+- 33.78)

2904 (-+ 25.79)

A / / /// //

t , MG, 9-THC/KG. 23H, AFTER 1 ML. SALINE/KG.

G C 2 T O ~ /KG

J 15 riG, PENTOBARIHTAL/KG, 23H. AFTER 100 riG, MA0-1/KG,

Fig. 1. Individual cumulative records of self-stimulation for animals. (A) pretreated with saline, followed by .5 mg THC/kg; (B)pretreated with 100mg Nialamide/kg, followed by 0.5 mg THC/kg, and; (C) pretreated with 100 mg Nialamide/kg followed by 25 mg pentobarbital/kg.


self-stimulation behavior. The record presented in B demonstrates the excitatory effect of THC following pretreatment with Nialamide. Record C shows the depressant effect of pentobarbital following Nialamide pretreatment.

Discussion

The results of our initial experiments indicated that doses of THC which normally inhibited self-stimulation would enhance this activity following MAO-I pretreatment. These data are consistent with the report of Sabelli et al.

that treatment with an MAO-I increased the general excitability of rats administered THC. Furthermore, during the course of our observations we consistently noted that subjects pretreated with Nialamide demonstrated marked straub tail reactions to the administration of a moderate or high dose of Ag-THC. Figure 2 presents representative examples. Animals presented in photographs A and B exhibit the tail flexion and striking ataxia typically noted after pretreatment with 100 rag. Nialamide/kg followed by the administration of 15 mg A9-THC/kg. Comparable effects have occasionally been reported after the injection of very high doses of THC to mice (Buxbaum et

al., 1969). These effects are also similar to those exhibited by rats dosed with

A

D

Fig. 2. (A and B) Ataxia and straub tail reactions accompanying 100 mg Niala- mide/kg pretreatment, followed by 15 mg THC/kg. (C and D) Absence of ataxia or straub tail in rats administered 100 mg Nialamide/kg followed by propylene-glycol.


reserpine following MAO-I pretreatment (Carlsson, 1966). Subjects depicted in photographs C and D demonstrate, the effects of pretreatment with Nialamide followed by propylene glycol administration. In these pictures there is no evidence of ataxia or straub tail. These data further indicate similarities between the action of A9-THC and compounds which facilitate the release of endogenous monoamines.

It is unlikely that the effects observed in this experiment are the result of Nialamide producing a general excitatory state during which any centrally active drug could produce stimulant effects. Subjects pretreated with Nialamide and then dosed with Nembutal, which like A9-THC typically inhibits self-stimulation, exhibited a considerable enhancement of these depressant effects. Similarly, it may be concluded from the work of Marks and Dark (1965) and Sjoqvist (1965) that pretreatment with an MAO-I accentuates the effects of centrally acting depressants, and enhances the excitatory effects of stimulants. Thus, the data from the current study might indicate that THC exerts an excitatory effect, involving the release of neurotransmitter substances, which is normally concealed by the action of MAO.

The results of this experiment, together with the data reported by Sofia and Dixit (1971) indicating that THC attenuates the effects of reserpine, might further indicate that A9-THC acts to facilitate the release of neural transmitter substances. Interpreted in this way, the excitatory effects of THC would be apparent either during the brief, initial stages of the drug's action, or following the inhibition of those processes responsible for the eventual metabolic deamination of monoamines.

EXPERIMENT 2

Further evaluation of this model of THC action attempted to establish a more direct association in the form of cross-tolerance, between cannabis and a compound known to facilitate turnover of monoamines. It would seem preferable to demonstrate cross-tolerance between THC and reserpine; however, repeated administration of reserpine does not produce tolerance, and the time course of reserpine action is unlike that of cannabis (Carlsson, 1966). Thus, we continued by examining the potential similarities between THC and tetrabenazene (TBZ), a compound which has a pharamacological action similar to reserpine (Haggendahl and Roos, 1968), but a time course comparable to cannabis (Haggendahl and Lindquist, 1963). The hypothermic effect of TBZ, like that of THC, is well documented (Carlsson, 1966). Prolonged treatment with either TBZ or A 9-THC leads to a form of tolerance to this hypothermic response (Haggendahl and Lindquist, 1963, Lomax, 1971a). If the action of cannabis is somewhat comparable to that of TBZ, it might be expected that cross-tolerance to these hypothermic effects could be demonstrated. Indeed


Englert et al. (1973) have demonstrated that subchronic injections of 1 mg THC/kg prevented the hypothermia produced by the subsequent administration of 15 mg reserpine/kg. However, these investigators failed to determine that intermittent injection of this relatively low dose of THC in fact produced tolerance to the hypothermic effects of THC; thus, cross- tolerance between THC and reserpine has yet to have been dearly demonstrated. Thus, we examined the effect of prior tolerance to THC on the hypothermic reaction to TBZ.

