Intralumenal docking of connexin 36 channels in the ER isolates mistrafficked protein.

The intracellular domains of connexins are essential for the assembly of gap junctions. For connexin 36 (Cx36), the major neuronal connexin, it has been shown that a dysfunctional PDZ-binding motif interferes with electrical synapse formation. However, it is still unknown how this motif coordinates the transport of Cx36. In the present study, we characterize a phenotype of Cx36 mutants that lack a functional PDZ-binding motif using HEK293T cells as an expression system. We provide evidence that an intact PDZ-binding motif is critical for proper endoplasmic reticulum (ER) export of Cx36. Removing the PDZ-binding motif of Cx36 results in ER retention and the formation of multimembrane vesicles containing gap junction-like connexin aggregates. Using a combination of site-directed mutagenesis and electron micrographs, we reveal that these vesicles consist of Cx36 channels that docked prematurely in the ER. Our data suggest a model in which ER-retained Cx36 channels reshape the ER membrane into concentric whorls that are released into the cytoplasm.

Loss of Panx1 function in zebrafish alters motor behavior in a lab-on-chip model of Parkinson's disease.

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 Roles of Calmodulin and CaMKII in Cx36 Plasticity.

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 (Cx36) 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 Cx36 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 Ca2+/calmodulin-dependent protein kinase II in integrating calcium signals to modulate the strength of electrical synapses through interactions with the gap junction protein Cx36.

Panx1b Modulates the Luminance Response and Direction of Locomotion in the Zebrafish.

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 panx1b 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.

Endocytosis of Connexin 36 is Mediated by Interaction with Caveolin-1.

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.

Pannexin-1 Deficiency Decreases Epileptic Activity in Mice.

OBJECTIVE: 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. RESULTS: 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. SIGNIFICANCE: 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.

Mechanisms of pannexin1 channel gating and regulation.

Pannexins are a family of integral membrane proteins with distinct post-translational modifications, sub-cellular localization and tissue distribution. Panx1 is the most studied and best-characterized isoform of this gene family. The ubiquitous expression, as well as its function as a major ATP release and nucleotide permeation channel, makes Panx1 a primary candidate for participating in the pathophysiology of CNS disorders. While many investigations revolve around Panx1 functions in health and disease, more recently, details started emerging about mechanisms that control Panx1 channel activity. These advancements in Panx1 biology have revealed that beyond its classical role as an unopposed plasma membrane channel, it participates in alternative pathways involving multiple intracellular compartments, protein complexes and a myriad of extracellular participants. Here, we review recent progress in our understanding of Panx1 at the center of these pathways, highlighting its modulation in a context specific manner. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

A microfluidic device for quantitative investigation of zebrafish larvae's rheotaxis.

Zebrafish is a model organism for various sensory-motor biological studies. Rheotaxis, or the ability of zebrafish to orient and swim against the water stream, is a common behavior that involves multiple sensory-motor processes such as their lateral line and visual systems. Due to the lack of a controllable and easy-to-use assay, zebrafish rheotaxis at larval stages is not well-understood. In this paper, we report a microfluidic device that can be used to apply the flow stimulus precisely and repeatedly along the longitudinal axis of individual zebrafish larvae to study their coaxial rheotaxis. We quantified rheotaxis in terms of the response rate and location along the channel at various flow velocities (9.5-38 mm.sec-1). The larvae effectively exhibited a similarly high rheotactic response at low and medium velocities (9.5 and 19 mm.sec-1); however, at high velocity of 38 mm.sec-1, despite sensing the flow, their rheotactic response decreased significantly. The flow velocity also affected the response location along the channel. At 9.5 mm.sec-1, responses were distributed evenly along the channel length while, at 19 and 38 mm.sec-1, the larvae demonstrated higher rheotaxis responses at the anterior and posterior ends of the channel, respectively. This result shows that although the response is similarly high at low and medium flow velocities, zebrafish larvae become more sensitive to the flow at medium velocity, demonstrating a modulated rheotactic behavior. Employing our device, further investigations can be conducted to study the sensory-motor systems involved in rheotaxis of zebrafish larvae and other fish species.

