Abstracts

Rationale: The CACNA1A gene encodes the α1A subunit of the neuronal voltage-gated Cav2.1 P/Q type calcium channel. Cav2.1 is critically important in mediating neurotransmission and is highly expressed presynaptically in cerebellar cells, prefrontal cortex and CA1 region of the hippocampus. These structures are known for generation, maintenance and spreading of discharges in generalized epilepsy. Mutations in the CACNA1A gene have been described in humans suffering from episodic ataxia type 2, infantile epilepsy with myoclonus, febrile or absence seizures. In zebrafish, cacna1a is duplicated, with two splice variants encoding the cacna1aa protein. Here, we aimed to describe the behavioral phenotype of larval zebrafish with partial cacna1aa loss-of-function. A special emphasis was set on EEG discharges in morphant brains. Methods: In order to induce partial loss-of-function of cacna1aa, two antisense morpholino oligomers (MO) designed to target the ATG sites of both cacna1aa splice variants were utilized (Gene Tools, USA). The combination of MOs (2.5 ng + 2.5 ng) was injected into fertilized one cell stage embryos. A randomized sequence MO was used as a control (Ctrl-MO, 5 ng). At 4 days post fertilization (4 dpf), the touch response of larvae was checked and locomotor activity tracking performed, in 5-min-long light and 10-min-long dark phases (Zebrabox, Viewpoint, France). At 118 hours post-fertilization (hpf) EEG analysis was carried out (MultiClamp 700B amplifier, Digidata 1440A digitizer, Axon instruments, USA). Morphants and controls were incubated with antiseizure drugs (ASDs) i.e. ethosuximide (10 mM), lamotrigine (200 µM), topiramate (100 µM), and valproate (100 µM) for 2 h. Next, larva was embedded in 2% low-melting-point agarose, the glass electrode filled with artificial cerebrospinal fluid, placed into the optic tectum and recordings were performed for 20 min. The number of EEG discharges was assessed using Clampfit 10.2 software (Molecular Devices Corporation, USA). For all experiments, larvae no older than 118 hpf were used - thus no ethical permission was needed. Results: Our study revealed that cacna1aa morphants were hypoactive, both under light and dark phases, compared to Ctrl-MO counterparts at 4 dpf (two-way ANOVA with repeated measures; group: F(1,89)=27.86, P<0.05, phase: F(2,89)=3.44, P<0.05, group x phase interaction: F(1,89)=1.79, P<0.05; n=44-48/group). Notably, the touch-evoked response was not disturbed (Student’s t-test; P>0.05; n=52-63) thus the observed differences in locomotor activity were not due to disturbances in neuromascular neurotransmission. The analysis of EEG discharges revealed spontaneous epileptiform-like events which occurred in 24 out of 26 of cacna1aa morphants, compare to 2 out of 19 Ctrl-MO which experienced a single short epileptiform-like event (P<0.05). Two-way ANOVA revealed statistically significant differences between the tested groups in the number of epileptiform discharges after exposure to ASDs (group: F[1,130]=14.94, P<0.05; treatment: F[4,130]=6.83, P<0.05; group x treatment interaction: F[4,130]=7.21, P<0.05; n=12-26/group). Herein, all used drugs were equally potent. Conclusions: Although the cacna1aa partial loss-of-function in larval zebrafish might mimic some symptoms of human CACNA1A deficiency, further validation with broad range of AEDs combined with an analysis of underlying changes in the larval brain is warranted to state if it may be a useful animal model of seizures. Funding: KG was funded by a mobility grant from the Polish Ministry of Science and Higher Education within the programme „Mobilność Plus V” (decision nr 1649/1/MOB/V/17/2018/0; 01.01.2018-31.12.2018). KG has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie (grant agreement No. 798703-GEMZ-H2020-MSCA-IF-2017; 01.01.2019-31.12.2020). This work was partially supported from start-up funds of NCMM (for CVE).The authors declare no conflict of interest.