Psychological Reportj, 1975, 37, 1051-1054.

@ Psycho!ogical Reporrs 1975

AVERSIVE EFFECTS OF REPEATED INJECTIONS OF THC I N RATS1 E D W I N J. KAY Lehigh University Summury.-Exposure of rats to a novel substance ( . I % sodium saccharin) was paired with 0 , .25, .5, 1.0, 2.0, and 4 . 0 mg/kg of dB-THC (tetrahydrocannabinol) for 1, 6 o r 12 days. In a subsequent test, all groups showed a decreased preference for saccharin as the dosage of T H C was increased. The results indicated that T H C is aversive in multiple as well as single injections.

Since the effective dose of T H C in man is in the 0.1 to 0.25 mg/kg range when inhaled (Weil, et a/., 1968), Elsmore and Fletcher ( 1972 ) pointed out that most studies of the effects of THC on rats may be studies of the toxic effects of the drug. Elsmore and Fletcher used a conditioned taste-aversion paradigm to demonstrate that THC is aversive over a range of dosages. Rats on a 23-hr. water deprivation schedule were given intraperitoneal injections of 0, .25, 1.0, 2.0, 4.0, 8.0, and 32.0 mg/kg of THC immediately after 1 hr. exposure to .1% saccharin solution, a novel substance. Subsequently, the rats were given simultaneous exposure to water and the saccharin solution in a preference test. There was a profound decrease in preference for saccharin as the dosage increased. Corcoran ( 1973) replicated these results using single pairings of T H C with saccharin, sodium chloride, and vanilla extract. The present study was ,intended to replicate the design of Elsmore and Fletcher for a lower range of dosages and to extend the design to include repeated daily pairings of saccharin with injections of the drug. The conditions with repeated injections were included so that the results could be applied to experiments with multiple injections of THC.

METHOD One-hundred and eighty-five experimentally naive, male Sprague-Dawley albino rats, 90 to 120 days old, served as subjects. The experiment was run while the animals were in their home cages. Each cage had two bottle holders mounted side by side on the front. One holder was for bottles of saccharin water ( 1 gm of sodium saccharin per liter of tap water) and the right holder was for bottles of tap water. THC, suspended in absolute alcohol, was furnished by the Narional lnstitute of Mental Health. Sufficient propylene glycol and absolute alcohol were 'This research was supported by National Insticute for Drug Abuse Grant No. lR03DA00964-01. I thank the National Insticute of Mental Health for providing the T H C used in rhis study. 1 also thank Aidan Altenor, Ned Heindel, Margaret Krawiec, Sidney Sanders and Monica Seech for their help in conducting this srudy.

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added to obtain concentrations of 0, $25,.5, 1.0, 2.0 and 4.0 mg of THC per ml in a vehicle consisting of 80% propylene glycol and 20% absolute alcohol. The drug doses were administered intraperitoneally in a volume of 1.0 ml/kg of body weight. The animals were run in three replications with 60 animals each in the first two replications and 65 animals in the third replication. The first 180 animals were assigned at random without regard to replication to one of 18 possible treatment conditions, with the restriction that there be 10 animals per condition. The remaining five animals were "extras" used to replace those animals from the first two replications who failed to complete the experiment. The 18 treatment groups were formed by taking all possible combinations of 6 levels of drug dosage (0.0, .25, .5, 1.0, 2.0, and 4.0 mg/kg) and 3 levels of number of days of injection (1, 6, and 1 2 ) . For example, 0.0-1 denotes the treatment group receiving 0 mg/kg for one day. Upon receipt, the rats were housed in individual cages and given free access to food and water. Six to 10 days before the start of each replication the racs were placed on 23-hr. water deprivation. On the fjrsr day of each replication, each animal was given 1 hr. access to saccharin. Immediately following the removal of the saccharin, each rat was given an injection at a dosage level corresponding to [he group to which he was assigned. The 1-day rats were given one day of this treatment, while the 6- and 12-day racs were given 6 and 12 consecutive days, respectively. On the day after the last treatment day, the rats were given 1 hr. access to water. Then followed three test days when each rat was given 1 hr. of simultaneous access toosaccharin and water. The weight of saccharin and the weight of water consumed on each test day by each rat was recorded. For each rat, the percent of saccharin solution consumed as compared to total liquid consumed was computed for each day and then averaged over the three days.