Differential expression of astrocytic connexins in a mouse model of prenatal alcohol exposure.

Maternal alcohol consumption during gestation can cause serious injury to the fetus, and may result in a range of physiological and behavioral impairments, including increased seizure susceptibility, that are collectively termed fetal alcohol spectrum disorder (FASD). The cellular mechanisms underlying increased seizure susceptibility in FASD are not well understood, but could involve altered excitatory coupling of neuronal populations mediated by gap junction proteins. We utilized a mouse model of the prenatal alcohol exposure (PAE) to study the expression pattern of connexin (Cx) major components of gap junctions, and pannexin proteins, which form membrane channels, in the brain of 2-3weeks old PAE and control postnatal offspring. PAE during the first trimester-equivalent period of pregnancy in mice resulted in significant up-regulation of Cx30 mRNA and Cx30 total protein in the hippocampus of PAE animals compared to age-matched controls. Surface level expression of both dimeric and monomeric Cx30 were also found to be significantly up-regulated in both hippocampus and cerebral cortex of PAE animals compared to age-matched controls. On the membrane surface, the fast migrating form of Cx43 was found to be up-regulated in the hippocampus of PAE mice. However, we did not see any up-regulation of the phosphorylated forms of Cx43 on the membrane surface. These results indicate that the expression and processing of astrocytic connexins (Cx30, Cx43) are up-regulated in the brain of PAE offspring, and these changes could play a role in the cerebral hyperexcitability observed in these animals.

A new mode of SAM domain mediated oligomerization observed in the CASKIN2 neuronal scaffolding protein.

BACKGROUND: CASKIN2 is a homolog of CASKIN1, a scaffolding protein that participates in a signaling network with CASK (calcium/calmodulin-dependent serine kinase). Despite a high level of homology between CASKIN2 and CASKIN1, CASKIN2 cannot bind CASK due to the absence of a CASK Interaction Domain and consequently, may have evolved undiscovered structural and functional distinctions. RESULTS: We demonstrate that the crystal structure of the Sterile Alpha Motif (SAM) domain tandem (SAM1-SAM2) oligomer from CASKIN2 is different than CASKIN1, with the minimal repeating unit being a dimer, rather than a monomer. Analytical ultracentrifugation sedimentation velocity methods revealed differences in monomer/dimer equilibria across a range of concentrations and ionic strengths for the wild type CASKIN2 SAM tandem and a structure-directed double mutant that could not oligomerize. Further distinguishing CASKIN2 from CASKIN1, EGFP-tagged SAM tandem proteins expressed in Neuro2a cells produced punctae that were distinct both in shape and size. CONCLUSIONS: This study illustrates a new way in which neuronal SAM domains can assemble into large macromolecular assemblies that might concentrate and amplify synaptic responses.

Internal ribosomal entry site (IRES) activity generates endogenous carboxyl-terminal domains of Cx43 and is responsive to hypoxic conditions.

Connexin43 (Cx43) is the most abundant gap junction protein in higher vertebrate organisms and has been shown to be involved in junctional and non-junctional functions. In addition to the expression of full-length Cx43, endogenously produced carboxyl-terminal segments of Cx43 have been described and have been suggested to be involved in manifold biological functions, such as hypoxic preconditioning and neuronal migration. Molecular aspects, however, behind the separate generation of carboxyl-terminal segments of Cx43 have remained elusive. Here we report on a mechanism that may play a key role in the separate production of these domains. First, stringent evidence derived from siRNA treatment and specific knockouts revealed significant loss of the low molecular weight fragments of Cx43. By applying a dicistronic vector strategy on transfected cell lines, we were able to identify putative IRES activity (nucleotides 442­637) in the coding region of Cx43, which resides upstream from the nucleotide sequence encoding the carboxyl terminus (nucleotides 637­1149). Functional responsiveness of the endogenous expression of Cx43 fragments to hypoxic/ischemic treatment was evaluated in in vitro and in vivo models, which led to a significant increase of the fastest migrating form (20 kDa) under conditions of metabolic deprivation. By nano-MS spectrometry, we achieved stringent evidence of the identity of the 20-kDa segment as part of the carboxyl-terminal domain of full-length Cx43. Our data prove the existence of endogenously expressed carboxyl-terminal domains, which may serve as valuable tools for further translational application in ischemic disorders.

