Neurochemical effects of KMO inhibition in rats

We tested whether systemic administration of the KMO inhibitor Ro 61-8048 would increase levels of KYNA in two brain regions implicated in rewarding effects of cannabinoids: the NAc shell and VTA. In vivo microdialysis experiments in freely moving rats showed that systemic administration of 30 and 100 mg per kilogram body weight intraperitoneally (i.p.) of Ro 61-8048 increased extracellular KYNA in the NAc shell by ∼150% and ∼225%, respectively (Fig. 1a; 30 mg per kg: F 11,55 = 28.59, P < 0.001; 100 mg per kg: F 11,55 = 15.03, P < 0.001). In the VTA, the 30 and 100 mg per kg doses of Ro 61-8048 elevated KYNA by ∼50% and ∼200%, respectively (Fig. 1b; 30 mg per kg: F 11,55 = 5.85, P < 0.001; 100 mg per kg: F 11,55 = 24.18, P < 0.001). Peak KYNA levels in both NAc and VTA were observed 80 min after injection of 100 mg per kg Ro 61-8048 and 140 min or later after injection of 30 mg per kg Ro 61-8048.

Figure 1: Effects of treatment with Ro 61-8048 on the extracellular concentration of kynurenic acid (KYNA) in NAc shell and VTA of freely moving rats. (a,b) Ro 61-8048 (30 and 100 mg per kg i.p.) significantly increased extracellular KYNA in the NAc shell (a; basal concentrations, 2.29 ± 0.18 and 1.51 ± 0.1 nM, respectively) and VTA (b; basal concentrations, 1.61 ± 0.1 and 1.29 ± 0.1 nM, respectively). KYNA reached peak concentrations 80 min after the injection of 100 mg per kg Ro 61-8048 in both brain areas. Arrows indicate time of Ro 61-8048 injection. Results are expressed as mean (± s.e.m.) percentage of basal values (n = 6 rats per group). *P < 0.05, **P < 0.01, ***P < 0.001, post hoc versus basal values. Full size image Download Excel source data

We then determined whether systemic administration of Ro 61-8048 would block THC-induced elevations of dopamine in the NAc shell and VTA in rats. In the NAc shell, THC (3 mg per kg, i.p.) significantly increased extracellular dopamine (Fig. 2a,b; treatment × time interaction, F 30,218 = 1.99, P < 0.003; area under the curve (AUC), F 3,22 = 6.06, P = 0.0036), but pretreatment with 30 or 100 mg per kg of Ro 61-8048 dose-dependently blocked this effect of THC (Fig. 2a,b). We saw similar effects in the VTA, where THC (3 mg per kg) also increased extracellular dopamine significantly (Fig. 2c,d; treatment × time interaction, F 30,188 = 4.25, P < 0.0001; AUC, F 3,19 = 22.01, P < 0.001), and pretreatment with either 30 or 100 mg per kg of Ro 61-8048 significantly reduced this effect of THC (Fig. 2c,d). When given alone, Ro 61-8048 (100 mg per kg) did not significantly affect dopamine levels in either the NAc (Fig. 2a; P = 0.99) or the VTA (Fig. 2c; P = 0.93).

Figure 2: Effects of elevated brain KYNA on THC-induced elevations of extracellular dopamine (that is, dopamine overflow) in NAc shell and VTA of freely moving rats. (a–d) THC (3 mg per kg) increased dopamine as compared to basal levels in both NAc shell (a; average basal concentrations, 37.60 ± 2.70 fmol per 10 μl; n = 7 rats) and VTA (c; average basal concentration, 26.18 ± 3.80 fmol per 10 μl; n = 7). The effects of THC on dopamine in NAc shell (a,b) were significantly attenuated by pretreatment with 100 mg per kg Ro 61-8048 (Ro; n = 6), and the effects of THC on dopamine in VTA (c,d) were significantly attenuated by pretreatment with 30 (n = 5) or 100 mg per kg Ro 61-8048 (n = 6). Ro 61-8048 alone (both n = 5) did not affect extracellular levels of dopamine in either area. (e,f) Local infusion of KYNA (500 nM) (n = 5) into NAc shell significantly reduced THC-induced dopamine elevations in NAc shell (e,f; n = 5). Arrows indicate time of THC, Ro 61-8048 or vehicle injection or local infusion of KYNA. Data are presented over the course of the session as a percentage of basal values (a,c,e) or as AUC during the first 120 min after THC or vehicle injection, expressed as a percentage of the mean value in the vehicle + THC group (b,d,f). Results are expressed as means ± s.e.m. *P < 0.05 versus baseline, #P < 0.05 versus vehicle + THC. Dosages are given in mg per kg; Veh, vehicle. Full size image Download Excel source data

