Time Course for the Induction and Maintenance of Tolerance to D9-Tetrahydrocannabinol in Mice
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Drug and Alcohol Dependence 60 (2000) 113–119 www.elsevier.com/locate/drugalcdep Time course for the induction and maintenance of tolerance to D9-tetrahydrocannabinol in mice Caroline E. Bass, Billy R. Martin * Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth Uni6ersity, P.O. Box 980613, Richmond, VA 23298, USA Received 4 August 1999; accepted 9 November 1999 Abstract The time course for the development of tolerance to delta-9-tetrahydrocannabinol (D9-THC) was studied in an effort to determine the role that length of dosing may have in the onset and maintenance of tolerance. Mice were chronically treated with either vehicle or 10 mg/kg of D9-THC subcutaneously twice a day. The mice were tested 24 h after the last injection for tolerance as assessed by the production of antinociception and suppression of spontaneous activity. Tolerance was first observed after three injections of D9-THC (1.5 days) resulting in a 7-fold and 23-fold decrease in potency for the measures of antinociception and hypoactivity, respectively. Seven injections of D9-THC (3.5 days of dosing) resulted in a 12-fold and 36-fold decrease in potency, respectively, while 13 injections of D9-THC (6.5 days of dosing) produced a 6.2-fold and 9.8-fold degree of tolerance. The time course for the recovery from D9-THC-induced tolerance was also determined with a separate group of animals. Mice were dosed for 6.5 days with 10 mg/kg of D9-THC and were not tested until 4.5, 7.5, and 11.5 days after cessation of drug treatment. After 4.5 days without drug treatment the mice exhibited a 7.5-fold and 2.3-fold degree of tolerance as measured by antinociception and hypoactivity, respectively. After 7.5 days without drug treatment a 3.4-fold degree of tolerance remained for the measure of antinociception, while no tolerance was detected for the measure of hypoactivity. No tolerance was observed for the measure of antinociception after 11.5 days without drug treatment. This time course indicates that the mechanisms responsible for either the production or maintenance of tolerance differ between the measures of antinociception and suppression of spontaneous activity. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Delta-9-tetrahydrocannabinol; D9-THC ; Cannabinoid; Tolerance; Time course 1. Introduction ligands, anandamide (Devane et al., 1992) and 2-arachi- donyl glycerol (Mechoulam et al., 1995), and the devel- 9 Delta-9-tetrahydrocannabinol (D -THC) is the major opment of a CB1 receptor antagonist (Rinaldi-Carmona psychoactive component of marijuana (Mechoulam et et al., 1994) have greatly advanced the understanding of al., 1970). The effects of this compound have been how these receptors produce the numerous effects of D9 under scrutiny for years, however, the molecular mech- -THC. Some of the basic roles of the CB1 receptor anisms underlying these events have only recently been and it’s subsequent signal transduction cascades have identified. This is primarily due to the discovery of two been elucidated, including the linkage of this receptor cannabinoid receptors designated CB1 (Matsuda et al., to inhibition of adenylyl cyclase (Howlett, 1984) and to 1990) and CB2 (Munro et al., 1993). The discovery of the MAP kinase system (Bouaboula et al., 1995). CB1 these receptors as well as two putative endogenous receptors play a role in pain modulation as evidenced by their ability to initiate the release of endogenous opioids (Pugh et al., 1996). Recent endeavors have focused on understanding the * Corresponding author. Tel.: +1-804-828-8407; fax: +1-804-828- 2117. regulation of the CB1 receptor in an effort to more E-mail address: [email protected] (B.R. Martin). clearly define the role of the cannabinoid system in the 114 C.E. Bass, B.R. Martin / Drug and Alcohol Dependence 60 (2000) 113–119 central nervous system. The ability of D9-THC to read- 2.2. Drug preparation and chronic administration ily produce tolerance that is easily measured (McMillan et al., 1971) has led to the use of this phenomena as a D9-THC was dissolved in a 1:1:18 solution of tool to study the biology of the cannabinoid receptor. ethanol, emulphor and saline, respectively. The mice This method is ideal for studying the regulation of the were injected subcutaneously once in the morning and cannabinoid receptor as it initiates natural regulatory once in the afternoon (approximately 09:00 and 16:00 processes of endogenous cannabinoid systems in an h) for varying lengths of time to establish the time intact animal. It has been known for some time that course of D9-THC-induced tolerance. On the last day of tolerance to D9-THC occurs in numerous species, in- dosing, the mice received