Abstract
Pannexin 1 (Panx1) forms ATP‐permeable membrane channels that play roles in purinergic signaling in the nervous system. A link between Panx1 activity and neurodegenerative disorders ...including Parkinson's disease (PD) has been suggested, but experimental evidence is limited. Here, a zebrafish model of PD was produced by exposing
panx1a
+/+
and
panx1a
−/−
zebrafish larvae to 6‐hydroxydopamine (6‐OHDA). Electrical stimulation in a microfluidic chip and quantitative real‐time‐qPCR of zebrafish larvae tested the role of Panx1 in both pathological and normal conditions. After 72‐h treatment with 6‐OHDA, the electric‐induced locomotor activity of 5 days post fertilization (5dpf)
panx1a
+/+
larvae were reduced, while the stimulus did not affect locomotor activity of age‐matched
panx1a
−/−
larvae. A RT‐qPCR analysis showed an increase in the expression of genes that are functionally related to dopaminergic signaling, like the tyrosine hydroxylase
(th2)
and the leucine‐rich repeat kinase 2
(lrrk2)
. Extending the 6‐OHDA treatment duration to 120 h caused a significant reduction in the locomotor response of 7dpf
panx1a
−/−
larvae compared to the untreated
panx1a
−/−
group. The RT‐qPCR data showed a reduced expression of dopaminergic signaling genes in both genotypes. It was concluded that the absence of Panx1a channels compromised dopaminergic signaling in 6‐OHDA‐treated zebrafish larvae and that the increase in the expression of dopaminergic genes was transient, most likely due to a compensatory upregulation. We propose that zebrafish Panx1a models offer opportunities to shed light on PD's physiological and molecular basis. Panx1a might play a role on the progression of PD, and therefore deserves further investigation.
The zebrafish is a powerful model to investigate the developmental roles of electrical synapses because many signaling pathways that regulate the development of the nervous system are highly ...conserved from fish to humans. Here, we provide evidence linking the mammalian connexin-36 (Cx36) ortholog
/Cx35.1, a major component of electrical synapses in the zebrafish, with a refractive error in the context of morphological, molecular, and behavioral changes of zebrafish larvae. Two abnormalities were identified. The optical coherence tomography analysis of the adult retina confirmed changes to the refractive properties caused by eye axial length reduction, leading to hyperopic shifts. The
/Cx35.1 depletion was also correlated with morphological changes to the head and body ratios in larvae. The differential expression of Wnt/ß-catenin signaling genes, connexins, and dopamine receptors suggested a contribution to the observed phenotypic differences. The alteration of visual-motor behavioral responses to abrupt light transitions was aggravated in larvae, providing evidence that cone photoreceptor cell activity was enhanced when
/Cx35.1 was depleted. The visual disturbances were reversed under low light conditions in
/Cx35.1
larvae. Since qRT-PCR data demonstrated that two rhodopsin genes were downregulated, we speculated that rod photoreceptor cells in
/Cx35.1
larvae were less sensitive to bright light transitions, thus providing additional evidence that a cone-mediated process caused the VMR light-ON hyperactivity after losing Cx35.1 expression. Together, this study provides evidence for the role of
/Cx35.1 in the development of the visual system and visually guided behaviors.
Pannexin1 (Panx1) can form ATP-permeable channels that play roles in the physiology of the visual system. In the zebrafish two ohnologs of Panx1, Panx1a and Panx1b, have unique and shared channel ...properties and tissue expression patterns. Panx1a channels are located in horizontal cells of the outer retina and modulate light decrement detection through an ATP/pH-dependent mechanisms and adenosine/dopamine signaling. Here, we decipher how the strategic localization of Panx1b channels in the inner retina and ganglion cell layer modulates visually evoked motor behavior. We describe a
knockout model generated by TALEN technology. The RNA-seq analysis of 6 days post-fertilization larvae is confirmed by real-time PCR and paired with testing of locomotion behaviors by visual motor and optomotor response tests. We show that the loss of Panx1b channels disrupts the retinal response to an abrupt loss of illumination and it decreases the larval ability to follow leftward direction of locomotion in low light conditions. We concluded that the loss of Panx1b channels compromises the final output of luminance as well as motion detection. The Panx1b protein also emerges as a modulator of the circadian clock system. The disruption of the circadian clock system in mutants suggests that Panx1b could participate in non-image forming processes in the inner retina.
