Visuomotor deficiency in panx1a knockout zebrafish is linked to dopaminergic signaling

Abstract

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play roles in the nervous system. The analysis of roles in both standard and pathological conditions benefits from a model organism with rapid development and early onset of behaviors. Such a model was developed by ablating the zebrafish panx1a gene using TALEN technology. Here, RNA-seq analysis of 6 days post fertilization larvae were confirmed by Real-Time PCR and paired with testing visual-motor behavior and in vivo electrophysiology. Results demonstrated that loss of panx1a specifically affected the expression of gene classes representing the development of the visual system and visual processing. Abnormal swimming behavior in the dark and the expression regulation of pre-and postsynaptic biomarkers suggested changes in dopaminergic signaling. Indeed, altered visuomotor behavior in the absence of functional Panx1a was evoked through D1/D2-like receptor agonist treatment and rescued with the D2-like receptor antagonist Haloperidol. Local field potentials recorded from superficial areas of the optic tectum receiving input from the retina confirmed abnormal responses to visual stimuli, which resembled treatments with a dopamine receptor agonist or pharmacological blocking of Panx1a. We conclude that Panx1a functions are relevant at a time point when neuronal networks supporting visualmotor functions undergo modifications preparing for complex behaviors of freely swimming fish. Pannexin 1 (Panx1) is an integral membrane glycoprotein forming ATP release channels in different tissues and cell types 1-5 , including neurons 6-8. In the CNS, evidence for physiological functions of Panx1 points at roles in the processing of sensory signals and learning and memory 9-11. For example, in Panx1 knockout mice, altered retinal contrast sensitivity 12 and hearing loss have been found 13,14. Also, performance in spatial learning and memory abilities such as object recognition and fear conditioning tasks are decreased 9,15,16. Intellectual disabilities, severe hearing loss, primary ovarian failure, kyphoscoliosis, and difficulties navigating in darkness were found in the first human patient identified with a homozygous Panx1 mutation 17. To form a better view of Panx1 functions in the processing of sensory information, we used the zebrafish as model organisms. Two Panx1 genes, panx1a and panx1b, originated from partial genome duplications during early teleost evolution 18,19. Although the two genes have been separated for more than 200 million years, principal channel functions are comparable to rodent or human Panx1 20. In the retina, the panx1a protein is expressed in horizontal cells 19,20 and plays essential roles in feedback from horizontal cells to cones in adult zebrafish 21-23. Here the panx1a gene was edited using transcription activator-like effector nucleases (TALEN). A loss of function mutation allowed to investigate Panx1a in 6 dpf old zebrafish larvae at a developmental stage when neuronal networks for visually guided locomotor behaviors were functional...

Panx1 channels promote both anti- and pro-seizure-like activities in the zebrafish via p2rx7 receptors and ATP signaling

“…RNA-seq data representing the transcriptomes of untreated 6dpf larvae 24,36 were analyzed next. Venn diagrams highlighted considerable differences in the number and regulation of genes when the FishEnrichR database 37,38 was data-mined for biological processes enriched in the nervous system (Supplementary Fig.…”

“…All animal work was performed at York University's zebrafish vivarium and in S2 biosafety laboratory in accordance with the Canadian Council for Animal Care guidelines after approval of the protocol by the Animal Care Committee (GZ: 2019-7-R2). The panx1 mutant lines were generated in house 24 .…”

“…In vivo electrophysiology. Published procedures were used to prepare anaesthetized 7 days post fertilization (7dpf) zebrafish larvae for in vivo electrophysiology 24,27 . Zebrafish larvae 7dpf were briefly anesthetized using 0.3mM Pancuronium bromide (Sigma-Aldrich) for 2-3 min until the touch response stopped.…”

“…Fosab's upregulation was reduced by 2-fold and jun had no significant upregulation with Pb treatment (Supplementary Table 2). RNA-seq data representing the transcriptomes of untreated 6dpf larvae 24,36 were analyzed next.…”

“…Gene-edited zebrafish lacking panx1a or panx1b 24,25 revealed opposite seizure-like phenotypes and PTZ-related morbidity. Targeted ablation of panx1b potentiates, while the absence of panx1a attenuates seizure manifestations according to recordings of in vivo local field potentials (ivLFP) and locomotor behavior.…”