a morning injection only. cluding rodents (Carlini, 1968), monkeys (Sassenrath Twenty-four hours later the mice were challenged with and Chapman, 1976), pigeons (McMillan et al., 1970), a single i.v. dose of either D9-THC or vehicle in the tail and dogs (Kaymakcalan et al., 1974). These studies vein followed by behavioral assays to assess tolerance. demonstrated tolerance to D9-THC through the use of a Dose–response curves were generated and the resultant variety of protocols. However, the data generated from ED50 values and potency ratios were calculated. Ani- many D9-THC tolerance studies have often been confl- mals were treated for either 1, 3, or 6 days of full icting and ambiguous. This may be due in part to the morning and afternoon dosing followed by one morn- manner in which tolerance was produced. The proto- ing injection. These regimens are represented as days cols often varied in the dose of D9-THC, the route of 1.5, 3.5, and 6.5, respectively. Day 0.5 represents one administration, and the number and timing of adminis- dose in the morning only followed by testing 24 h later. trations. One important facet of the different dosing The time course by which D9-THC-induced tolerance protocols, and a possible source for descrepancies subsides was assessed by dosing for 6.5 days followed among studies, involves the length of exposure to D9- by 4.5, 7.5, or 11.5 days without drug treatment. The THC during tolerance production. mice were challenged after this recovery period with an In order to ascertain the effect that length of expo- i.v. injection of D9-THC in the tail vein. Again, dose– D9 sure to -THC may have on the production and response curves, ED50 values, and potency ratios were degree of tolerance generated, we sought to determine generated. the time course through which D9-THC induces toler- ance. This effort will not only allow the quantification 2.3. Beha6ioral e6aluations of the degree of tolerance to D9-THC but will also help determine when the onset of tolerance occurs as well as All animals were allowed to acclimate to the observa- when full tolerance is reached. The time required for tion room overnight. Twenty-four hours after the last D9-THC-induced tolerance to abate was also deter- injection the mice in both drug- and vehicle-treated mined. The mouse was chosen as an appropriate model groups received a challenge dose of D9-THC and were for these experiments due to their ease of maintenance evaluated for hypomotility and antinociception. The and the ability of D9-THC to produce a distinctive 24-h time period was chosen in an effort to allow the profile of effects. This profile includes antinociception, animal to clear any residual drug from the last injec- hypomotility, catalepsy, and hypothermia (Martin et tion. Antinociception was determined using the tail- al., 1991). Initial studies in this laboratory have deter- flick reaction time to a heat stimulus (Dewey et al., mined that D9-THC administered twice a day for 6.5 1970). The baseline latency period (2–4 s) was first days produces a robust and reliable tolerance to these determined prior to i.v. administration of vehicle or four measures in mice (Abood et al., 1993; Fan et al., drug. The mice were then administered D9-THC 1994). This established protocol was modified to deter- through tail vein injection and placed in individual mine the time course of D9-THC-induced tolerance for photocell activity chambers 5 min later. Spontaneous this study. activity was monitored for 10 min in a Digiscan Animal Activity Monitor (Omnitech Electronic Inc., Columbus, OH) as measured by the number of interruptions of 16 2. Materials and methods photocell beams per chamber. The mice were then assessed at 20 min post-injection for tail-flick latency 2.1. Subjects and the difference in control and test latencies were calculated. A 10-s maximum latency was used in order Male ICR mice (Harlan Laboratories, Indianapolis, to avoid tail injury. IN) weighing between 24 and 30 g were used in all experiments. Mice were maintained on a 14:10 h light/ 2.4. Data analysis dark cycle with food and water available ad lib. D9- THC was obtained from the National Institute on Drug Suppression of spontaneous activity was expressed as Abuse (Bethesda, MD). the percentage of the activity of mice treated repeti- C.E. Bass, B.R. Martin / Drug and Alcohol Dependence 60 (2000) 113–119 115 tively with vehicle and challenged with vehicle. Anti- nociception was expressed as percent of maximum pos- sible effect (% MPE=[(test latency−control latency)/(10 s−control latency)]×100). Dose–re- sponse curves were generated based upon increasing D9 -THC challenge doses in groups of 6–12 mice. ED50 values were calculated based upon least squares linear regression followed by calculation of 95% confidence limits (Bliss, 1967).