Anatomical and electrophysiological evidence that gap junctions and electrical coupling occur between neurons was initially confined to invertebrates and nonmammals and was thought to be a primitive ...form of synaptic transmission. More recent studies revealed that electrical communication is common in the mammalian central nervous system (CNS), often coexisting with chemical synaptic transmission. The subsequent progress indicated that electrical synapses formed by the gap junction protein connexin-36 (
) and its paralogs in nonmammals constitute vital elements in mammalian and fish synaptic circuitry. They govern the collective activity of ensembles of coupled neurons, and
gap junctions endow them with enormous adaptive plasticity, like that seen at chemical synapses. Moreover, they orchestrate the synchronized neuronal network activity and rhythmic oscillations that underlie the fundamental integrative processes, such as memory and learning. Here, we review the available mechanistic evidence and models that argue for the essential roles of calcium, calmodulin, and the Ca
/calmodulin-dependent protein kinase II in integrating calcium signals to modulate the strength of electrical synapses through interactions with the gap junction protein
.
The gap junctional protein connexin 36 (Cx36) has been co-purified with the lipid raft protein caveolin-1 (Cav-1). The relevance of an interaction between the two proteins is unknown. In this study, ...we explored the significance of Cav-1 interaction in the context of intracellular and membrane transport of Cx36. Coimmunoprecipitation assays and Förster resonance energy transfer analysis (FRET) were used to confirm the interaction between the two proteins in the Neuro 2a cell line. We found that the Cx36 and Cav-1 interaction was dependent on the intracellular calcium levels. By employing different microscopy techniques, we demonstrated that Cav-1 enhances the vesicular transport of Cx36. Pharmacological interventions coupled with cell surface biotinylation assays and FRET analysis revealed that Cav-1 regulates membrane localization of Cx36. Our data indicate that the interaction between Cx36 and Cav-1 plays a role in the internalization of Cx36 by a caveolin-dependent pathway.
•A device to study the electric response of semi-mobile zebrafish larva in detail.•Zebrafish larvae sense the electric field (EF) direction and respond differently.•Voltage drop across larva’s body ...affects their response duration and tail frequency.•Exposure to multiple EF pulses result in decay in zebrafish larva’s response.•Two studies show that zebrafish larvae show habituation to EF pulses in the device.
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We previously showed that electric current induces zebrafish larvae to move towards the anode pole along a microchannel. For a larva with a fixed head and a moving tail, we observed that the response to electricity depended on the current magnitude. The effects of electric signal direction, voltage magnitude and habituation to repeated exposures to electric pulses were not characterized. Here, this knowledge gap was addressed by exploiting them in a microfluidic device with a head-trap to immobilize a zebrafish larva and a downstream chamber for tail movement phenotypic characterization based on response duration (RD) and tail beat frequency (TBF). We first assessed larvae’s response to electric current direction (at 3 µA) and voltage magnitude. Changing the current direction significantly altered the RD and TBF with long and low-frequency responses seen when the anode was positioned at the larvae’s tail. The electric voltage had a significant effect on larvae’s locomotion with long RD and low TBF observed at 5.6 V in the range of 1.3–9 V. We also demonstrated that the zebrafish locomotor response to repeated 3 µA current pulses diminished with dependency on the interstimulus interval. However, the diminished response was fully recovered after a 5-min resting period or introduction of a novel light stimulus (i.e., habituation-dishabituation strategy). Therefore, electric response suppression in zebrafish was attributed to the habituation as a form of non-associative learning. Our microfluidic platform has a broad application potential in behavioral neuroscience to study cognitive phenotypes, fundamental studies on the biological roots of electric response, and in pharmacological screening.
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 visual-motor functions undergo modifications preparing for complex behaviors of freely swimming fish.