This finding that systemic administration of Ro 61-8048 blocked the effects of THC on dopamine in reward-related brain areas, coupled with the finding that Ro 61-8048 increased KYNA in these areas, led us to determine whether the effects of THC on dopamine could be blocked by infusing KYNA directly into the NAc shell. We observed that THC (3 mg per kg, i.p.) significantly increased extracellular dopamine in the NAc shell of freely moving rats when the local tissue was continuously infused with vehicle, but not when the tissue was continuously infused with KYNA (500 nM) (Fig. 2e,f: treatment × time interaction, F 14, 91 = 3.61, P < 0.0001; AUC: F 2,13 = 13.64, P = 0.0006). In the absence of THC, local infusion of KYNA (500 nM) into the NAc shell had no effect on dopamine levels (Fig. 2e,f).

To verify that the ability of Ro 61-8048 to block THC-induced dopamine elevations in the NAc shell was due its actions at α7nAChRs, we reversed the effects of Ro 61-8048 with galantamine and PNU120596, both agonists at the allosteric potentiating site of α7nAChRs where KYNA acts23,42. In these two experiments (one with galantamine and one with PNU120596), we again found that systemic Ro 61-8048 (100 mg per kg, i.p.) significantly decreased the ability of THC (3 mg per kg, i.p.) to raise dopamine in the NAc shell (replicating the effect seen in Fig. 2a,b), reducing the AUC by about 60 to 70% (Fig. 3a; F 5,27 = 8.34, P < 0.001; Fig. 3b; F 4,17 = 9.87, P < 0.0003). Furthermore, we observed that pretreatment with galantamine (Fig. 3a; 3 mg per kg, i.p.) or PNU120596 (Fig. 3b; 1 mg per kg, i.p.) reversed this effect of Ro 61-8048. Neither galantamine nor PNU120596 altered dopamine levels when given alone, nor did they alter the effects of THC (Fig. 3a,b). Thus, we confirmed that α7nAChRs were involved in the ability of Ro 61-8048 to block THC-induced dopamine elevations in rats.

Figure 3: Prevention of the neurochemical effects of Ro 61-8048 by two agonists at the allosteric potentiating site of the α7nAChR, galantamine and PNU120596. (a,b) THC administration (3 mg per kg i.p.; a, n = 7 rats; b, n = 4) elevated extracellular dopamine (that is, dopamine overflow) in NAc shell (average basal concentration, 39.69 ± 5.05 fmol per 10 μl) of freely moving rats. These THC-induced increases in dopamine were blocked by Ro 61-8048 (100 mg per kg, i.p., 40 min before THC; a, n = 6; b, n = 5), and this blockade was prevented by pretreatment with galantamine (a; 3 mg per kg, i.p., 60 min before THC; n = 5) or PNU120596 (b; 1 mg per kg, i.p., 60 min before THC; n = 4). Galantamine (n = 5), PNU120596 (n = 4) or Ro 61-8048 (n = 5) alone did not increase dopamine, and neither galantamine nor PNU120596 (both n = 5) affected THC-induced dopamine increases. Dopamine levels are expressed as AUC (relative to the mean value in the vehicles + THC 3 mg per kg condition) over the 180 min following THC or vehicle injection. Bars represent means ± s.e.m. *P < 0.05, **P < 0.01, post hoc versus vehicles + THC 3 mg per kg; Dunnett's test. Dosages are given in mg per kg; values of 0 represent vehicle. Full size image Download Excel source data