Characterization of cytoplasmic polyadenylation element binding 2 protein expression and its RNA binding activity.

Cytoplasmic polyadenylation element binding (CPEB) proteins are translational regulators that are involved in the control of cellular senescence, synaptic plasticity, learning, and memory. We have previously found all four known CPEB family members to be transcribed in the mouse hippocampus. Aside from a brief description of CPEB2 in mouse brain, not much is known about its biological role. Hence, this study aims to investigate CPEB2 expression in mouse brain. With reverse transcription polymerase chain reaction (RT-PCR) of total mouse brain cDNA, we identified four distinct CPEB2 splice variants. Single-cell RT-PCR showed that CPEB2 is predominantly expressed in neurons of the juvenile and adult brain and that individual cells express different sets of splice variants. Staining of brain slices with a custom-made CPEB2 antibody revealed ubiquitous expression of the protein in many brain regions, including hippocampus, striatum, thalamus, cortex, and cerebellum. We also found differential expression of CPEB2 protein in excitatory, inhibitory, and dopaminergic neurons. In primary hippocampal cultures, the subcellular localization of CPEB2 in neurons and astrocytes resembled that of CPEB1. Electrophoretic mobility shift assay and RNA coimmunoprecipitation revealed CPEB2 interaction with ß-catenin and Ca(2+) /calmodulin-dependent protein kinase II (both established CPEB1 targets), indicating an overlap in RNA binding specificity between CPEB1 and CPEB2. Furthermore, we identified ephrin receptor A4 as a putative novel target of CPEB2. In conclusion, our study identifies CPEB2 splice variants to be differentially expressed among individual cells and across cell types of the mouse hippocampus, and reveals overlapping binding specificity between CPEB2 and CPEB1.

ATOH8, a regulator of skeletal myogenesis in the hypaxial myotome of the trunk.

The embryonic muscles of the axial skeleton and limbs take their origin from the dermomyotomes of the somites. During embryonic myogenesis, muscle precursors delaminate from the dermomyotome giving rise to the hypaxial and epaxial myotome. Mutant studies for myogenic regulatory factors have shown that the development of the hypaxial myotome differs from the formation of the epaxial myotome and that the development of the hypaxial myotome depends on the latter within the trunk region. The transcriptional networks that regulate the transition of proliferative dermomyotomal cells into the predominantly post-mitotic hypaxial myotome, as well as the eventual patterning of the myotome, are not fully understood. Similar transitions occurring during the development of the neural system have been shown to be controlled by the Atonal family of helix-loop-helix transcription factors. Here, we demonstrate that ATOH8, a member of the Atonal family, is expressed in a subset of embryonic muscle cells in the dermomyotome and myotome. Using the RNAi approach, we show that loss of ATOH8 in the lateral somites at the trunk level results in a blockage of differentiation and thus causes cells to be maintained in a predetermined state. Furthermore, we show that ATOH8 is also expressed in cultured C2C12 mouse myoblasts and becomes dramatically downregulated during their differentiation. We propose that ATOH8 plays a role during the transition of myoblasts from the proliferative phase to the differentiation phase and in the regulation of myogenesis in the hypaxial myotome of the trunk.

Synaptic transmission from horizontal cells to cones is impaired by loss of connexin hemichannels.

