Ca2+ Dynamics of Gap Junction Coupled and Uncoupled Deiters' Cells in the Organ of Corti in Hearing BALB/c Mice.

ATP, as a paracrine signalling molecule, induces intracellular Ca2+ elevation via the activation of purinergic receptors on the surface of glia-like cochlear supporting cells. These cells, including the Deiters' cells (DCs), are also coupled by gap junctions that allow the propagation of intercellular Ca2+ waves via diffusion of Ca2+ mobilising second messenger IP3 between neighbouring cells. We have compared the ATP-evoked Ca2+ transients and the effect of two different gap junction (GJ) blockers (octanol and carbenoxolone, CBX) on the Ca2+ transients in DCs located in the apical and middle turns of the hemicochlea preparation of BALB/c mice (P14-19). Octanol had no effect on Ca2+ signalling, while CBX inhibited the ATP response, more prominently in the middle turn. Based on astrocyte models and using our experimental results, we successfully simulated the Ca2+ dynamics in DCs in different cochlear regions. The mathematical model reliably described the Ca2+ transients in the DCs and suggested that the tonotopical differences could originate from differences in purinoceptor and Ca2+ pump expressions and in IP3-Ca2+ release mechanisms. The cochlear turn-dependent effect of CBX might be the result of the differing connexin isoform composition of GJs along the tonotopic axis. The contribution of IP3-mediated Ca2+ signalling inhibition by CBX cannot be excluded.

DUSP2 deletion with CRISPR/Cas9 promotes Mauthner cell axonal regeneration at the early stage of zebrafish.

Axon regeneration of central neurons is a complex process that is tightly regulated by multiple extrinsic and intrinsic factors. The expression levels of distinct genes are changed after central neural system (CNS) injury and affect axon regeneration. A previous study identified dusp2 as an upregulated gene in zebrafish with spinal cord injury. Here, we found that dual specificity phosphatase 2 (DUSP2) is a negative regulator of axon regeneration of the Mauthner cell (M-cell). DUSP2 is a phosphatase that mediates the dephosphorylation of JNK. In this study, we knocked out dusp2 by CRISPR/Cas9 and found that M-cell axons of dusp2-/- zebrafish had a better regeneration at the early stage after birth (within 8 days after birth), while those of dusp2+/- zebrafish did not. Overexpression of DUSP2 in Tg (Tol 056) zebrafish by single-cell electroporation retarded the regeneration of M-cell axons. Western blotting results showed that DUSP2 knockout slightly increased the levels of phosphorylated JNK. These findings suggest that knocking out DUSP2 promoted the regeneration of zebrafish M-cell axons, possibly through enhancing JNK phosphorylation.

Visualizing the Intracellular Trafficking in Zebrafish Mauthner Cells.

Axonal transport is crucial for the development and survival of neurons and maintenance of neuronal function. Disruption in this active process causes diverse neurological diseases. Thus, study of the intracellular trafficking as one way to gain the knowledge of the kinetics of axonal transport is essential to understand the mechanisms underlying the neuropathology. A lot of studies have been completed in vitro with neuron cultures and tissues, which may not accurately replicate the in vivo situation. Therefore, intravital manipulations are essential to achieve this goal. Here we introduce a technique that has been widely used in our lab to study the cargo trafficking in zebrafish at single-cell resolution. We use mitochondria as a representative neuronal cargo and provide step-by-step instructions on how to label specific cargoes within zebrafish Mauthner cells. This method can also be expanded to study the kinetics of other cargoes as well as the role of molecular regulators in axonal transport.

Deep Learning-Assisted Automated Single Cell Electroporation Platform for Effective Genetic Manipulation of Hard-to-Transfect Cells.

Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human-induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP-E) system, a nanopipette-based single-cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell-nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP-E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation.

Monosynaptic rabies virus tracing from projection-targeted single neurons.