Method

Subjects

Twenty-five male, Sprague-Dawley rats, over 90 days old at the beginning of the experiment, were randomly assigned to each of three groups. The subjects were housed individually, with food and water freely available at all times, in a laboratory in which the atmospheric temperature was consistently maintained at 22°C (+0.5).

Procedure

The THC tolerant group (n = 10) received daily administrations of 5 mg A9-THC/kg. One control group (n = 10) received an equal volume of propylene glycol, while a third group (n = 5) received chronic administrations of 5 mg THC/kg. Core temperature was then monitored, to the nearest tenth of a degree, at 30 rain intervals by means of a thermistor probe inserted rectally at least 5 cm. Tolerance to THC was assumed by the absence of hypothermia up to 1 hr after drug injection. This criterion was achieved by all subjects within 7 days.

On the day immediately following the achieving of THC tolerance by all subjects, experimental animals (n = 10) were injected intraperitoneally with 50 mg TBZ/kg. One Control group (N = 10) received an identical dose of TBZ, while the other Control group (N = 5) was similarly injected with a comparable volume of propylene glycol. Core temperature was monitored each half hour to determine the time required for each animal to achieve maximal hypothermia, and the net temperature reduction over time. Testing was concluded when all subjects in any experimental group failed to exhibit a further reduction in temperature for 2 consecutive hours. The maximal hypothermic response was determined by the greatest decrement in temperature observed in each animal at any 30 minute interval. Core temperature of animals administered propylene glycol was monitored throughout.

Ag-THC was prepared as before in a concentration of 5 mg/ml. Tetra- benazene was supplied by Hoffmann LaRoche Laboratories. This compound


was dissolved in distilled water to yield a concentration of 50mg/ml. Propylene glycol and physiological saline were prepared as before. All compounds were administered by intraperitoneal injection.

Following testing all subjects were sacrificed with pentobarbital sodium to detect any evidence of peritonitis, necrosis, resin deposits, or cyst formation on the peritoneal wall. No evidence of these pathologies was observed during postmortem examination.

Results

Figure 3 presents the mean core temperature of Treatment and Control subjects after being dosed with either tetrabenazene or propylene glycol. Rats pretreated with A9-THC and then administered propylene glycol exhibited only slight and inconsistent variations in core temperature. Mann-Whitney nonparametric analysis revealed that animals pretreated with propylene glycol and then administered TBZ exhibited a greater decrease in temperature than animals first made tolerant to THC and then dosed with TBZ (U = 16, P < 0.05). Further, animals tolerant to THC took significantly less time to exhibit maximal hypothermia after drug treatment than subjects pretreated with propylene glycol and then administered TBZ (U = 18, P < 0.05).

L,J

O

37i , - , ,

33- PG-TBZ (N-IO)

o I ~ ~ ,~ ~ ~ ~ ~ TIME (hours)

Fig. 3. Mean core temperature, as a function of time, in rats either: tolerant to 5 mg A9-THC prior to propylene glycol injection; tolerant to 5 mg/kg Ag-THC prior to 25 mg/kg TBZ administration, and; administered 25 mg TBZ/kg following propylene- glycol pretreatment.


Discussion

The data demonstrate cross-tolerance between the hypothermic effects of THC and TBZ. Subjects made tolerant to THC showed hypothermic reactions to TBZ of less magnitude and shorter duration than those observed in nontolerant rats.

It is doubtful that these results reflect ineffective absorption of tetrabenazene from the injection site, of the sort described by Ho et al. (1971), resulting from gross tissue damage accompanying chronic injection of a caustic and viscous THC solution (Mazaleski, 1971, Sodetz et al., 1972). First, investigators have observed no gross tissue damage following a treatment regimen of this sort (Phillips et al., 1971). Second, no observable visceral pathology was detected upon subsequent postmortem examination of our subjects.

