ABSTRACT
Of the three primary cannabinoids in cannabis: Δ9-Tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD) and cannabinol (CBN), very little is known about the actions of CBN, the primary oxidative metabolite of THC. Our goal was to determine if CBN exposure during gastrulation alters embryonic development, and if so, does it act via the canonical cannabinoid receptors. Zebrafish embryos were exposed to CBN during gastrulation and exhibited dose-dependent malformations, increased mortality, decreased locomotion and a reduction in motor neuron branching. Moreover, larva showed a significant reduction in the response to sound stimuli. CBN exposure altered the development of hair cells associated with otic vesicles and the lateral line. Pharmacological block of Cb2rs with AM 630 or JTE 907 prevented many of the CBN-induced developmental defects, while block of Cb1rs with AM 251 or CP 945598 had little or no effect. Altogether we show that embryonic exposure to CBN results in alterations in embryonic growth, neuronal and hair cell development, physiology and behavior via Cb2r-mediated mechanisms.
Subject(s)
Cannabinoids , Cannabinol , Animals , Cannabinol/metabolism , Zebrafish/metabolism , Cannabinoids/pharmacology , Cannabinoids/metabolism , Dronabinol/pharmacology , Dronabinol/metabolism , Receptors, CannabinoidABSTRACT
Fast excitatory synaptic transmission in the CNS is mediated by the neurotransmitter glutamate, binding to and activating AMPA receptors (AMPARs). AMPARs are known to interact with auxiliary proteins that modulate their behavior. One such family of proteins is the transmembrane AMPAR-related proteins, known as TARPs. Little is known about the role of TARPs during development or about their function in nonmammalian organisms. Here, we report on the presence of TARP γ-4 in developing zebrafish. We find that zebrafish express 2 forms of TARP γ-4: γ-4a and γ-4b as early as 12 h post-fertilization. Sequence analysis shows that both γ-4a and γ-4b shows great level of variation particularly in the intracellular C-terminal domain compared to rat, mouse, and human γ-4. RT-qPCR showed a gradual increase in the expression of γ-4a throughout the first 5 days of development, whereas γ-4b levels were constant until day 5 when levels increased significantly. Knockdown of TARP γ-4a and γ-4b via either splice-blocking morpholinos or translation-blocking morpholinos resulted in embryos that exhibited deficits in C-start escape responses, showing reduced C-bend angles. Morphant larvae displayed reduced bouts of swimming. Whole-cell patch-clamp recordings of AMPAR-mediated currents from Mauthner cells showed a reduction in the frequency of mEPCs but no change in amplitude or kinetics. Together, these results suggest that γ-4a and γ-4b are required for proper neuronal development.