To determine whether treatment with Ro 61-8048 alters the effects of cannabinoid CB 1 receptor agonists other than THC, we studied dopamine elevations induced by the synthetic agonist WIN 55,212-2. Like THC, WIN 55,212-2 (0.3 mg per kg intravenously (i.v.)) significantly increased extracellular dopamine in the NAc shell (Fig. 4a; treatment × time interaction, F 28,98 = 3.28, P < 0.0001). Although Ro 61-8048 (30 or 100 mg per kg) alone did not affect dopamine levels (Fig. 4b; both P values = 0.99), pretreatment with 30 or 100 mg per kg of Ro 61-8048 significantly reduced the ability of WIN 55,212-2 to increase dopamine in the NAc shell (Fig. 4a,c; AUC: F 4,14 = 3.73, P = 0.0288).

Figure 4: Effects of treatment with Ro 61-8048 on abuse-related effects of the synthetic CB 1 agonist WIN 55,212-2 in rats. (a,b) Ro 61-8048 (Ro; 30 or 100 mg per kg) reduced the increases in extracellular dopamine levels produced by WIN 55,212-2 (WIN; 0.3 mg per kg) in NAc shell (a). Ro 61-8048 alone did not significantly affect dopamine levels in NAc shell (b). Results expressed as a percentage of basal values over time (n = 4 rats in all groups). Arrows indicate time of injection. (c) Dopamine levels expressed as AUC (relative to mean value in vehicle (Veh) + WIN condition) over the 120 min following WIN or vehicle injection. *P < 0.05 versus baseline, #P < 0.05 versus Veh + WIN, simultaneous confidence intervals. (d,e) Presses on active and inactive levers per session (n = 5). Ro 61-8048 (sessions T1–T3), but not its vehicle (sessions B1–B3; recovery sessions R1 and R2), dose-dependently decreased the number of WIN injections (12.5 μg per kg; 1 injection per active-lever response) self-administered over 2-h sessions. *P < 0.05,**P < 0.01, post hoc versus average of sessions B1–B3, Tukey test. (f) Presses on active and inactive levers per session. Ro 61-8048 (100 mg per kg) blocked reinstatement of extinguished drug-seeking induced by injection of WIN (0.3 mg per kg) (n = 5). **P < 0.01, post hoc versus vehicles; ##P < 0.01, post hoc versus vehicle + WIN 0.3 mg per kg condition, Tukey test. All points or bars represent means ± s.e.m. Dosages are given in mg per kg; Veh or values of 0 represent vehicle. Full size image Download Excel source data

Behavioral effects of KMO inhibition in rats

Having determined that the KMO inhibitor Ro 61-8048 could block the effects of cannabinoid CB 1 agonists in reward-related brain areas, we tested the effects of this treatment in behavioral models of cannabinoid abuse. We first turned to a rodent model of cannabinoid reinforcement in which rats intravenously self-administer WIN 55,212-2 (12.5 μg per kg per injection). This synthetic cannabinoid had a clear reinforcing effect, causing rats to respond significantly more on the lever that delivered the drug than on an inactive control lever (Fig. 4d,e; F 1,4 = 23.95, P = 0.008). Treatment with 30 or 100 mg per kg Ro 61-8048 40 min before each session significantly decreased self-administration of WIN 55,212-2 (Fig. 4d, 30 mg per kg: F 3,12 = 19.5, P < 0.001; Fig. 4e, 100 mg per kg: F 3,12 = 6.92, P = 0.006). The 100 mg per kg dose of Ro 61-8048 decreased self-administration responses (on the active lever) over all 3 d of testing, but it also significantly affected responses on the inactive lever (Fig. 4e; F 3,12 = 18.35, P < 0.001). This effect on inactive-lever responses was not seen with 30 mg per kg Ro 61-8048 (Fig. 4d; F 3,12 = 1.76, P = 0.21), yet this lower dose was effective in blocking the self-administration of WIN 55,212-2. Self-administration quickly recovered to baseline levels when Ro 61-8048 treatment was discontinued.