A single neuron integrates inputs from thousands of presynaptic neurons to generate outputs. Circuit tracing using G-deleted rabies virus (RVΔG) vectors permits the brain-wide labeling of presynaptic inputs to targeted single neurons. However, the experimental procedures are complex, and the success rate of circuit labeling is low because of the lack of validation to increase the accuracy and efficiency of monosynaptic RVΔG tracing from targeted single neurons. We established an efficient RVΔG tracing method from projection target-defined single neurons using TVA950, a transmembrane isoform of TVA receptors, for initial viral infection. Presynaptic neurons were transsynaptically labeled from 80 % of the TVA950-expressing single starter neurons that survived after infection with EnvA-pseudotyped RVΔG in the adult mouse brain. We labeled single neuronal networks in the primary visual cortex (V1) and higher visual areas, namely the posteromedial area (PM) and anteromedial area (AM), as well as the single neuronal networks of PM-projecting V1 single neurons. Monosynaptic RVΔG tracing from projection-targeted single neurons revealed the input-output organization of single neuronal networks. Single-neuron network analysis based on RVΔG tracing will help dissect the heterogeneity of neural circuits and link circuit motifs and large-scale networks across scales, thereby clarifying information processing and circuit computation in the brain.

In vivo genome editing in single mammalian brain neurons through CRISPR-Cas9 and cytosine base editors.

Gene manipulation is a useful approach for understanding functions of genes and is important for investigating basic mechanisms of brain function on the level of single neurons and circuits. Despite the development and the wide range of applications of CRISPR-Cas9 and base editors (BEs), their implementation for an analysis of individual neurons in vivo remained limited. In fact, conventional gene manipulations are generally achieved only on the population level. Here, we combined either CRISPR-Cas9 or BEs with the targeted single-cell electroporation technique as a proof-of-concept test for gene manipulation in single neurons in vivo. Our assay consisted of CRISPR-Cas9- or BEs-induced gene knockout in single Purkinje cells in the cerebellum. Our results demonstrate the feasibility of both gene editing and base editing in single cells in the intact brain, providing a tool through which molecular perturbations of individual neurons can be used for analysis of circuits and, ultimately, behaviors.

Promotion of Dendritic Differentiation of Cerebellar Purkinje Cells by Ca2+/calmodulin-dependent Protein Kinase IIα, IIß and IV and Possible Involvement of CREB Phosphorylation.

Cerebellar Purkinje cells develop the most elaborate dendritic trees among neurons in the brain. To examine the role of Ca2+/calmodulin-dependent protein kinase (CaMK) IIα, IIß and IV in the dendritic differentiation of Purkinje cells, we introduced siRNA against these CaMKs into Purkinje cells in cerebellar cell cultures using a single-cell electroporation technique. Single-cell electroporation enables us to transfer siRNA into specific cells within a heterogeneous cell population. In addition, we can easily and reliably transfer multiple types of siRNA into a cell simply by loading them together in one micropipette. Any one of the siRNA against CaMKIIα, IIß and IV (single knockdown) or any combinations of two of the siRNA against these CaMKs (double knockdown) had no significant effects on the dendritic differentiation of Purkinje cells. However, the combination of all three siRNA against these CaMKs (triple knockdown) inhibited the branching of Purkinje cell dendrites. Furthermore, the triple knockdown reduced the phosphorylation of CREB in Purkinje cells. These findings suggest the promotion of dendritic differentiation of Purkinje cells by CaMKIIα, IIß and IV and the possible involvement of phosphorylation of CREB as a common substrate of these CaMKs.

Single-Cell Visualization Deep in Brain Structures by Gene Transfer.

A projection neuron targets multiple regions beyond the functional brain area. In order to map neuronal connectivity in a massive neural network, a means for visualizing the entire morphology of a single neuron is needed. Progress has facilitated single-neuron analysis in the cerebral cortex, but individual neurons in deep brain structures remain difficult to visualize. To this end, we developed an in vivo single-cell electroporation method for juvenile and adult brains that can be performed under a standard stereomicroscope. This technique involves rapid gene transfection and allows the visualization of dendritic and axonal morphologies of individual neurons located deep in brain structures. The transfection efficiency was enhanced by directly injecting the expression vector encoding green fluorescent protein instead of monitoring cell attachment to the electrode tip. We obtained similar transfection efficiencies in both young adult (≥P40) and juvenile mice (P21-30). By tracing the axons of thalamocortical neurons, we identified a specific subtype of neuron distinguished by its projection pattern. Additionally, transfected mOrange-tagged vesicle-associated membrane protein 2-a presynaptic protein-was strongly localized in terminal boutons of thalamocortical neurons. Thus, our in vivo single-cell gene transfer system offers rapid single-neuron analysis deep in brain. Our approach combines observation of neuronal morphology with functional analysis of genes of interest, which can be useful for monitoring changes in neuronal activity corresponding to specific behaviors in living animals.

Single-Cell Electroporation with Real-Time Impedance Assessment Using a Constriction Microchannel.

