Early suppression of the endocannabinoid degrading enzymes FAAH and MAGL alters locomotor development in zebrafish

Abstract

The fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) enzymes are the predominant catabolic regulators of the major endocannabinoids (eCBs), anadamide (AEA) and 2-arachidonoylglycerol (2-AG), respectively. The expression and roles of eCBs during early embryogenesis remain to be fully investigated. Here, we inhibited FAAH and MAGL in zebrafish embryos during the first 24 hours of life and examined motor neuron and locomotor development at 2 and 5 days post fertilization (dpf). Application of the dual FAAH/MAGL inhibitor, JZL195 (2 µmol l−1) resulted in a reduction in primary motor neuron (PMN) and secondary motor neuron (SMN) axonal branching. JZL195 also reduced nicotinic acetylcholine receptor (nAChR) expression at neuromuscular junctions (NMJs). Application of URB597 (5 µmol l−1), a specific inhibitor of the FAAH enzyme also decreased primary motor neuron branching but did not affect secondary motor neuron branching and nAChR expression. Interestingly, JZL184 (5 µmol l−1), a specific inhibitor of MAGL showed no effects on motor neuron branching or nAChR expression. Co-treatment of the enzyme inhibitors with the CB1R inhibitor AM 251 confirmed the involvement of CB1R in motor neuron branching. Disruption of FAAH or MAGL reduced larval swimming activity, and AM251 attenuated the JZL195 and URB597 induced locomotor changes, but not the effects of JZL184. Together, these findings indicate that inhibition of FAAH, or augmentation of AEA acting through CB1R during early development may be responsible for locomotor deficiencies.

The endocannabinoid systemʼs involvement in motor development relies on cannabinoid receptors, TRP channels, and Sonic Hedgehog signaling

“…It was shown previously that zebrafish embryos exposed to the FAAH and MAGL inhibitors URB 597, JZL 184, and JZL 195 experience deficits in locomotion, and so here we examined the locomotor alterations caused by inhibiting these eCS catabolic enzymes (Khara et al, 2022 ; Sufian et al, 2021 ). Because FAAH and MAGL inhibition are linked to increased eCB levels, our investigation began by targeting the canonical cannabinoid receptors: CB1R and CB2R (Griebel et al, 2015 ; Zou & Kumar, 2018 ).…”

“…Along with these compounds, animals were co‐treated with the CB1R antagonist AM 251 (Selleck Chemicals), the CB2R antagonist AM630 (Adooq Bioscience), the TRPV1/TRPA1/TRPM8 inhibitor AMG 9090 (Alomone Labs), or the SMO agonist Purmorphamine (Sigma‐Aldrich). The concentrations used for these four drugs were 10 nM for AM 251, 1 μM for AM 630, 0.01 μM for AMG 9090, and 5 μM for purmorphamine and were determined based on previous studies (Khara et al, 2022 ; Sufian et al, 2021 ) and dose–response relationships performed for survival, hatching, morphology, and locomotion in embryos and larva. All compounds listed here were dissolved in dimethyl sulfoxide (DMSO), which was used as the vehicle control.…”

“…All compounds listed here were dissolved in dimethyl sulfoxide (DMSO), which was used as the vehicle control. The drug exposure took place immediately after egg fertilization until 24 h post‐fertilization (hpf), effectively spanning from ~0–24 hpf as done previously (Khara et al, 2022 ; Sufian et al, 2021 ). Embryos were exposed to the compounds via the embryo media.…”

“…For instance, the CB1R is important for the development of hindbrain reticulospinal neurons in zebrafish, which are crucial for sensorimotor function (Watson et al, 2008). Previous work also demonstrates that perturbations of the eCS in embryonic zebrafish lead to deficits in the development of motor neurons and locomotor activities, and these alterations were demonstrated to be mediated through the CB1R (Ahmed et al, 2018;Sufian et al, 2019;Sufian et al, 2021). Meanwhile, SHH signaling is crucial for neural tube formation and is required for motor neuron differentiation (Ericson et al, 1996).…”

“…eCB signaling can be perturbed by blocking the activity of FAAH and MAGL, leading to over‐activation of the eCS (Gobbi et al, 2005 ; Griebel et al, 2015 ; Long et al, 2009 ). Inhibiting the enzymes FAAH and MAGL has been shown to decrease locomotion and alter motor neuron development (Boa‐Amponsem et al, 2019 ; Seillier et al, 2014 ; Sufian et al, 2021 ). Furthermore, we have recently observed that FAAH/MAGL inhibition alters functional locomotor and sensorimotor development in zebrafish (Khara et al, 2022 ).…”