As relapse to drug use after long periods of abstinence represents one of the greatest challenges for the treatment of addiction, we also investigated whether Ro 61-8048 would block reinstatement of drug seeking by abstinent rats in an animal model of relapse. When WIN 55,212-2 delivery was discontinued, rats' drug-seeking behavior decreased to low levels (Fig. 4f). A noncontingent priming injection of WIN 55,212-2 (0.3 mg per kg., i.p., 10 min before the session) reinstated drug-seeking behavior, but this relapse-like effect was completely blocked by pretreatment with 100 mg per kg Ro 61-8048 (Fig. 4f; F 5,20 = 231.13, P < 0.001). None of these treatments significantly affected responses on the inactive lever (Fig. 4f; both treatment P values = 0.51). Thus, Ro 61-8048 prevented the relapse-like effect induced by reexposure to cannabinoids in rats.

KMO inhibition and THC reward in squirrel monkeys

Because THC self-administration in squirrel monkeys provides the most congruent animal model of human cannabinoid abuse31, we used this model to examine the effects of Ro 61-8048. We also tested the effects of Ro 61-8048 in monkeys trained to self-administer food and cocaine under the same schedule of reinforcement (fixed-ratio 10) to determine whether the effects of Ro 61-8048 are specific to cannabinoid reward.

At the peak of the THC self-administration dose-effect curve (4 μg per kg per injection THC; Fig. 5a), squirrel monkeys self-administered an average of 50.80 ± 1.90 injections per session and lever-pressed at an average rate of 1.20 ± 0.25 responses per second in the presence of a green light signaling THC availability. We investigated self-administration of this THC dose in monkeys across 3 consecutive days of treatment with Ro 61-8048 (10 or 20 mg per kg, 40 min before each session). Ro 61-8048 was always administered intramuscularly (i.m.) in monkeys. Ro 61-8048 significantly and dose-dependently reduced THC self-administration during all three sessions (Fig. 5a; 10 mg per kg Ro 61-8048: F 3,12 = 4.07, P = 0.033; 20 mg per kg Ro 61-8048: F 3,12 = 30.93, P < 0.001). Self-administration behavior returned to baseline levels when Ro 61-8048 treatment ended.

Figure 5: Effects of Ro 61-8048 on THC, food and cocaine self-administration in squirrel monkeys. (a–f) Ro 61-8048 (10 and 20 mg per kg, i.m.) significantly decreased the number of THC injections self-administered during 1-h sessions (a) and decreased overall response rates (d) by squirrel monkeys under a fixed-ratio 10 schedule at a THC dose of 4 μg per kg per injection (n = 5 monkeys, means ± s.e.m.). Ro 61-8048 did not significantly affect food-reinforced behavior (b,e) or cocaine (30 μg per kg per injection) self-administration behavior (c,f) in monkeys under conditions that paralleled the THC self-administration procedure (n = 4 for food, n = 3 for cocaine, means ± s.e.m.). The Ro 61-8048 vehicle was given 40 min before each baseline session. (g–i) Number of THC injections per session (g), overall response rates in the presence of the green light signaling THC availability (h) and total THC intake per session (i) as a function of the THC dose. Each data point represents the mean ± s.e.m. of the last three sessions under each THC condition and under vehicle conditions (n = 3–5 monkeys). Pretreatment with Ro 61-8048 (20 mg per kg) caused a rightward shift of the THC dose-response curves compared to vehicle pretreatment. *P < 0.05, **P < 0.01, post hoc versus the last session with vehicle pretreatment (session 3) (a–f) or vehicle conditions (g,h), Bonferroni test. “V” represents THC vehicle. Full size image Download Excel source data

In the food self-administration model (Fig. 5b), monkeys self-administered 53.46 ± 1.29 food pellets per session on average, with a response rate of 1.97 ± 0.66 responses per second in the presence of a green light signaling food availability. During three daily sessions with Ro 61-8048 pretreatment, food-reinforced response in monkeys was not affected by either 10 or 20 mg per kg of Ro 61-8048, under testing conditions that paralleled those used to evaluate THC self-administration (Fig. 5b; 10 mg per kg Ro 61-8048: F 3,9 = 1.76, P = 0.22; 20 mg per kg Ro 61-8048: F 3,9 = 1.77, P = 0.22). Thus, Ro 61-8048 did not produce a nonspecific disruption of behavior.