The data further demonstrate similarities between the effects THC and compounds known to facilitate the release of functional monoamines. How- ever, the unilateral nature of this cross-tolerance demonstration requires comment. Rats administered daily doses of TBZ in an effort to establish tolerance to the drug, suffered additive respiratory depression necessitating the abandoning of continued treatment; thus symmetrical cross tolerance could not be examined. Englert et al. were, however, able to detect a bilateral component in their examination of THC and reserpine's mutually antagonistic and synergistic effects. In order to increase the generality of our demonstration of cross tolerance, we explored the possible behavioral cross-tolerance between A 9-THC and TBZ.

EXPERIMENT 3

Behavioral tolerance to cannabis has been well established (McMillan e t al., 1970). Potvin and Fried (1972) demonstrated that the chronic administration of THC enhanced the unlearned ability of rats to swim while under the influence of the drug. We attempted to extend our observations of cross tolerance between THC and TBZ to a behavioral measure in a swimming escape test.

M e t h o d

Subjects

Sixty male, naive, Sprague-Dawley rats over the age of 90 days at the beginning of the experiment were used. All subjects were housed individually with food and water freely available throughout.


Apparatus

Swimming tests were conducted in a galvanized steel tub measuring 60 cm in diameter with an aluminum panel placed around the rim to produce a continuous wall 50 cm in height. One wall was equipped with a plywood platform measuring 10 × 15 cm, placed level with the water edge, 30 cm above the tub's floor. Water temperature was maintained at room temperature (approximately 22°C).

Procedure

Two groups of 30 subjects were administered either 5 mg A 9-THC/kg or a comparable volume of propylene glycol in the home cage for 5 consecutive days. Testing with alternate rats established that behavioral tolerance to THC had developed by that time.

On the sixth day of the experiment 10 rats from each group of 30 were dosed with either: 5 nag THC/kg, 25 mg TBZ/kg, or 1 ml propylene glycol/kg. Each animal was tested on the swimming task only once, a half hour after receiving the appropriate drug treatment. The rat was placed in the middle of the tub and positioned in the opposite direction of the platform. The time required for each subject to reach and mount the platform was recorded. A maximum of 60 sec was allowed; subjects not reaching the platform were removed at that time and a latency of 60 sec recorded. While not all subjects accomplished this task in the allotted time, all were able to swim or float above water and thus none were removed prematurely.

Ag-THC, TBZ, propylene glycol, and saline solutions were prepared prior to the initiation of the experiment as before, placed in sealed and sterile vials suitable for injection and refrigerated throughout. All drug administrations were by intraperitoneal injection.

Immediately following testing all subjects were removed to their home cages. Animals were subsequently sacrificed with pentobarbital and autopsies performed to determine the condition of the peritoneum following chronic drug treatment. No pathology was detected.

Results

Figure 4 presents the mean time required for subjects to perform the swimming task. The time required by rats administered either THC or TBZ, following pretreatment with propylene glycol, was significantly greater than for all other groups [F(1, 8) = 9.37, P < 0.01]. These animals typically floated in a motionless position or swam slowly in a counterclockwise direction. Their hind legs were characteristically motionless and entangled, suggesting marked ataxia. Upon encountering the platform, they would avoid contact or occasionally attempt to mount; being unable to coordinate body movements

326 CARDER AND DEIKEL 6o] "~ 50

I -

o 401

~ 3O- <

~ 2 0 -

G g

z o-

5,2.7

150

rE] n PG THC PG PG PG THC

46.8

3 !

i 2Z 2 i

L..:

THC PG THC THC TBZ TBZ

P R E - T R E A T M E N T C O N D I T I O N

Fig. 4. Mean time (in seconds) to reach and mount platform following 5 mg 2x 9-THC/kg or propylene-glycol pretreatment prior to 5 mg Ag-THC/kg, 25 mg TBZ/kg, or propylene-glycol administration.

sufficiently to climb the platform, these subjects would continue slowly moving about the tub.

As further depicted in Fig. 4, rats pretreated with THC, and then dosed with either THC or TBZ, took significantly less time to complete the swimming task than animals pretreated with propylene glycol before the administration of THC or TBZ IF(l, 18) = 4.76, P < 0.05). These subjects were noticeably less ataxic; their movements were well coordinated and purposefully directed. They typically swam vigorously, seldom floated, and showed minimal difficulty in mounting the platform. Of all groups tested, subjects injected with propylene glycol swam the fastest regardless of pretreatment; however, differences between subjects administered propylene glycol, and rats pretreated with Ag-THC and then administered either THC or TBZ were not statistically significant IF = 3.98, P < 0.07].