In the vertebrate retina, horizontal cells generate the inhibitory surround of bipolar cells, an essential step in contrast enhancement. For the last decades, the mechanism involved in this inhibitory synaptic pathway has been a major controversy in retinal research. One hypothesis suggests that connexin hemichannels mediate this negative feedback signal; another suggests that feedback is mediated by protons. Mutant zebrafish were generated that lack connexin 55.5 hemichannels in horizontal cells. Whole cell voltage clamp recordings were made from isolated horizontal cells and cones in flat mount retinas. Light-induced feedback from horizontal cells to cones was reduced in mutants. A reduction of feedback was also found when horizontal cells were pharmacologically hyperpolarized but was absent when they were pharmacologically depolarized. Hemichannel currents in isolated horizontal cells showed a similar behavior. The hyperpolarization-induced hemichannel current was strongly reduced in the mutants while the depolarization-induced hemichannel current was not. Intracellular recordings were made from horizontal cells. Consistent with impaired feedback in the mutant, spectral opponent responses in horizontal cells were diminished in these animals. A behavioral assay revealed a lower contrast-sensitivity, illustrating the role of the horizontal cell to cone feedback pathway in contrast enhancement. Model simulations showed that the observed modifications of feedback can be accounted for by an ephaptic mechanism. A model for feedback, in which the number of connexin hemichannels is reduced to about 40%, fully predicts the specific asymmetric modification of feedback. To our knowledge, this is the first successful genetic interference in the feedback pathway from horizontal cells to cones. It provides direct evidence for an unconventional role of connexin hemichannels in the inhibitory synapse between horizontal cells and cones. This is an important step in resolving a long-standing debate about the unusual form of (ephaptic) synaptic transmission between horizontal cells and cones in the vertebrate retina.

Gap junction Delta-2b (gjd2b/Cx35.1) depletion causes hyperopia and visual-motor deficiencies in the zebrafish.

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 gjd2b/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 gjd2b/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 gjd2b/Cx35.1 was depleted. The visual disturbances were reversed under low light conditions in gjd2b -/- /Cx35.1-/- larvae. Since qRT-PCR data demonstrated that two rhodopsin genes were downregulated, we speculated that rod photoreceptor cells in gjd2b/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 gjd2b/Cx35.1 in the development of the visual system and visually guided behaviors.

Pannexin 1 constitutes the large conductance cation channel of cardiac myocytes.

A large conductance (∼300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca(2+) release from the sarcoplasmic reticulum, particularly when augmented by caffeine, has been described previously in isolated cardiac myocytes. A potential candidate for this channel is pannexin 1 (Panx1), which has been shown to form large ion channels when expressed in Xenopus oocytes and mammalian cells. Panx1 function is implicated in ATP-mediated auto-/paracrine signaling, and a crucial role in several cell death pathways has been suggested. Here, we demonstrate that after culturing for 4 days LCC activity is no longer detected in myocytes but can be rescued by adenoviral gene transfer of Panx1. Endogenous LCCs and those related to expression of Panx1 share key pharmacological properties previously used for identifying and characterizing Panx1 channels. These data demonstrate that Panx1 constitutes the LCC of cardiac myocytes. Sporadic openings of single Panx1 channels in the absence of Ca(2+) release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.

A phosphodiesterase 2A isoform localized to mitochondria regulates respiration.

Mitochondria are central organelles in cellular energy metabolism, apoptosis, and aging processes. A signaling network regulating these functions was recently shown to include soluble adenylyl cyclase as a local source of the second messenger cAMP in the mitochondrial matrix. However, a mitochondrial cAMP-degrading phosphodiesterase (PDE) necessary for switching off this cAMP signal has not yet been identified. Here, we describe the identification and characterization of a PDE2A isoform in mitochondria from rodent liver and brain. We find that mitochondrial PDE2A is located in the matrix and that the unique N terminus of PDE2A isoform 2 specifically leads to mitochondrial localization of this isoform. Functional assays show that mitochondrial PDE2A forms a local signaling system with soluble adenylyl cyclase in the matrix, which regulates the activity of the respiratory chain. Our findings complete a cAMP signaling cascade in mitochondria and have implications for understanding the regulation of mitochondrial processes and for their pharmacological modulation.

