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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., Competing Interests: Conflicts of interests The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

This paper presents an energy-efficient mm-scale self-contained bidirectional optogenetic neuro-stimulator, which employs a novel highly-linear μLED driving circuit architecture as well as inkjet-printed custom-designed optical μlenses for light directivity enhancement. The proposed current-mode μLED driver performs linear control of optical stimulation for the entire target range ( 10 mA) while requiring the smallest reported headroom, yielding a significant boost in the energy conversion efficiency. A 30.46× improvement in the power delivery efficiency to the target tissue is achieved by employing a pair of printed optical μlenses. The fabricated SoC also integrates two recording channels for LFP recording and digitization, as well as power management blocks. A micro-coil is also embedded on the chip to receive inductive power and our experimental results show a PTE of 2.24 % for the wireless link. The self-contained system including the μLEDs, μlenses and the capacitors required by the power management blocks is sized 6 mm 3 and weighs 12.5 mg. Full experimental measurement results for electrical and optical circuitry as well as in vitro measurement results are reported.

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.

Pannexin 1 (Panx1) is a ubiquitously expressed hexameric integral membrane protein known to function as an adenosine triphosphate (ATP) release channel. Panx1 proteins exist in unglycosylated core form (Gly0). They undergo critical post-translational modifications forming the high mannose glycosylation state (Gly1) in the endoplasmic reticulum (ER) and the complex glycosylation state (Gly2) in the Golgi apparatus. The regulation of transition from the ER to the cell membrane is not fully understood. Using site-specific mutagenesis, dye uptake assays, and interaction testing, we identified two conserved aromatic residues, Trp123 and Tyr205, in the transmembrane domains 2 and 3 of the zebrafish panx1a protein . Results suggest that both residues primarily govern the assembly of panx1a subunits into channels, with mutant proteins failing to interact. The results provide insight into a mechanism enabling regulation of Panx1 oligomerization, glycosylation, and trafficking., Competing Interests: The authors declare no conflict of interest.

Behavioral responses of zebrafish larvae to environmental cues are important functional readouts that should be evoked on-demand and studied phenotypically in behavioral, genetical and developmental investigations. Very recently, it was shown that zebrafish larvae execute a voluntary and oriented movement toward the positive electrode of an electric field along a microchannel. Phenotypic characterization of this response was not feasible due to larva's rapid movement along the channel. To overcome this challenge, a microfluidic device was introduced to partially immobilize the larva's head while leaving its mid-body and tail unrestrained in a chamber to image motor behaviors in response to electric stimulation, hence achieving quantitative phenotyping of the electrically evoked movement in zebrafish larvae. The effect of electric current on the tail-beat frequency and response duration of 5-7 days postfertilization zebrafish larvae was studied. Investigations were also performed on zebrafish exposed to neurotoxin 6-hydroxydopamine and larvae carrying a pannexin1a (panx1a) gene knockout, as a proof of principle applications to demonstrate on-demand movement behavior screening in chemical and mutant assays. We demonstrated for the first time that 6-hydroxydopamine leads to electric response impairment, levodopa treatment rescues the response and panx1a is involved in the electrically evoked movement of zebrafish larvae. We envision that our technique is broadly applicable as a screening tool to quantitatively examine zebrafish larvae's movements in response to physical and chemical stimulations in investigations of Parkinson's and other neurodegenerative diseases, and as a tool to combine recent advances in genome engineering of model organisms to uncover the biology of electric response., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Connexin-36 (Cx36) electrical synapses strengthen transmission in a calcium/calmodulin (CaM)/calmodulin-dependent kinase II (CaMKII)-dependent manner similar to a mechanism whereby the N-methyl-D-aspartate (NMDA) receptor subunit NR2B facilitates chemical transmission. Since NR2B-microtubule interactions recruit receptors to the cell membrane during plasticity, we hypothesized an analogous modality for Cx36. We determined that Cx36 binding to tubulin at the carboxy-terminal domain was distinct from Cx43 and NR2B by binding a motif overlapping with the CaM and CaMKII binding motifs. Dual patch-clamp recordings demonstrated that pharmacological interference of the cytoskeleton and deleting the binding motif at the Cx36 carboxyl-terminal (CT) reversibly abolished Cx36 plasticity. Mechanistic details of trafficking to the gap-junction plaque (GJP) were probed pharmacologically and through mutational analysis, all of which affected GJP size and formation between cell pairs. Lys279, Ile280, and Lys281 positions were particularly critical. This study demonstrates that tubulin-dependent transport of Cx36 potentiates synaptic strength by delivering channels to GJPs, reinforcing the role of protein transport at chemical and electrical synapses to fine-tune communication between neurons., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