Moreover, Ro 61-8048 (20 mg per kg, 40 min before the session) reversed the disruptive effects of THC (0.56 mg per kg i.v., immediately before the session) on food-maintained self-administration behavior (Supplementary Fig. 1a: pellets per session F 3,6 = 23.29, P = 0.001; post hoc analysis, THC 0.56 mg per kg versus Ro 61-8048 20 mg per kg + THC 0.56 mg per kg, P = 0.003; Supplementary Fig. 1b: response rate F 3,6 = 15.37, P = 0.003; post hoc analysis, THC 0.56 mg per kg versus Ro 61-8048 20 mg per kg + THC 0.56 mg per kg, P = 0.018). Treatment with 0.56 mg per kg THC alone significantly reduced both the food-maintained response rate (from 0.90 ± 0.17 to 0.09 ± 0.03 responses per second) and the number of food pellets per session (from 49.5 ± 1.76 to 19.00 ± 4.51) as compared to values observed after vehicle treatment (post hoc analysis, vehicle versus THC 0.56 mg per kg: response rate, P = 0.003; pellets per session, P = 0.002). Pretreatment with Ro 61-8048 had no effect by itself (post hoc analysis, vehicle versus Ro 61-8048 20 mg per kg: rate. P = 0.28; pellets. P = 0.93), but it completely reversed the effects of THC on response rate (to 0.62 ± 0.08 responses per second; post hoc analysis, vehicle versus Ro 61-8048 20 mg per kg + THC 0.56 mg per kg, P = 0.22) and food intake (to 46.67 ± 1.45 pellets per session; post hoc analysis, P = 0.91).

In monkeys trained to self-administer cocaine (Fig. 5c), the dose of Ro 61-8048 (20 mg per kg) that effectively decreased THC self-administration did not alter cocaine self-administration (30 μg per kg per injection). Monkeys averaged 44.56 ± 0.62 injections per session with a mean response rate of 0.54 ± 0.04 responses per second in the presence of a green light signaling cocaine availability. Pretreatment with Ro 61-8048 40 min before each of three daily sessions did not significantly affect cocaine self-administration during those sessions (Fig. 5c; F 3,6 = 1.33, P = 0.35). Lever response rates were significantly affected during Ro 61-8048 treatment only in the group self-administering THC (Fig. 5d; 10 mg per kg Ro 61-8048: F 3,12 = 9.82, P = 0.001; 20 mg per kg Ro 61-8048: F 3,12 = 15.92, P < 0.001). In monkeys self-administering food or cocaine, response rates were not affected by treatment with Ro 61-8048 (Fig. 5e, food self-administration: 10 mg per kg Ro 61-8048, F 3,9 = 1.1, P = 0.40; 20 mg per kg Ro 61-8048, F 3,9 = 1.61, P = 0.26; Fig. 5f, cocaine self-administration: F 3,6 = 0.69, P = 0.59).

To further characterize the nature of the effects of Ro 61-8048 on THC self-administration, we varied the dose of THC and obtained classic inverted U-shaped dose-effect curves (Fig. 5g,h). THC maintained significantly more self-injections (Fig. 5g; F 6,22 = 29.34, P < 0.001) than vehicle at 0.5, 1, 2, 4 and 8 μg per kg per injection and significantly higher response rates (Fig. 5h; F 6,22 = 36.76, P < 0.001) than vehicle at 4 μg per kg per injection. We found that pretreatment with 20 mg per kg of Ro 61-8048 significantly shifted the THC dose-response curve for injections per session down and to the right (Fig. 5g; interaction of THC and Ro 61-8048, F 5,17 = 35.45, P < 0.001), consistent with a decrease in THC's rewarding effects. This Ro 61-8048 dose also produced a significant downward and rightward shift for response rates (Fig. 5h; interaction of THC and Ro 61-8048, F 5,17 = 16.10, P < 0.001). Post hoc pairwise comparisons revealed significant differences in the effects of 1, 2, 4 and 16 μg THC per kg per injection after Ro 61-8048 pretreatment on the number of self-administered injections per session (all P < 0.001) and significant differences in effects of 2, 4 and 16 μg per kg THC per injection after Ro 61-8048 pretreatment on response rates (2 μg per kg, P = 0.04; 4 μg per kg, P < 0.001; 16 μg per kg, P = 0.012). The total amount of THC received during the session was significantly decreased by Ro 61-8048 across most of the dose-effect function (Fig. 5i; F 5,17 = 59.4, P < 0.001), but it increased at the highest dose per injection.

