Objective: Voltage-gated potassium channels of the KCNQ (Kv7) family are targeted by a variety of activator compounds with therapeutic potential for treatment of epilepsy. Exploration of this drug class has revealed a variety of effective compounds with diverse mechanisms. In this study, we aimed to clarify functional criteria for categorization of Kv7 activator compounds, and to compare the effects of prototypical drugs in a zebrafish larvae model. Methods: In vitro electrophysiological approaches with recombinant ion channels were used to highlight functional properties important for classification of drug mechanisms. We also benchmarked the effects of representative antiepileptic Kv7 activator drugs using behavioral seizure assays of zebrafish larvae and in vivo Ca 2+ imaging with the ratiometric Ca 2+ sensor CaMPARI. Results: Drug effects on channel gating kinetics, and drug sensitivity profiles to diagnostic channel mutations, were used to highlight properties for categorization of Kv7 activator drugs into voltage sensor-targeted or pore-targeted subtypes. Quantifying seizures and ratiometric Ca 2+ imaging in freely swimming zebrafish larvae demonstrated that while all Kv7 activators tested lead to suppression of neuronal excitability, pore-targeted activators (like ML213 and retigabine) strongly suppress seizure behavior, whereas ICA-069673 triggers a seizure-like hypermotile behavior. Significance: This study suggests criteria to categorize antiepileptic Kv7 activator drugs based on their underlying mechanism. We also establish the use of in vivo CaMPARI as a tool for screening effects of anticonvulsant drugs on neuronal excitability in zebrafish. In summary, despite a shared ability to suppress neuronal excitability, our findings illustrate how mechanistic differences between Kv7 activator subtypes influence their effects on heteromeric channels and lead to vastly different in vivo outcomes.

Traumatic brain injury (TBI) is a prominent risk factor for dementias including tauopathies like chronic traumatic encephalopathy (CTE). The mechanisms that promote prion-like spreading of Tau aggregates after TBI are not fully understood, in part due to lack of tractable animal models. Here, we test the putative role of seizures in promoting the spread of tauopathy. We introduce ‘tauopathy reporter’ zebrafish expressing a genetically encoded fluorescent Tau biosensor that reliably reports accumulation of human Tau species when seeded via intraventricular brain injections. Subjecting zebrafish larvae to a novel TBI paradigm produced various TBI features including cell death, post–traumatic seizures, and Tau inclusions. Bath application of dynamin inhibitors or anticonvulsant drugs rescued TBI-induced tauopathy and cell death. These data suggest a role for seizure activity in the prion-like seeding and spreading of tauopathy following TBI. Further work is warranted regarding anti-convulsants that dampen post-traumatic seizures as a route to moderating subsequent tauopathy.

In light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) until the end of gastrulation (1–10 h post-fertilization) and analyzed later in development (4–5 days post-fertilization). In order to measure neural activity, we implemented Calcium-Modulated Photoactivatable Ratiometric Integrator (CaMPARI) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and their receptors to understand the implications of cannabis consumption on fetal development.

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Highlights:• Introduces first Traumatic Brain Injury (TBI) model in larval zebrafish, and its easy • TBI induces clinically relevant cell death, haemorrhage & post-traumatic seizures • Ca 2+ imaging during TBI reveals spike in brain activity concomitant with seizures • Tau-GFP Biosensor allows repeated in vivo measures of prion-like tau aggregation • post-TBI, anticonvulsants stop tauopathies akin to Chronic Traumatic Encephalopathy 3 Summary:Traumatic brain injury (TBI) is a prominent risk factor for neurodegenerative diseases 1 and dementias including chronic traumatic encephalopathy (CTE). TBI and CTE, like all 2 tauopathies, are characterized by accumulation of Tau into aggregates that 3 progressively spread to other brain regions in a prion-like manner. The mechanisms that 4 promote spreading and cellular uptake of tau seeds after TBI are not fully understood, in 5 part due to lack of tractable animal models. Here, we test the putative roles for excess 6 neuronal activity and dynamin-dependent endocytosis in promoting the in vivo spread of 7 tauopathy. We introduce 'tauopathy reporter' zebrafish expressing a genetically-8 encoded fluorescent Tau biosensor that reliably reports accumulation of human tau 9 species when seeded via intra-ventricular brain injections. Subjecting zebrafish larvae to 10 a novel TBI paradigm produced various TBI symptoms including cell death, 11hemorrhage, blood flow abnormalities, post-traumatic seizures, and Tau inclusions. 12Bath application of anticonvulsant drugs rescued TBI-induced tauopathy and cell death; 13 these benefits were attributable to inhibition of post-traumatic seizures because co-14 application of convulsants reversed these beneficial effects. However, one convulsant 15 drug, 4-Aminopyridine, unexpectedly abrogated TBI-induced tauopathy -this was due to 16 its inhibitory action on endocytosis as confirmed via additional dynamin inhibitors. These 17 data suggest a role for seizure activity and dynamin-dependent endocytosis in the prion-18 like seeding and spreading of tauopathy following TBI. Further work is warranted 19 regarding anti-convulsants that dampen post-traumatic seizures as a route to 20 moderating subsequent tauopathy. Moreover, the data highlight the utility of deploying 21 in vivo Tau biosensor and TBI methods in larval zebrafish, especially regarding drug 22 screening and intervention. 23 24 4 Introduction: