stim2b Knockout Induces Hyperactivity and Susceptibility to Seizures in Zebrafish Larvae

Abstract

In neurons, stromal interaction molecule (STIM) proteins regulate store-operated Ca2+ entry (SOCE) and are involved in calcium signaling pathways. However, STIM activity in neurological diseases is unclear and should be clarified by studies that are performed in vivo rather than in cultured cells in vitro. The present study investigated the role of neuronal Stim2b protein in zebrafish. We generated stim2b knockout zebrafish, which were fertile and had a regular lifespan. Using various behavioral tests, we found that stim2b−/− zebrafish larvae were hyperactive compared with wild-type fish. The mutants exhibited increases in mobility and thigmotaxis and disruptions of phototaxis. They were also more sensitive to pentylenetetrazol and glutamate treatments. Using lightsheet microscopy, a higher average oscillation frequency and higher average amplitude of neuronal Ca2+ oscillations were observed in stim2b−/− larvae. RNA sequencing detected upregulation of the annexin 3a and gpr39 genes and downregulation of the rrm2, neuroguidin, and homer2 genes. The latter gene encodes a protein that is involved in several processes that are involved in Ca2+ homeostasis in neurons, including metabotropic glutamate receptors. We propose that Stim2b deficiency in neurons dysregulates SOCE and triggers changes in gene expression, thereby causing abnormal behavior, such as hyperactivity and susceptibility to seizures.

Knockout of stim2a Increases Calcium Oscillations in Neurons and Induces Hyperactive-Like Phenotype in Zebrafish Larvae

“…The zebrafish genome has many duplicated genes, including stim2a and stim2b [ 10 , 45 ]. Thus, in contrast to early lethality in Stim2 knockout mouse lines, the knockout of one zebrafish isoform yields a viable animal, which we demonstrated for stim2a knockout fish in the present study and stim2b knockout fish in our recent study [ 50 ].…”

“…Pentylenetetrazol induces seizures, and zebrafish were shown to exhibit behavioral reactions in response to this treatment beginning at 3 dpf [ 53 , 54 ]. Glutamate increases neuronal activity and locomotor activity [ 50 , 55 ]. Importantly, glutamate was shown to penetrate zebrafish larvae and reach brain neurons.…”

“…Importantly, glutamate was shown to penetrate zebrafish larvae and reach brain neurons. Thus, the main effect of this drug on animal behavior is primarily attributable to an increase in brain activity [ 50 , 55 ]. We performed PTZ treatment from a lower to a higher dose [ 53 ].…”

“…Thus, complex behavior cannot be analyzed in S tim2 knockout mice [ 31 , 32 ]. In zebrafish, the nervous system develops at an early stage and allows larvae to exhibit complex behavior [ 42 , 50 , 59 ]. Additionally, because of larval transparency, in vivo Ca 2+ measurements can be performed in neurons [ 45 , 60 ].…”

“…Zebrafish Stim1 was shown to be crucial for the axon guidance of motor neurons and the regulation of calcium signaling [ 49 ]. We recently showed that the knockout of one orthologue of Stim2 , stim2b , caused hyperactivity and increased the susceptibility to seizures in zebrafish larvae [ 50 ]. Considering that Stim2 isoforms might have diverse effects on brain activity, the role of Stim2a should also be investigated.…”