RESULTS Six rats died during the experiment. Further, two rats from the 2.0-12 group and one rat from the 4.0-12 group were withdrawn from the experiment because they stopped consuming saccharin solution, their only source of water, and appeared unable to survive the rigors of the experiment. The analysis of variance performed on the data ignored replications as a to condirions within treatmenc. This is statistically sound since the assignment replications was random. Although this procedure tends to lower the power of the various F tests, the loss turns out to be negligible. Since the analysis of variance involved an unequal number of observations per cell, an unweightedmeans analysis of variance (Winer, 1962, p. 374) was used. Table 1 shows the saccharin preference data averaged over the three test days for all the groups of rats. A two-way factorial analysis of variance was

AVERSIVE EFFECTS O F THC

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TABLE 1

PERCENT SACCHARIN

FOLLOWING 1, 6, OR

THC No. daily Pairings

AVERAGED OVER THREETESTDAYS, BY 12 DAILY PAIRINGS OF SACCHARIN WITH

PREFERENCE,

AS A FUNCTION OF

RATS

DOSE"

Dosage of THC (mg/kg)

0

1/4

1/2

1

2

4

23.5 28.7 69.5 68.2 63.8 35.8 13.0 10.7 28.0 41.1 6 75.8 50.1 65.5 44.1 42.6 46.0 30.1 12 72.4 *n = 9 for the 1.0-12 and 2.0-12 groups, n = 8 for the 4.0-12 group, and n = 10 for all other groups.

1

'

applied. The levels of one factor were the six dosages, and the levels of the other factor were the three differenc numbers of treatment days. The main effect for days of treatment was significant (F2,158 = 8.48, p < .001); the main effect for dosage was significant (F?,158 = 28.32, p < .001), and the interaction was significant (P10,158 = 2.63, p < .01). The significant effect due to dosage was caused by a decrease in preference for saccharin as dosage increased. The significant effect due to days of treatment was due to reliable differences between the three means: 48% for the 1-day rats, 37% for the 6-day rats, and 50% for the 12-day rats. Duncan's multiple-range test was applied to these three means. The 1- and 12-day rats did not differ significantly from each other, but both groups differed from the 6-day rats at the .O1 level. The significant interaction was apparently caused by the relatively greater decrease in preference for saccharin over different dosages for the 6-day groups and the relatively smaller decrease in preference over differenc dosages for the 12-day groups. Duncan's multiple-range test was applied to the individual group means to determine, within a particular level of days of treatment, which levels of drug dose led to a difference from the corresponding control group, e.g., whether the 2.0-6 rats differed from the 0.0-6 rats. With the exception of the .25-1, .5-1 and .25-12 groups, all groups differed from their corresponding control groups at the .O1 level.

DISCUSSION The data for the 1-day groups are in excellent agreement with the data of Elsmore and Fletcher. Point by point, the two sets of data are almost identical. In general, the effect of multiple pairings of THC with saccharin is similar to that of one pairing. The greater the dosage of THC the greater is the acquired aversiveness of saccharin. Any dosage in excess of .5 mg/kg is clearly aversive under any of the three conditions. While a stronger aversion is developed with six acquisition days as compared to one day, the aversion is reduced with 12 acquisition days. This last result is suggestive of a mild tolerance effect. The

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rats may become used to the ill effects of THC with repeated pairings. This suggestion must be treated quite cautiously because of a bias in the data. The data from two rats in the 2.0-12 group and one rat in the 4.0-12 group were lost because the rats refused to drink saccharin and were withdrawn from the experiment. Presumably, if they had survived, they would have shown a very low preference for saccharin during testing. Thus, the data for the 2.0-12 and 4.0-12 groups were chosen in a biased manner. This bias can produce a significant interaction. The I-, 6-, and 12-day rats have different dosage-dependent saccharin preference curves. Nevertheless, in all three cases T H C was shown to have aversive properties. Thus, most behavioral studies of the effect of T H C on rats, whether using one or more daily injections of THC, have been studies, in part, of the aversive properties of THC. REFERENCES C O R C O ~M. N , E. Role of drug novelty and metabolism in the aversive effects of hashish ~njections in rats.

ELSMORE, T. F.,

&

Life Sciences, 1973, 12, 63-72.

FLETCHER, G. V. A'-tetrahydrocannabinol: aversive effects in rats

high doses. Science, 1972, 175, 911-912. & NELSON, J. M. Clinical and psychological effects of marijuana in man. Science, 1968, 162, 1234-1242. WINER,B. J. Stalistical principles in experimental design. New York: McGraw-Hill, 1962. at

WEIL,A. T., ZINBERG, W. E.,

Accepted September 4, 1975.

Aversive effects of repeated injections of THC in rats.

Psychological Reportj, 1975, 37, 1051-1054. @ Psycho!ogical Reporrs 1975 AVERSIVE EFFECTS OF REPEATED INJECTIONS OF THC I N RATS1 E D W I N J. KAY L...
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