Elucidating the roles of endocannabinoid signalling pathway in motor neuron and locomotor development in zebrafish early life

• Author / Creator

  Sufian, Md Shah

• The major endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG) mediate their effects through their actions on cannabinoid receptors (CBRs), mainly via CB1R and CB2Rs. The predominant catabolic regulators that degrade AEA and 2-AG are fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL also known as MGLL), respectively. Although the eCB signalling pathway is present from very early stages of development, little is known about their mechanisms that underlie embryonic growth, and the onset of locomotion and motor neuron development. In my thesis, I sought to investigate the role of the eCB system in zebrafish embryonic development by pharmacologically inhibiting the CB1 and CB2 receptors (with AM251 and AM630, respectively) for the first two days of post-fertilization (dpf). Selective inhibition of CB1R and CB2Rs upon treatment with AM251 and AM630 resulted in embryos with axial malformations, shorter trunks, pericardial edema and reduced heart rates, particularly at higher concentrations at 2 dpf. Locomotion studies at 5 dpf revealed a change in their activity, swim velocity, the number of swim bouts and the cumulative duration of swim bouts. The morphological and locomotor deficits observed upon inhibition of the CBRs during the first 48 hours of development led me to examine in more detail the role of CB1R and CB2R during development. Therefore, I further investigated the role of the CBRs by pharmacologically inhibiting CB1R and CB2R (with AM251 and AM630, respectively) in either the first or second day of development. Motor neuron morphology and neuromuscular outputs were then examined and quantified in 5 dpf larvae. Although blocking both CB1R and CB2R resulted in gross morphological deficits and reductions in heart rate, the effects of CB2Rs inhibition were more pronounced when compared to
  CB1Rs. Blocking CB1Rs from 0 to 24 h post-fertilization (hpf) resulted in an increase in the number of secondary and tertiary branches of primary motor neurons, whereas blocking CB2Rs had the opposite effect. Both treatments resulted in reduced levels of swimming. Additionally, blocking CB1Rs resulted in greater instances of non-inflated and partially inflated swim bladders compared with AM630 treatment, suggesting that at least some of the deficits in locomotion may result from an inability to adjust buoyancy.
  Finally, I studied the effects of inhibiting the enzymes (FAAH and MAGL) that break down the eCBs, in the early part of development. My study analyzed the functional roles of increased accumulation of AEA and 2-AG levels upon inhibition of FAAH and MAGL, and examined their effects on development. In vivo application of a dual FAAH/MAGL inhibitor, JZL 195, resulted in a reduction in primary and secondary motor neuronal axon branching, as well as in a reduction of nicotinic acetylcholine receptors (nAChRs) expression at neuromuscular junctions (NMJs). Application of the specific inhibitor of the FAAH enzyme, URB 597, also resulted in a decrease in motor neuron branching of the primary motor neurons only. However, secondary motor neuron branching and nAChR expression remained unaffected following URB 597 treatment in the first 24 hrs of development. Furthermore, I also confirmed that the effects of JZL 195 and URB 597 were mediated through CB1Rbecause co-treatment with the selective CB1R inhibitor, AM 251, prevented the aberrant branching of motor neurons and reduced nAchRs expression. Interestingly, JZL 184, a selective MAGL inhibitor, showed no effects throughout the study when examining motor neuron development and nAChRs expression at NMJs. Locomotion studies revealed that any disruption of FAAH or MAGL activity in the first 24 hrs of development by JZL 195, URB 597 and JZL 184 can reduce larval swimming activity. Although CB1R inhibition with AM 251 attenuated JZL 195 and URB 597 induced behavioural changes, it was unable to block the effects of JZL 184. Therefore, my study strongly indicates that accumulation of AEA preferentially acting through CB1R at early life is responsible for motor neuronal changes associated locomotor deficiency.

• Subjects / Keywords

  • Endocannabinoids

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-mnaf-pm46

• License

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