Mutants of the Zebrafish K+ Channel Hcn2b Exhibit Epileptic-like Behaviors

Abstract

Epilepsy is a chronic neurological disorder that affects 50 million people worldwide. The most common form of epilepsy is idiopathic, where most of the genetic defects of this type of epilepsy occur in ion channels. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization, and are mainly expressed in the heart and central and peripheral nervous systems. In humans, four HCN genes have been described, and emergent clinical data shows that dysfunctional HCN channels are involved in epilepsy. Danio rerio has become a versatile organism to model a wide variety of diseases. In this work, we used CRISPR/Cas9 to generate hcn2b mutants in zebrafish, and characterized them molecularly and behaviorally. We obtained an hcn2b mutant allele with an 89 bp deletion that produced a premature stop codon. The mutant exhibited a high mortality rate in its life span, probably due to its sudden death. We did not detect heart malformations or important heart rate alterations. Absence seizures and moderate seizures were observed in response to light. These seizures rarely caused instant death. The results show that mutations in the Hcn2b channel are involved in epilepsy and provide evidence of the advantages of zebrafish to further our understanding of the pathogenesis of epilepsy.

Toxicological and Sedative Effects of Chipilin (Crotalaria longirostrata) Leaf Extracts Obtained by Maceration and Supercritical Fluid Extraction

“…The response of the larvae to dark–light–dark transitions was evaluated according to a reported protocol. 22 Larvae at 7 days post fertilization ( n = 16) that were exposed to working solutions with daily replacement from 2 hpf were individually transferred to a 24-well plate with 1.5 mL of E3 buffer. The plate was set above a light chamber and covered with black conical plastic that held a video camera on top.…”

In Search of a Target Gene for a Desirable Phenotype in Aquaculture: Genome Editing of Cyprinidae and Salmonidae Species

“…Genome editing has been used to model human tumors such as liver cancer [70], paraganglioma [71], skin melanoma [72], and epithelioid sarcoma [73]. Among neurologic diseases, amyotrophic lateral sclerosis [74], epilepsy [75][76][77][78][79] [105,106], inner ear [107][108][109][110][111], vestibular apparatus [111], otoliths [112], brain [89, [113][114][115][116][117], neural tissue [118][119][120][121][122][123][124][125][126][127] and nervous system [128][129][130], heart [131][132][133][134][135][136][137][138][139], cardiomyocytes [140][141]…”

“…Genome editing has been used to model human tumors such as liver cancer [ 70 ], paraganglioma [ 71 ], skin melanoma [ 72 ], and epithelioid sarcoma [ 73 ]. Among neurologic diseases, amyotrophic lateral sclerosis [ 74 ], epilepsy [ 75 , 76 , 77 , 78 , 79 ], Hirschprung’s disease [ 80 ], autism [ 81 , 82 , 83 , 84 ], spastic paraplegia [ 85 ], restless legs syndrome [ 86 ], insomnia [ 87 ], microcephaly [ 46 ], neurotransmitter function of dopamine [ 88 ], monoamine [ 89 ], and others [ 90 , 91 ]. Cleft lip and cleft palate mutants also have been tested [ 92 ].…”

Zebrafish as a Model for Neurological Disorders

“…Interestingly, the results suggested that in contrast to the human gene, fbxl3a is not essential for brain development, but is a key component of the circadian clock, as is the case in mice. Using the same technology, Rodriguez-Ortiz and Martinez-Torres [6] produced a loss-of-function mutant of hcn2b, one of the two zebrafish paralogs of the HCN2 gene encoding a hyperpolarization-activated cyclic nucleotide gated (HCN) channel. This gene has been found mutated in humans presenting generalized epilepsy, and the zebrafish hcn2b mutant exhibits epileptiform seizures and epilepsy-related behaviors, highlighting the strong evolutionary conservation of the neurochemical processes underlying neuron excitation.…”