A bstract: This paper focuses on cytotoxicity examination of superparamagnetic iron oxide nanoparticles (SPIONs) using different methods, including impedance spectroscopy. Recent advances of SPIONs for clinical and research applications have triggered the need to understand their effects in cells. Despite the great advances in adapting various biological and chemical methods to assess in-vitro toxicity of SPIONs, less attention has been paid on the development of a high throughput label-free screening platform to study the interaction between the cells and nanoparticles including SPIONs. In this paper, we have taken the first step toward this goal by proposing a label-free impedimetric method for monitoring living cells treated with SPIONs. We demonstrate the effect of SPIONs on the adhesion, growth, proliferation, and viability of neuroblastoma 2A (N2a) cells using impedance spectroscopy as a label-free method, along with other standard microscopic and cell viability testing methods as control methods. Our results have shown a decreased viability of the cells as the concentration of SPIONs increases with percentages of 59%, 47%, and 40% for 100 µg/mL (C4), 200 µg/mL (C5), 300 µg/mL (C6), respectively. Although all SPIONs concentrations have allowed the growth of cells within 72 hours, C4, C5, and C6 showed slower growth compared to the control (C1). The growth and proliferation of N2a cells are faster in the absence or low concentration of SPIONS. The percent coefficient of variation (% CV) was used to compare cell concentrations obtained by TBDE assay and a Scepter cell counter. Results also showed that the lower the SPIONs concentration, the lower the impedance is expected to be in the sensing electrodes without the cells. Meanwhile, the variation of surface area (∆S) was affected by the concentration of SPIONs. It was observed that the double layer capacitance was almost constant because of the higher attachment of cells, the lower surface area coated by SPIONs. In conclusion, impedance changes of electrodes exposed to the mixture of cells and SPIONs offer a wide dynamic range (>1 MΩ using Electric Cell-substrate Impedance electrodes) suitable for cytotoxicity studies. Based on impedance based, viability testing and microscopic methods' results, SPIONs concentrations higher than 100 ug/mL and 300 ug/mL cause minor and major effects, respectively. We propose that a high throughput impedance-based label-free platform provides great advantages for studying SPIONs in a cell-based context, opening a window of opportunity to design and test the next generation of SPIONs with reduced toxicity for biomedical or medical applications., Competing Interests: The authors declare no conflict of interest.