We then asked whether positive allosteric modulators of α7nAChRs (galantamine and PNU120596) would prevent the effects of Ro 61-8048 on THC self-administration in monkeys. Galantamine (0.3–3 mg per kg, i.m.) dose-dependently prevented the effects of Ro 61-8048 (20 mg per kg, i.m.) on THC self-administration in monkeys (Fig. 6a; F 4,12 = 36.48, P < 0.0001), but galantamine alone (0.3–3 mg per kg, i.m.) had no significant effect (Fig. 6b; F 3,9 = 3.41, P = 0.067). Like galantamine, PNU120596 (0.3–3 mg per kg, i.m.) also dose-dependently prevented the effects of Ro 61-8048 (20 mg per kg, i.m.) on THC self-administration in monkeys (Fig. 6c; F 4,12 = 35.71, P < 0.0001) but had no significant effect when given alone (Fig. 6d: F 3,9 = 1.98, P = 0.19). Thus, we confirmed that α7nAChRs were involved in the ability of Ro 61-8048 to block the reinforcing effects of THC in nonhuman primates.

Figure 6: Reversal of behavioral effects of Ro 61-8048 by positive allosteric modulators of α7nAChRs. (a–d) Galantamine (a) or PNU120596 (c) dose-dependently reversed the blockade of THC (4 μg per kg per injection) self-administration caused by pretreatment with Ro 61-8048 (20 mg per kg). Galantamine (b) and PNU120596 (d) alone had no significant effect. **P < 0.01, post hoc versus vehicles + THC 4 μg per kg; #P < 0.05, ##P < 0.01, post hoc versus vehicle + Ro 61-8048 20 mg per kg + THC 4 μg per kg, Tukey test. (e,f) Treatment with Ro 61-8048 blocked the reinstatement of extinguished THC-seeking responses produced by a priming injection of THC (40 μg per kg, i.v.), and this effect was prevented by pretreatment with galantamine (e) or PNU120596 (f). *P < 0.05, **P < 0.01, post hoc versus vehicles; ##P < 0.01, post hoc versus vehicles + THC 40 μg per kg, $$P < 0.01, post hoc versus vehicle + Ro 61-8048 20 mg per kg + THC 40 μg per kg, Tukey test. (g,h) Treatment with Ro 61-8048 also blocked the reinstatement of extinguished THC-seeking responses induced by reintroduction of cues previously associated with THC, and this effect of Ro 61-8048 was reversed by pretreatment with galantamine (g) or PNU120596 (h). **P < 0.01, post hoc versus vehicles + no cues; ##P < 0.01, post hoc versus vehicles + cues; $P < 0.05, $$P < 0.01, post hoc versus vehicle + Ro 61-8048 20 mg per kg + cues, Tukey test. n = 4 monkeys for all conditions, except n = 3 in h. Bars represent means ± s.e.m. “0” represents vehicle in all panels. THC dosages expressed in μg per kg per injection; all other dosages expressed in mg per kg. Full size image Download Excel source data

