The effects of handling stress on Cannabinoid receptor expression in killfish, Fundulus heteroclitus
LE3 .A278 2019
Bachelor of Science
The endocannabinoid system is composed of protein receptors, enzymes, and lipid-based neurotransmitters. The system is located within the central nervous system and peripheral organs in vertebrates, and is implicated in many physiological and cognitive functions, including neural development, memory, anxiety and immune cell proliferation. ∆9-tetrahydrocannabinol and cannabidiol are ligands known to interact with the endocannabinoid system which induce the psychoactive and medicinal effects seen in users. This project looked to examine how stress affects gene expression and protein synthesis of the two cannabinoid receptors, CB1 and CB2, in the brain and spleen of killifish (Fundulus heteroclitus). Killifish were exposed to a handling stress for 30 sec, and sampled at 1, 6, and 24 hours following the stress. RNA was extracted from the brain and spleen, and protein was extracted from the brain. RNA was reverse transcribed into cDNA, and qPCR was used to examine cb1 expression in the brain and cb 2expression in the spleen. The DDCq method was used to determine fold regulation and standard error between the stressed and non-stressed groups. Western blotting was used to explore protein production in the brain. Blot volumes and densities of CB1 and CB2 were normalized to b-actin to compare relative protein production. cb2was upregulated at 6 and 24 hours following exposure to stress, while cb1 was upregulated at 24 hours. CB1 and CB2 synthesis was significantly increased at 6 and 24 hours following the handling stress. This study demonstrated that cannabinoid receptor gene expression and protein synthesis are upregulated in response to anaerobic handling stress in F.heteroclitus. Future research can examine how cannabinoid receptor expression is affected by exposure to exogenous cannabinoids.
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