Selective effects of ethanol, heroin, and cocaine self-administration on microdialysate AEA and 2-AG levels collected from the NAc. In each panel the shaded bar reflects the period of drug self-administration. Panel A: Dialysate 2-AG levels were significantly increased by voluntary oral EtOH self-administration (10% w/v; 0.39 ± 0.03 g/kg total EtOH intake during 30 min. self-administration session; n = 9). There was no significant effect of EtOH intake on AEA levels measured from these same dialysate samples. Panel B: Dialysate AEA levels were significantly increased by voluntary heroin self-administration (20 μg/infusion; 443 ± 62 μg/kg total intake during 120 min self-administration session; n = 7). In contrast, dialysate 2-AG levels were subtly, but significantly decreased during the latter portions of the self-administration session. Panel C: Dialysate AEA and 2-AG levels were not altered by cocaine self-administration (0.25 mg/infusion; 18 ± 0.9 mg/kg total intake during 120 min self-administration; n = 8). Data are modified from [82].

Effects of A) acute WIN 55212-2 and B) repeated WIN 55212-2 exposures on acute paraoxon toxicity. Rats were treated with paraoxon (0.4 mg/kg, sc) either just before a single dose of WIN (1.5 mg/kg, ip) or 2 hours after the seventh daily dose of WIN. Functional signs of toxicity (involuntary movements, tremors) were scored (1 = normal; 2 = slight tremors in neck and head; 3 = moderate tremors involving more caudal aspects of body; 4 = severe whole body tremors) by a blinded observer from 0.5–4 hours after paraoxon challenge. Data represent median ± interquartile range. No signs of toxicity were noted in rats receiving vehicles or WIN only (data not shown). An asterisk indicates a significant difference between groups. Data are modified from [129].

Effects of chlorpyrifos and parathion on extracellular hippocampal endocannabinoid levels. Cannula were placed in dorsal hippocampus three days prior to treatment. Rats (n=4/treatment group) were given either vehicle (peanut oil), chlorpyrifos (280 mg/kg, sc) or parathion (27 mg/kg, sc). Dialysis probes were inserted four days later and were perfused with buffer containing 30% hydroxypropyl β-cyclodextrin to increase eCB recovery [82]. After pre-perfusion, five 10-min samples were collected, each separated by 50 min. Dialysate levels of AEA and 2-AG were determined by HPLC-MS, average values from replicates over time calculated, and mean ± SE values by treatment reported [82].

Chemical structures of 9-tetrahydrocannabinol, anandamide and 2-arachidonoyl glycerol.

Localized infusion of the CB1 receptor antagonist SR141716A (1 and 3 μg/0.5 μl/side) into the rat nucleus accumbens significantly reduces the operant self-administration of EtOH (Panel A: 10% w/v; n = 11) and heroin (Panel B: 20 μg/infusion; n = 10), but not cocaine (Panel C: 0.25 mg/infusion; n = 7). Thus, intra-NAc CB1 antagonist administration reduced the self-administration of the two drugs whose intake significantly increases interstitial NAc EC levels (e.g. EtOH and heroin, see Fig. 2) but did not alter the self-administration of the drug that did not significantly alter interstitial NAc EC levels (e.g. cocaine). Data are modified from [69, 82].

Comparative in vitro inhibition of rat hippocampal monoacylglycerol lipase activity by chlorpyrifos oxon and paraoxon. Rat hippocampus was homogenized on ice in 0.32 M sucrose, pH 8.0 with a Polytron at 27,000 rpm. Tissues were centrifuged at 100,000 × g for 60 minutes to obtain a soluble fraction. Aliquots of the soluble fraction were preincubated for 30 minutes with vehicle or one of a range of concentrations of either paraoxon (PO) or chlorpyrifos oxon (CPO) prior to evaluating residual monoacylglycerol lipase (MAGL) activity with 2-oleoyl [³H]glycerol as the substrate (10 μM final concentration).

Possible interactions between cholinergic and eCB signaling in the toxicity of organophosphorus anticholinesterases. OPs may enhance eCB signaling either indirectly through acetylcholinesterase inhibition or directly by binding to components of the eCB pathway. Extensive acetylcholinesterase inhibition will increase synaptic acetylcholine levels and prolong cholinergic receptor activation to elicit cholinergic signs of toxicity. Depolarization of postsynaptic neurons can lead to release of eCBs and modulation of neurotransmitter release. Prolonged cholinergic receptor activation can stimulate downstream non-cholinergic neurons. Glutamatergic neuron activation through NMDA receptors can increase intracellular Ca⁺⁺ levels and cellular toxicity, or through metabotropic MGluR receptors can lead to eCB release that may attenuate further recruitment of downstream signaling and toxicity. Postsynaptic muscarinic M1/M3 receptor activation leads to receptor-mediated eCB release to activate CB1 receptors and inhibit ACh release at the presynaptic cholinergic terminal. Upon release, AEA can activate CB1 receptors (extracellularly) to inhibit neurotransmitter release and TRPV1 receptors (intracellularly) to increase Ca⁺⁺ influx, while 2-AG acts at the CB1 receptor but not at the TRPV1 site. In the direct pathway, some OPs may bind to molecular components of eCB signaling to alter CB1 receptor activation, eCB synthesis, eCB degradation or eCB uptake and modulate cholinergic and/or non-cholinergic neurotransmitter release. Direct FAAH inhibition by some OPs can increase intracellular AEA, and in some neurons, this may lead to increased TRPV1 activation, elevated intracellular Ca⁺⁺ and decreased 2-AG signaling. Reduced 2-AG signaling could in turn reduce eCB mediated inhibition of neurotransmitter release, enhancing cholinergic and non-cholinergic signaling. Direct inhibition of MAGL by some OPs may in turn selectively increase 2-AG signaling.