KMO inhibition and relapse in squirrel monkeys

To further study the effects of KMO inhibition in animal models of relapse to THC seeking, we determined whether Ro 61-8048 blocked reinstatement induced by reexposure to THC or THC-associated cues in squirrel monkeys and whether α7nAChRs were involved in this blockade. When lever-press responses for THC had been extinguished by discontinuing THC delivery, administration of a noncontingent priming injection of THC (40 μg per kg, i.v.) before the session reinstated drug-seeking (Fig. 6e, F 5,14 = 34.37, P < 0.001; Fig. 6f, F 5,13 = 77.81, P < 0.001). Treatment with Ro 61-8048 (20 mg per kg) blocked this THC-induced reinstatement (Fig. 6e,f; both P < 0.001 versus THC), and pretreatment with either galantamine (3 mg per kg) or PNU120596 (1 mg per kg) prevented this blockade (Fig. 6e,f). Ro 61-8048 alone did not reinstate drug-seeking behavior (Fig. 6e,f). Both galantamine and PNU120596 produced a low level of reinstatement of drug-seeking behavior (Fig. 6e, P = 0.033 versus vehicles; Fig. 6g, P = 0.012 versus vehicles), but this effect was significantly smaller than the reinstatement produced by a priming injection of THC (Fig. 6e, P < 0.001 versus THC; Fig. 6f, P < 0.001 versus THC).

Because relapse can be triggered by reexposure to drug-related environmental cues, we looked at cue-induced reinstatement of THC seeking. When both THC delivery and presentation of cues signaling delivery of THC were discontinued, THC seeking by the monkeys decreased to very low levels (Fig. 6g,h). When visual cue presentation was restored and i.v. vehicle was delivered contingent on lever response, THC-seeking behavior was reinstated (Fig. 6g, F 5,13 = 21.16, P < 0.001; Fig. 6h, F 5,10 = 57.87, P < 0.001). This cue-induced reinstatement was significantly decreased by Ro 61-8048 (20 mg per kg) (Fig. 6g,h). Pretreatment with either galantamine (3 mg per kg) or PNU120596 (1 mg per kg) prevented these effects of Ro 61-8048 (Fig. 6g,h). When Ro 61-8048, galantamine or PNU120596 were given with the THC vehicle without presentation of cues, THC-seeking behavior was not reinstated (Fig. 6g, post hoc analysis versus vehicle + no cues: Ro 61-8048, P = 0.95; galantamine, P = 1.00; Fig. 6h: Ro 61-8048, P = 0.79; PNU120596, P = 0.69). These results suggest that treatment with a KMO inhibitor could prevent relapse caused by reexposure to THC or to THC-associated cues, and that this effect of KMO inhibition occurs through an α7nAChR-mediated mechanism.

Effects of KMO inhibition and THC on working memory

Because excessive levels of KYNA may be associated with cognitive impairment43,44 and because THC is well known to impair memory, we tested the effects of Ro 61-8048 on working memory in rats trained with a delayed nonmatching-to-position procedure and in squirrel monkeys trained with a delayed matching-to-sample procedure. In rats, THC (3 or 5.6 mg per kg) and Ro 61-8048 (100 mg per kg) were administered alone and in combination. Ro 61-8048 had no effect on memory when given alone (Fig. 7a), but THC decreased accuracy in a delay-dependent manner, consistent with a selective impairment of working memory (Fig. 7b). Ro 61-8048 did not alter the effects of THC (Fig. 7c,d). The main effects of THC (F 2,13 = 13.56, P < 0.0001) and delay (F 4,28 = 47.34, P < 0.0001) were both significant, but the effects of Ro 61-8048 were not (P = 0.85). Paired comparisons indicated that both doses of THC significantly impaired working memory (P < 0.0035).

Figure 7: Effects of Ro 61-8048 and THC on working memory in rats and squirrel monkeys. (a–d) The 100 mg per kg dose of Ro 61-8048, which was effective in blocking the effects of THC in reward-related brain areas in rats, did not have deleterious effects on short-term memory in rats when given alone (a) or in combination with THC (3 or 5.6 mg per kg, i.p.; c,d) in a delayed nonmatching-to-position model of working memory. Both doses of THC significantly decreased accuracy (b; P < 0.007), but this was not exacerbated by Ro 61-8048 (c,d). (e,f) The 20 mg per kg dose of Ro 61-8048, which was effective in blocking the effects of THC in reward-related brain areas in monkeys, did not have deleterious effects on short-term memory in monkeys when given alone (e) or in combination with THC (f) in a delayed matching-to-sample model of working memory. THC (0.1 mg per kg, i.m.) significantly decreased accuracy (f), and this was reversed by Ro 61-8048 (f). Accuracy (percentage of trials with a correct response) is shown (means ± s.e.m.; n = 8 rats, n = 3 monkeys) as a function of delay and of drug treatment. Dosages are given in mg per kg. Full size image Download Excel source data