In mammals, the pannexin gene family consists of three members (Panx1, 2, 3), which represent a class of integral membrane channel proteins sharing some structural features with chordate gap junction proteins, the connexins. Since their discovery in the early 21st century, pannexin expression has been detected throughout the vertebrate body including eye, ear, nose and tongue, making the investigation of the roles of this new class of channel protein in health and disease very appealing. The localization in sensory organs, coupled with unique channel properties and associations with major signaling pathways make Panx1, and its relative's, significant contributors for fundamental functions in sensory perception. Until recently, cell-based studies were at the forefront of pannexin research. Lately, the availability of mice with genetic ablation of pannexins opened new avenues for testing pannexin functions and behavioural phenotyping. Although we are only at the beginning of understanding the roles of pannexins in health and disease, this review summarizes recent advances in elucidating the various emerging roles pannexins play in sensory systems, with an emphasis on unresolved conflicts., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Pannexin-1 (Panx1) expression is raised in several animal seizure models and in resected human epileptic brain tissue, suggesting relevance to epilepsy. Multiple factors that are characteristic of seizures are thought to regulate Panx1 channel opening, including elevated levels of extracellular K + . Panx1, when open, 1) releases ATP, glutamate, and other metabolites into the extracellular medium, and 2) may depolarize the membrane due to a channel reversal potential around 0mV. Resultant ATP release from stimulated Panx1 can activate purinergic receptors, including P2X7 receptors. Glutamate and other signaling molecules released by Panx1 opening may have both excitatory and inhibitory actions on seizure generation. This review examines the critical and complex roles of Panx1 channels in epilepsy, which could provide a basis for future therapeutics., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Pannexins (Panx) are integral membrane proteins, with Panx1 being the best-characterized member of the protein family. Panx1 is implicated in sensory processing, and knockout (KO) animal models have become the primary tool to investigate the role(s) of Panx1 in sensory systems. Extending previous work from our group on primary olfaction, the expression patterns of Panxs in the vomeronasal organ (VNO), an auxiliary olfactory sense organ with a role in reproduction and social behavior, were compared. Using qRT-PCR and Immunohistochemistry (IHC), we confirmed the loss of Panx1, found similar Panx2 expression levels in both models, and a significant upregulation of Panx3 in mice with a global ablation of Panx1. Specifically, Panx3 showed upregulated expression in nerve fibers of the non-sensory epithelial layer in juvenile and adult KO mice and in the sensory layer of adults, which overlaps with Panx1 expression areas in WT populations. Since both social behavior and evoked ATP release in the VNO was not compromised in KO animals, we hypothesized that Panx3 could compensate for the loss of Panx1. This led us to compare Panx1 and Panx3 channels in vitro , demonstrating similar dye uptake and ATP release properties. Outcomes of this study strongly suggest that Panx3 may functionally compensate for the loss of Panx1 in the VNO of the olfactory system, ensuring sustained chemosensory processing. This finding extends previous reports on the upregulation of Panx3 in arterial walls and the skin of Panx1 KO mice, suggesting that roles of Panx1 warrant uncharacterized safeguarding mechanisms involving Panx3.

The zebrafish is a lower vertebrate model organism offering multiple applications for both fundamental and biomedical research into the nervous system from genes to behaviour. Investigation of zebrafish larvae's movement in response to various stimuli, which involves the dopaminergic system, is of interest in the field of sensory-motor integration. Nevertheless, the conventional methods of movement screening in Petri dishes and multi-well plates are mostly qualitative, uncontrollable, and inaccurate in terms of stimulus delivery and response analysis. We recently presented a microfluidic device built as a versatile platform for fluid flow stimulation and high speed time-lapse imaging of rheotaxis behaviour of zebrafish larvae. Here, we describe for the first time that this microfluidic device can also be used to test zebrafish larvae's sense of the electric field and electrotaxis in a systemic manner. We further show that electrotaxis is correlated with the dopamine signalling pathway in a time of day dependent manner and by selectively involving the D2-like dopamine receptors. The primary outcomes of this research opens avenues to study the molecular and physiological basis of electrotaxis, the effects of known agonist and antagonist compounds on the dopaminergic system, and the screen of novel pharmacological tools in the context of neurodegenerative disorders. We propose that this microfluidic device has broad application potential, including the investigation of complex stimuli, biological pathways, behaviors, and brain disorders.

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Retinal horizontal cells (HCs) feed back negatively to cone photoreceptors and in that way generate the center/surround organization of bipolar cell receptive fields. The mechanism by which HCs inhibit photoreceptors is a matter of debate. General consensus exists that horizontal cell activity leads to the modulation of the cone Ca-current. This modulation has two components, one fast and the other slow. Several mechanisms for this modulation have been proposed: a fast ephaptic mechanism, and a slow pH mediated mechanism. Here we test the hypothesis that the slow negative feedback signal from HCs to cones is mediated by Panx1 channels expressed at the tips of the dendrites of horizontal cell. We generated zebrafish lacking Panx1 and found that the slow component of the feedback signal was strongly reduced in the mutants showing that Panx1 channels are a fundamental part of the negative feedback pathway from HCs to cones.