Electrical stimulation of brain or muscle activities has gained attention for studying the molecular and cellular mechanisms involved in electric‐induced responses. We recently showed zebrafish's ...response to electricity. Here, we hypothesized that this response is affected by the dopaminergic signaling pathways. The effects of multiple dopamine agonists and antagonists on the electric response of 6 days‐postfertilization zebrafish larvae were investigated using a microfluidic device with enhanced control of experimentation and throughput. All dopamine antagonists decreased locomotor activities, while dopamine agonists did not induce similar behaviors. The D2‐selective dopamine agonist quinpirole enhanced the movement. Exposure to nonselective and D1‐selective dopamine agonists apomorphine and SKF‐81297 caused no significant change in the electric response. Exposing larvae that were pretreated with nonselective and D2‐selective dopamine antagonists butaclamol and haloperidol to apomorphine and quinpirole, respectively, restored the electric locomotion. These results reveal a correlation between electric response and dopamine signaling pathway. Furthermore, they demonstrate that electric‐induced zebrafish larvae locomotion can be conditioned by modulating dopamine receptor functions. Our electrofluidic assay has profound application potential for fundamental electric‐induced response research and brain disorder studies especially those related to the dopamine imbalance and as a chemical screening method when investigating biological pathways and behaviors.
Graphical and Lay Summary
The current work shows the first application of our multi‐fish microfluidic platform in chemical screening to answer novel questions such as examining if dopaminergic pathways are involved in zebrafish larvae electric response. Our chip can also be used by many in the fields of organism‐on‐a‐chip and zebrafish to investigate fundamental questions about zebrafish behavior or to perform larger‐scale neurobehavioral monitoring with quantitative phenotype readouts.
Pannexin-1 (Panx1) is suspected of having a critical role in modulating neuronal excitability and acute neurological insults. Herein, we assess the changes in behavioral and electrophysiological ...markers of excitability associated with Panx1 via three distinct models of epilepsy. Methods Control and Panx1 knockout C57Bl/6 mice of both sexes were monitored for their behavioral and electrographic responses to seizure-generating stimuli in three epilepsy models-(1) systemic injection of pentylenetetrazol, (2) acute electrical kindling of the hippocampus and (3) neocortical slice exposure to 4-aminopyridine. Phase-amplitude cross-frequency coupling was used to assess changes in an epileptogenic state resulting from Panx1 deletion.
Seizure activity was suppressed in Panx1 knockouts and by application of Panx1 channel blockers, Brilliant Blue-FCF and probenecid, across all epilepsy models. In response to pentylenetetrazol, WT mice spent a greater proportion of time experiencing severe (stage 6) seizures as compared to Panx1-deficient mice. Following electrical stimulation of the hippocampal CA3 region, Panx1 knockouts had significantly shorter evoked afterdischarges and were resistant to kindling. In response to 4-aminopyridine, neocortical field recordings in slices of Panx1 knockout mice showed reduced instances of electrographic seizure-like events. Cross-frequency coupling analysis of these field potentials highlighted a reduced coupling of excitatory delta-gamma and delta-HF rhythms in the Panx1 knockout.
These results suggest that Panx1 plays a pivotal role in maintaining neuronal hyperexcitability in epilepsy models and that genetic or pharmacological targeting of Panx1 has anti-convulsant effects.
Background
Microfluidic devices are being used for phenotypic screening of zebrafish larvae in fundamental and pre‐clinical research. A challenge for the broad use of these microfluidic devices is ...their low throughput, especially in behavioral assays. Previously, we introduced the tail locomotion of a semi‐mobile zebrafish larva evoked on‐demand with electric signal in a microfluidic device. Here, we report the lessons learned for increasing the number of specimens from one to four larvae in this device.
Methods and Results
Multiple parameters including loading and testing time per fish and loading and orientation efficiencies were refined to optimize the performance of modified designs. Flow and electric field simulations within the final device provided insight into the flow behavior and functionality of traps when compared to previous single‐larva devices. Outcomes led to a new design which decreased the testing time per larva by ≈60%. Further, loading and orientation efficiencies increased by more than 80%. Critical behavioral parameters such as response duration and tail beat frequency were similar in both single and quadruple‐fish devices.
Conclusion
The developed microfluidic device has significant advantages for greater throughput and efficiency when behavioral phenotyping is required in various applications, including chemical testing in toxicology and gene screening.
Graphical and Lay Summary
Monitoring the behavior of multiple zebrafish larvae on‐demand and at the same time is important in biological investigations. A quadruple‐fish electro‐fluidic device for behavioral phenotyping of zebrafish larvae in response to electricity is provided in this paper. This methodology can be used in many assays involving zebrafish as a model organism for disease studies and drug screening.
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