Behavioral sequelae following acute diisopropylfluorophosphate intoxication in rats: comparative effects of atropine and cannabinomimetics

Abstract

The comparative effects of atropine and the indirect cannabinomimetics URB597 (a fatty acid amide hydrolase inhibitor) and URB602 (a monoacylglycerol lipase inhibitor) on functional and neurobehavioral endpoints following acute diisopropylfluorophosphate intoxication were studied. Male Sprague-Dawley rats were treated with vehicle or DFP (2.5mg/kg, sc), immediately post-treated with either vehicle, atropine (16mg/kg), URB597 (3mg/kg), URB602 (10mg/kg) or a combination of URB597 and URB602, and functional signs of toxicity as well as nocturnal motor activity were measured daily for seven consecutive days. Performance in the elevated plus maze (for anxiety-like behavior) and the forced swimming test (for depression-like behavior) was measured at days 6-8 and 27-29 after dosing. Twenty-four hours after dosing, DFP markedly reduced cholinesterase activity in selected brain regions and peripheral tissues (diaphragm and plasma). Substantial recovery of cholinesterase activity was noted at both 8 and 29days after dosing but significant inhibition was still noted in some brain regions at the latest time-point. DFP elicited body weight reductions and typical signs of cholinergic toxicity, and reduced nocturnal ambulation and rearing. Atropine and the cannabinomimetics (alone and in combination) partially attenuated DFP-induced functional signs of toxicity. None of the post-treatments reversed the DFP-induced reduction in ambulation or rearing, however. No significant treatment-related effects on elevated plus maze performance were noted. DFP-treated rats exhibited decreased swimming and increased immobility in the forced swimming test at both time-points. None of the post-treatments had any effect on DFP-induced changes in immobility or swimming at day 8. At day 29, atropine and the combination of URB597/URB602 significantly blocked DFP-induced changes in immobility, while URB597 and the combination reversed DFP-induced changes in swimming. The results suggest that early blockade of muscarinic receptors and enhancement of eCB signaling can attenuate both acute and delayed effects elicited by DFP.

Figure 1. Effects of DFP and post-treatment with vehicle, atropine, URB597, URB602 or the combination of URB597 and URB602 on body weight

Rats were injected with DFP and immediately treated with vehicle (DFP/VEH, n = 17), atropine (DFP/ATR, n = 10), URB597 (DFP/URB597, n = 8), URB602 (DFP/URB602, n = 5) or the combination (DFP/COMBO, n = 5). Control rats (PNO/VEH, n = 9) were injected with peanut oil and immediately treated with vehicle. Body weight was recorded at 1, 6 and 29 days post-injection, and data are expressed as mean + standard error of the mean (SEM). All DFP-treated groups showed significant body weight reductions at both 1 and 6 days after dosing, while no significant treatment-related differences were noted at day 29.

Figure 2. Effects of post-treatment with vehicle, atropine, URB597, URB602 or the combination of URB597 and URB602 on functional signs of toxicity elicited by DFP

Rats were injected with DFP and immediately treated with vehicle (DFP/VEH, n = 17), atropine (DFP/ATR, n = 10), URB597 (DFP/URB597, n = 8), URB602 (DFP/URB602, n = 5) or the combination (DFP/COMBO, n = 5). Control rats (PNO/VEH, n = 9) were injected with peanut oil and immediately treated with vehicle. Functional signs of toxicity [i.e., involuntary movements (A) and SLUD signs (B)] were measured at 1, 2, 4 and 8 h post-injection as well as daily for seven consecutive days. Data are expressed as median + interquartile ratio. An asterisk indicates a significant difference compared to the PNO/VEH group, and a pound sign indicates a significant difference compared to the DFP/VEH group.

Figure 3. Effects of DFP and post-treatment with vehicle, atropine, URB597, URB602 or the combination of URB597 and URB602 on nocturnal motor activity

Rats were injected with DFP and immediately treated with vehicle (DFP/VEH, n = 7), atropine (DFP/ATR, n = 3), URB597 (DFP/URB597, n = 6), URB602 (DFP/URB602, n = 5) or the combination (DFP/COMBO, n = 5). Control rats (PNO/VEH, n = 6) were injected with peanut oil and immediately treated with vehicle. Nocturnal motor activity [i.e., ambulation (A) and rearing (B)] was measured daily for seven consecutive days post-injection. Data are expressed as mean + SEM. An asterisk indicates a significant difference between the PNO/VEH and DFP/VEH groups.

Figure 4. Effects of DFP and post-treatment with vehicle, atropine, URB597, URB602 or the combination of URB597 and URB602 on performance in the elevated plus maze

A) Rats were injected with DFP and immediately treated with vehicle (DFP/VEH, n = 15), atropine (DFP/ATR, n = 9), URB597 (DFP/URB597, n = 8), URB602 (DFP/URB602, n = 5) or the combination (DFP/COMBO, n = 5). Control rats (PNO/VEH, n = 9) were injected with peanut oil and immediately treated with vehicle. Performance in the elevated plus maze (i.e., percentage of time spent in the open arms) was then measured at six days after dosing. B) Rats were injected with DFP and immediately treated with vehicle (DFP/VEH, n = 9), atropine (DFP/ATR, n = 3), URB597 (DFP/URB597, n = 8), URB602 (DFP/URB602, n = 5) or the combination (DFP/COMBO, n = 5). Control rats (PNO/VEH, n = 9) were injected with peanut oil and immediately treated with vehicle. Percentage of time spent in the open arms was then measured at 27 days after dosing. Data are expressed as mean + SEM.

Figure 5. Effects of DFP and post-treatment with vehicle, atropine, URB597, URB602 or the combination of URB597 and URB602 on performance in the forced swimming test

A) Rats were injected with DFP and immediately treated with vehicle (DFP/VEH, n = 17), atropine (DFP/ATR, n = 10), URB597 (DFP/URB597, n = 8), URB602 (DFP/URB602, n = 5) or the combination (DFP/COMBO, n = 5). Control rats (PNO/VEH, n = 9) were injected with peanut oil and immediately treated with vehicle. Performance in the forced swimming test (i.e., time spent climbing, swimming or immobile) was then measured at eight days after dosing. B) Rats were injected with DFP and immediately treated with vehicle (DFP/VEH, n = 9), atropine (DFP/ATR, n = 3), URB597 (DFP/URB597, n = 8), URB602 (DFP/URB602, n = 5) or the combination (DFP/COMBO, n = 5). Control rats (PNO/VEH, n = 9) were injected with peanut oil and immediately treated with vehicle. Performance in the forced swimming test was then measured at 29 days after dosing. Data are expressed as mean + SEM. An asterisk indicates a significant difference compared to the PNO/VEH group, and a pound sign indicates a significant difference compared to the DFP/VEH group.