The zebrafish larva is an important vertebrate model for sensory-motor integration studies, genetic screening, and drug discovery because of its excellent characteristics such as optical transparency, genetic manipulability, and genetic similarity to humans. Operations such as precise manipulation of zebrafish larvae, controlled exposure to chemicals, and behavioural monitoring are of utmost importance to the abovementioned studies. In this work, a novel microfluidic device is presented to easily stabilize an individual larva's head using a microfluidic trap while leaving the majority of the body and the tail unhindered to move freely in a downstream chamber. The device is equipped with a microvalve to prevent the larva's escape from the trap and a microchannel beside the larva's head to expose it to chemicals at desired concentrations and times, while investigating multiple behaviours such as the tail, eye, and mouth movement frequencies. An in situ air bubble removal module was also incorporated to increase the yield of experiments. The functionality of our device in comparison to a conventional droplet-based technique was tested using l-arginine exposure and viability assays. We found that the larvae in the device and the droplet exhibit similar tail and eye response trends to nM-mM concentrations of l-arginine, and that the survival of the larvae is not affected by the device. However, the tail responses in the device were numerically higher than the droplet-tested larvae at nM-mM l-arginine concentrations. In the future, our device has the potential to be used for conducting simultaneous whole-brain functional imaging, upon optimized immobilization of the brain, and behavioural analysis to uncover differences between diseased and healthy states in zebrafish.

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.

Functional plasticity of neuronal gap junctions involves the interaction of the neuronal connexin36 with calcium/calmodulin-dependent kinase II (CaMKII). The important relationship between Cx36 and CaMKII must also be considered in the context of another protein partner, Ca 2+ loaded calmodulin, binding an overlapping site in the carboxy-terminus of Cx36. We demonstrate that CaM and CaMKII binding to Cx36 is calcium-dependent, with Cx36 able to engage with CaM outside of the gap junction plaque. Furthermore, Ca 2+ loaded calmodulin activates Cx36 channels, which is different to other connexins. The NMR solution structure demonstrates that CaM binds Cx36 in its characteristic compact state with major hydrophobic contributions arising from W277 at anchor position 1 and V284 at position 8 of Cx36. Our results establish Cx36 as a hub binding Ca 2+ loaded CaM and they identify this interaction as a critical step with implications for functions preceding the initiation of CaMKII mediated plasticity at electrical synapses.

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.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Prenatal alcohol exposure (PAE) can lead to long-lasting neurological alterations that may predispose individuals to seizures and neurobehavioral dysfunction. To date, there exists limited information regarding the underlying pathophysiological mechanisms. The hippocampal CA3 region generates excitatory population activity, called sharp waves (SPWs), that provide an ideal model to study perturbations in neuronal excitability at the network and cellular levels. In the present study, we utilized a mouse model of PAE and used dual extracellular and whole-cell patch-clamp recordings from CA3 hippocampal pyramidal cells to evaluate the effect of 1st trimester-equivalent ethanol exposure (10% v/v) on SPW activity and excitatory/inhibitory balance. We observed that PAE significantly altered in vitro SPW waveforms, with an increased duration and amplitude, when compared to controls. In addition, PAE slices exhibited reduced pharmacological inhibition by the GABA-A receptor antagonist bicuculline (BMI) on SPW activity, and increased population spike paired-pulse ratios, all indicative of network disinhibition within the PAE hippocampus. Evaluation of PAE CA3 pyramidal cell activity associated with SPWs, revealed increased action potential cell firing, which was accompanied by an imbalance of excitatory/inhibitory synaptic drive, shifted in favor of excitation. Moreover, we observed intrinsic changes in CA3 pyramidal activity in PAE animals, including increased burst firing and instantaneous firing rate. This is the first study to provide evidence for hippocampal dysfunction in the ability to maintain network homeostasis and underlying cellular hyperexcitability in a model of PAE. These circuit and cellular level alterations may contribute to the increased propensity for seizures and neurobehavioral dysfunction observed in patients with a history of PAE., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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 β-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., (© 2014 Wiley Periodicals, Inc.)