The electroporation system can serve as a tool for the intracellular delivery of foreign cargos. However, this technique is presently limited by the inaccurate electric field applied to the single cells and lack of a real-time electroporation metrics subsystem. Here, we reported a microfluidic system for precise and rapid single-cell electroporation and simultaneous impedance monitoring in a constriction microchannel. When single cells (A549) were continuously passing through the constriction microchannel, a localized high electric field was applied on the cell membrane, which resulted in highly efficient (up to 96.6%) electroporation. During a single cell entering the constriction channel, an abrupt impedance drop was noticed and demonstrated to be correlated with the occurrence of electroporation. Besides, while the cell was moving in the constriction channel, the stabilized impedance showed the capability to quantify the electroporation extent. The correspondence of the impedance variation and electroporation was validated by the intracellular delivery of the fluorescence indicator (propidium iodide). Based on the obtained results, this system is capable of precise control of electroporation and real-time, label-free impedance assessment, providing a potential tool for intracellular delivery and other biomedical applications.

A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture.

BACKGROUND: Exogenous gene introduction by transfection is one of the most important approaches for understanding the function of specific genes at the cellular level. Electroporation has a long-standing history as a versatile gene delivery technique in vitro and in vivo. However, it has been underutilized in vitro because of technical difficulty and insufficient transfection efficiency. NEW METHOD: We have developed an electroporation technique that combines the use of large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses. Here, we describe the technique and compare it with existing methods. RESULTS: Our method achieves a high transfection efficiency (∼80 %) in both excitatory and inhibitory neurons with no detectable side effects on their function. We demonstrate this method is capable of transferring at least three different genes into a single neuron. In addition, we demonstrate the ability to transfect different genes into neighboring cells. COMPARISON WITH EXISTING METHODS: The majority of existing methods use fine-tipped glass electrodes (i.e. > 10 MΩ) and apply high voltage (10 V) pulses with high frequency (100 Hz) for 1 s. These parameters contribute to practical difficulties thus lowering the transfection efficiency. Our unique method minimizes electrode clogging and therefore procedure duration, increasing transfection efficiency and cellular viability. CONCLUSIONS: Our modifications, relative to current methods, optimize electroporation efficiency and cell survival. Our approach offers distinct research strategies not only in elucidating cell-autonomous functions of genes but also for assessing genes contributing to intercellular functions, such as trans-synaptic interactions.

Postnatal Development of the Subcellular Structures and Purinergic Signaling of Deiters' Cells along the Tonotopic Axis of the Cochlea.

Exploring the development of the hearing organ helps in the understanding of hearing and hearing impairments and it promotes the development of the regenerative approaches-based therapeutic efforts. The role of supporting cells in the development of the organ of Corti is much less elucidated than that of the cochlear sensory receptor cells. The use of our recently published method of single-cell electroporation loading of a fluorescent Ca2+ probe in the mouse hemicochlea preparation provided an appropriate means to investigate the Deiters' cells at the subcellular level in two different cochlear turns (apical, middle). Deiters' cell's soma and process elongated, and the process became slimmer by maturation without tonotopic preference. The tonotopically heterogeneous spontaneous Ca2+ activity less frequently occurred by maturation and implied subcellular difference. The exogenous ATP- and UTP-evoked Ca2+ responses were maturation-dependent and showed P2Y receptor dominance in the apical turn. By monitoring the basic structural dimensions of this supporting cell type as well as its spontaneous and evoked purinergic Ca2+ signaling in the hemicochlea preparation in different stages in the critical postnatal P5-25 developmental period for the first time, we showed that the soma and the phalangeal process of the Deiters' cells go through age- and tonotopy-dependent changes in the morphometric parameters and purinergic signaling.

Targeted single-cell electroporation loading of Ca2+ indicators in the mature hemicochlea preparation.