An Implantable Optogenetic Neuro-Stimulator SoC With Extended Optical Pulse-Width Enabled by Supply-Variation-Immune Cycled Light-Toggling Stimulation.

The design, development, and experimental validation of an inductively-powered four-channel optical neuro-stimulator system on a chip (SoC) with on-chip neural recording, temperature monitoring, signal processing, and bidirectional wireless data communication are presented. A biologically-inspired optical stimulation approach is employed that extends the limitations on the stimulation pulse-width and frequency (i.e., enabling wirelessly-powered optical stimulation at very low frequencies (e.g., 10 Hz)) while significantly reducing the required on-device storage capacitor size. The biological efficacy of the proposed approach is validated and compared with conventional stimulation through in vitro experiments. The stimulator's energy efficiency is enhanced by employing a high-gain (850 A/A) current amplifier/driver in each channel that steers up to 10 mA into the optical source with an excellent linearity ( 0.5LSB), while 1) yielding the lowest-in-literature required voltage headroom, and 2) being insensitive to large (up to 12%) supply voltage drops, which is ideal for battery-less implantable devices. Additionally, to maximize the percentage of the generated optical power that reaches the targeted cells (thus, further energy efficiency enhancement), inkjet printing is utilized to fabricate custom-designed optical µlenses that are placed directly on top of the silicon SoC to enhance the generated light's directivity by > 30×. An electrophysiological recording channel for real-time monitoring of the stimulation efficacy and a high-precision (0.1 °C resolution) temperature readout circuit for shutting off stimulation upon detection of an unsafe temperature increase are also integrated on the chip. Additionally, the SoC hosts an ASK receiver and an LSK transmitter for downlink and uplink wireless data communication, respectively. The SoC is fabricated in a standard 130 nm CMOS process and occupies 6 mm 2. Measurement results for different sensory and communication blocks are presented, as well as in vitro experimental validation results showing simultaneous optical stimulation, electrical recording, and calcium imaging.

Dopaminergic signaling regulates zebrafish larvae's response to electricity.

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.

Simultaneous screening of zebrafish larvae cardiac and respiratory functions: a microfluidic multi-phenotypic approach.

Multi-phenotypic screening of multiple zebrafish larvae plays an important role in enhancing the quality and speed of biological assays. Many microfluidic platforms have been presented for zebrafish phenotypic assays, but multi-organ screening of multiple larvae, from different needed orientations, in a single device that can enable rapid and large-sample testing is yet to be achieved. Here, we propose a multi-phenotypic quadruple-fish microfluidic chip for simultaneous monitoring of heart activity and fin movement of 5-7-day postfertilization zebrafish larvae trapped in the chip. In each experiment, fin movements of four larvae were quantified in the dorsal view in terms of fin beat frequency (FBF). Positioning of four optical prisms next to the traps provided the lateral views of the four larvae and enabled heart rate (HR) monitoring. The device's functionality in chemical testing was validated by assessing the impacts of ethanol on heart and fin activities. Larvae treated with 3% ethanol displayed a significant drop of 13.2 and 35.8% in HR and FBF, respectively. Subsequent tests with cadmium chloride highlighted the novel application of our device for screening the effect of heavy metals on cardiac and respiratory function at the same time. Exposure to 5 $\mu$g/l cadmium chloride revealed a significant increase of 8.2% and 39.2% in HR and FBF, respectively. The device can be employed to monitor multi-phenotypic behavioral responses of zebrafish larvae induced by chemical stimuli in various chemical screening assays, in applications such as ecotoxicology and drug discovery.