In squirrel monkeys, working memory was also impaired by THC alone (0.1 mg per kg) but not by Ro 61-8048 (20 mg per kg) alone (Fig. 7e,f). THC at short delay values also decreased accuracy slightly in monkeys, suggesting that impairments might have been due in part to nonselective disruption by THC, similar to the disruptions in food-maintained behavior described above. THC-induced impairments in monkeys were reversed by Ro 61-8048 (Fig. 7f; main effect of Ro 61-8048, F 1,2 = 20.46, P < 0.05; main effect of THC, F 1,2 = 42.42, P < 0.05; main effect of delay, F 4,8 = 59.65, P < 0.0001).

KMO inhibition and discriminative-stimulus effects of THC

To determine whether Ro 61-8048 could affect not only the reinforcing effects of THC but also its subjective effects, we studied effects of Ro 61-8048 in rodent and primate models of cannabinoid discrimination. In rats trained to detect whether they had been injected with THC or its vehicle, lever selection was dose dependent, with maximal selection of the drug lever (99.66%) at the 3 mg per kg training dose of THC (Fig. 8a; F 5,40 = 28.03, P < 0.001). Notably, the THC dose-effect curve was not significantly shifted by treatment with either 30 or 100 mg per kg of Ro 61-8048 (Fig. 8a; F 2,45 = 2.66, P = 0.10), indicating that Ro 61-8048 does not block all of the subjective effects of cannabinoid CB 1 agonists in rats. Also in this procedure, Ro 61-8048 did not disrupt food-reinforced behavior when given alone (Fig. 8b; F 2,16 = 0.53, P = 0.59) or in combination with different doses of THC (Fig. 8b; F 2,45 = 0.24, P = 0.79), indicating that decreases in WIN 55,212-2 self-administration produced by Ro 61-8048 in rats (Fig. 4d,e) were not due to nonspecific behavioral disruption.

Figure 8: Effects of Ro 61-8048 on discriminative-stimulus effects of THC in rats and squirrel monkeys. (a,b) Rats trained to discriminate THC (3 mg per kg, i.p.) from vehicle (V) under a fixed-ratio 10 schedule of food delivery were tested with various doses of THC. The percentage of responses on the CB 1 agonist–appropriate lever was a monotonically increasing function of dose. Treatment with Ro 61-8048 (30 or 100 mg per kg, i.p.) did not significantly alter this curve (a), and Ro 61-8048 did not significantly alter the food-reinforced response rate after THC or vehicle administration (b). Abscissae, dose, log scale; ordinate, percentage of responses on the THC-associated lever (a) or response rate (b). (c,d) When monkeys, trained under a stimulus-shock termination schedule to discriminate injection of the selective cannabinoid CB 1 agonist AM4054 (10 μg per kg, i.m.) from vehicle, were injected i.m. with various doses of THC, the percentage of responses on the CB 1 -appropriate lever was a monotonically increasing function of cumulative dose (c). Ro 61-8048 (20 mg per kg, i.m.) reduced the monkeys' ability to detect interoceptive effects of THC in the cannabinoid CB 1 discrimination procedure (c). Ro 61-8048 did not significantly affect response rates after THC administration in this procedure (d). Abscissae, cumulative THC dose, log scale; ordinate, percentage of responses on the AM4054-associated lever (c) or response rate (d). Symbols left of the abscissa breaks indicate performance during vehicle and AM4054 control sessions. All data are presented as means ± s.e.m. (n = 9 rats, n = 3 monkeys). Full size image Download Excel source data