GENERAL DISCUSSION

The main results of the present series of experiments were: (1)Pretreat- ment with Nialamide, followed by A9-THC produced an excitatory effect on self-stimulation behavior in rats. The same dose of THC, without MAO-I pretreatment, impaired self-stimulation. While stimulant effects have consistently been reported accompanying MAO-I pretreatment followed by reserpine (Carlsson, 1966), these effects were not observed with MAO-I


pretreatment followed by Nembutal; (2)rats made tolerant to A9-THC that were then given TBZ exhibited hypothermia, which was of lower magnitude and shorter duration when compared to animals not tolerant to THC. Thus the data demonstrate cross-tolerance between THC and the reserpine-congener, tetrabenazene, and; (3)Subjects made tolerant to A9-THC demonstrated behavioral cross-tolerance to the effects of TBZ on a swimming-escape task. Taken together, these data further demonstrate consistent similarities between cannabis and compounds known to facilitate the turnover of neurotransmitter substances from neuronal stores.

The biochemical actions of TBZ and other reserpine-congeners have been well documented. A primary effect is the relatively nonselective depletion of monoamines from presynatpic stores (Lewis, 1963; Carlsson, 1966). While many of the effects of TBZ are different from those of THC, the data reported by Sofia et al. (1971) as well as the effects demonstrated here, suggest consistent similarities between these compounds. The results we have obtained would further support the interpretation that A 9-THC acts, in part, by a mechanism involving the release of functional neurotransmitter substances. The initial phase of cannabis action might involve a release of functional neurotransmitter substances leading to behavioral stimulation. Eventually, however, levels of these monoamines would be depleted to such an extent that behavioral depression would result. A variety of studies reporting the behavioral effects of Ag-THC suggest this action, as marijuana elicits a fundamentally biphasic effect on motor activity (Joachimoglu, 1965; Moreton and Davis, 1970); furthermore, the stimulant effects, when observed, typically precede the more prolonged depressant phase (Carlini and Kramer, 1965).

While reserpine also depletes serotonergic stores, several investigators have observed increases in endogenous concentrations of 5-HT following Ag-THC administration (Welch et al., 1971; Sofia et al., 1971). Those measures employed in the present series of experiments, principally self- stimulation and motor activity, have all been established as behaviors that are particularly sensitive to alterations in catecholaminergic systems (German and Bowden, 1974). Furthermore, Gallagher et al. (1972) concluded that doses of THC which produce significant behavioral effects fail to alter the dynamics of central serotonergic systems.

It may be that the actions of A 9-THC result in the relatively selective activation of adrenergic mechanism; indeed, there is in the literature a number of studies which indicate that the catecholamines play a prominent role in the mediation of cannabis action. Indeed, the prolongation of amphetamine- elicited stimulation by cannabinols (Forney, 1971), as well as marijuana induced facilitation of aggression (Carlini and Masur, 1969; Carder and Olson, 1972), dearly suggest the activation of catecholaminergic systems accompanying the administration of Ag-THC. Finally, the similarities between the


behavioral effects of a-methyl-dopa and high doses of A 9-THC (Sjoerdsma, pp. 635-636, 1973; Domino, 1971), including hypotension and sedation, are consistent with a model of cannabis action involving, in part, the eventual depletion of functional catecholamines.

The inability of several biochemical investigations to consistently detect the changes in central catecholamine levels predicted by this hypothesis of dxg-THC action, may reflect the cannabinols' preferential depletion of that fraction of stored catecholamines which is the most functionally significant in neuronal transmission. Dahlstrom (1973) has reviewed several lines of evidence suggesting the existence of various intraneuronal pools of neurotransmitter substance. One such collection (Store II, in Dahlstrom's terminology) repre- sents a small easily releasable pool of neurotransmitter, which is the most significant fraction in the mediation of behavioral activity. Depletion of monoamines included in such a pool, as suggested by the data we have obtained, might only produce minimal effects in absolute transmitter levels, as revealed by biochemical assay, while significantly altering neuronal transmission; these effects would be more readily detected with the use of behavioral assay techniques such as those we have employed.

It is not the purpose of this paper to outline the biochemical effects of cannabis; neither is it our intent to establish an identity between zxg-THC and TBZ. Rather, we have attempted to demonstrate some of the initial similarities between dxg-THC and TBZ, and propose that the behavioral effects of THC administration may be importantly related to the modulation of catecholamine neurotransmitter substances. This model of cannabis action offers a .useful perspective which warrants considerable investigation and refinement by future studies.

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