Investigating the effects of cannabinoid exposure on zebrafish development
Creators: Amin, MD Ruhul
Publisher: name: University of Alberta Library
Year: 2022

<p>Cannabis is one of the most commonly used illicit recreational drugs and is widely used for medicinal purposes. Cannabis contains three primary cannabinoids: 9-Tetrahydrocannabinol (THC), Cannabidiol (CBD) and Cannabinol (CBN). The psychoactive ingredient in cannabis is THC, whereas the major non-psychoactive ingredients are CBD and CBN. In recent times, cannabis use during pregnancy has increased worldwide, and studies have reported that cannabis and many of its constituent compounds can cross the placenta (Fried, 1995; George &amp; Vaccarino, 2015). Hence, exposure to cannabis has the potential to pose a severe risk to developing offspring. Importantly, there is a lack of information on the effects of cannabinoids on developing embryos. Therefore, in this thesis I investigated the effect of brief exposure of THC (a racemic mixture of THC), CBD and CBN during gastrulation on zebrafish development. I found that THC-, CBD- and CBN-treated embryos exhibited reduced heart rates, axial malformations and shorter trunks. These treatments also altered synaptic activity at neuromuscular junctions and reduced the number of motor neuron axonal branches. Locomotion studies show that larvae exhibited reductions in the number of escape responses to sound stimuli, but not to touch stimuli. When investigating the effects on neurons, I specifically focused on motor neurons, and reticulospinal neurons known as the Mauthner cells (M-cells) that are involved in escape response movements. Cannabinoid-treated embryos exhibited subtle alterations in M-cells morphology, which might contribute towards the escape response dynamics. An examination of free-swimming activity showed an alteration in the swimming output of cannabinoid-treated embryos. Because locomotion was altered, I also examined muscle fiber development. The fluorescent labelling and transmission electron microscopy images showed that skeletal muscle fibers were largely intact, but the red and white muscle fibers were slightly disorganized. I also observed significant changes in the pattern of expression of nicotinic acetylcholine receptors at neuromuscular junctions. <br>
Since cannabinoid-treated embryos exhibited a reduced response to sound, scanning electron microscopy analysis of CBN exposure showed a defective hair cell development along the lateral line. The pharmacological block of cannabinoid receptor 2 (CB2R) with AM630 or JTE907 prevented many of the CBN-induced developmental defects, while the block of cannabinoid receptor 1 (CB1R) with AM251 or CP945598 had little or no effect indicating that CBN-induced developmental defects are mainly CB2R-mediated. Next, I sought to examine the effect of stereoselectivity on zebrafish embryo development because THC has four stereoisomers. (-) trans 9THC (referred as (-) THC afterwards) is one of those isomers and predominantly present in natural cannabis. I found that (-) THC affects the gross morphology, heart rate, MN branching and locomotion similar to other cannabinoids. Most importantly, (-) THC (&gt;0.5 mg/L)-treated zebrafish did not survive past 15 days of development. Since embryos treated with (-) THC did not rise in the water column or swim, I investigated swimbladder development and found that the (-) THC treatment negatively impacted swimbladder development in a sonic hedgehog-dependent manner. In support of this, RNAseq analysis revealed downregulation of hedgehog genes, including ptch2, smo, gli1 and gili2b. In addition, more than 1000 and 1400 genes were downregulated and upregulated respectively in (-) THC-treated embryos. <br>
I asked whether the brief exposure of cannabinoids had persistent effects into later life stages. Therefore, I reared the exposed embryos to adulthood and carried out open field, novel object approach and shoaling tests. Both (-) THC and CBD-treated adult zebrafish exhibited anxiety-like behavior compared to controls. Finally, I found that brief exposure to (-) THC and CBD has a transgenerational impact because F1 embryos collected from (-) THC and CBD parents exhibited reduced locomotion, alterations in primary MN branching and changes in the expression of the neuronal marker genes, including c-fos and bdnf. Together, these findings indicate that zebrafish exposed to cannabinoids during gastrulation exhibit changes in various aspects of development (including, neuronal, muscle and swimbladder development) that may impact embryonic locomotion; a number of these changes are associated with alteration in gene expression.  In addition, these brief exposures to cannabinoids had a persistent effect in adult and into the next generation.</p>