Ca2+ is an important intracellular messenger and regulator in both physiological and pathophysiological mechanisms in the hearing organ. Investigation of cellular Ca2+ homeostasis in the mature cochlea is hampered by the special anatomy and high vulnerability of the organ. A quick, straightforward and reliable Ca2+ imaging method with high spatial and temporal resolution in the mature organ of Corti is missing. Cell cultures or isolated cells do not preserve the special microenvironment and intercellular communication, while cochlear explants are excised from only a restricted portion of the organ of Corti and usually from neonatal pre-hearing murines. The hemicochlea, prepared from hearing mice allows tonotopic experimental approach on the radial perspective in the basal, middle and apical turns of the organ. We used the preparation recently for functional imaging in supporting cells of the organ of Corti after bulk loading of the Ca2+ indicator. However, bulk loading takes long time, is variable and non-selective, and causes the accumulation of the indicator in the extracellular space. In this study we show the improved labeling of supporting cells of the organ of Corti by targeted single-cell electroporation in mature mouse hemicochlea. Single-cell electroporation proved to be a reliable way of reducing the duration and variability of loading and allowed subcellular Ca2+ imaging by increasing the signal-to-noise ratio, while cell viability was retained during the experiments. We demonstrated the applicability of the method by measuring the effect of purinergic, TRPA1, TRPV1 and ACh receptor stimulation on intracellular Ca2+ concentration at the cellular and subcellular level. In agreement with previous results, ATP evoked reversible and repeatable Ca2+ transients in Deiters', Hensen's and Claudius' cells. TRPA1 and TRPV1 stimulation by AITC and capsaicin, respectively, failed to induce any Ca2+ response in the supporting cells, except in a single Hensen's cell in which AITC evoked transients with smaller amplitude. AITC also caused the displacement of the tissue. Carbachol, agonist of ACh receptors induced Ca2+ transients in about a third of Deiters' and fifth of Hensen's cells. Here we have presented a fast and cell-specific indicator loading method allowing subcellular functional Ca2+ imaging in supporting cells of the organ of Corti in the mature hemicochlea preparation, thus providing a straightforward tool for deciphering the poorly understood regulation of Ca2+ homeostasis in these cells.

Projection Patterns of Corticofugal Neurons Associated with Vibrissa Movement.

Rodents actively whisk their vibrissae, which, when they come in contact with surrounding objects, enables rodents to gather spatial information about the environment. Cortical motor command of whisking is crucial for the control of vibrissa movement. Using awake and head-fixed rats, we investigated the correlations between axonal projection patterns and firing properties in identified layer 5 neurons in the motor cortex, which are associated with vibrissa movement. We found that cortical neurons that sent axons to the brainstem fired preferentially during large-amplitude vibrissa movements and that corticocallosal neurons exhibited a high firing rate during small vibrissa movements or during a quiet state. The differences between these two corticofugal circuits may be related to the mechanisms of motor-associated information processing.

MicroRNA-133b Negatively Regulates Zebrafish Single Mauthner-Cell Axon Regeneration through Targeting tppp3 in Vivo.

Axon regeneration, fundamental to nerve repair, and functional recovery, relies on rapid changes in gene expression attributable to microRNA (miRNA) regulation. MiR-133b has been proved to play an important role in different organ regeneration in zebrafish, but its role in regulating axon regeneration in vivo is still controversial. Here, combining single-cell electroporation with a vector-based miRNA-expression system, we have modulated the expression of miR-133b in Mauthner-cells (M-cells) at the single-cell level in zebrafish. Through in vivo imaging, we show that overexpression of miR-133b inhibits axon regeneration, whereas down-regulation of miR-133b, promotes axon outgrowth. We further show that miR-133b regulates axon regeneration by directly targeting a novel regeneration-associated gene, tppp3, which belongs to Tubulin polymerization-promoting protein family. Gain or loss-of-function of tppp3 experiments indicated that tppp3 was a novel gene that could promote axon regeneration. In addition, we observed a reduction of mitochondrial motility, which have been identified to have a positive correlation with axon regeneration, in miR-133b overexpressed M-cells. Taken together, our work provides a novel way to study the role of miRNAs in individual cell and establishes a critical cell autonomous role of miR-133b in zebrafish M-cell axon regeneration. We propose that up-regulation of the newly founded regeneration-associated gene tppp3 may enhance axonal regeneration.

Expression of the GluA2 subunit of glutamate receptors is required for the normal dendritic differentiation of cerebellar Purkinje cells.

Cerebellar Purkinje cells differentiate the most elaborate dendritic trees among neurons in the brain and constitute the principal part of cerebellar neuronal circuitry. In the present study, we examined the role of the GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors in the dendritic differentiation of Purkinje cells. Since mature Purkinje cells express the GluA2 subunit, AMPA receptors on them exhibit a low Ca2+ permeability. Does this expression of GluA2, leading to the loss of Ca2+ permeability of AMPA receptors, have a positive significance in the dendritic differentiation of Purkinje cells? To answer this question, we introduced GluA2 siRNA into immature Purkinje cells in cerebellar cell cultures using a single-cell electroporation technique. The dendritic elongation and branching, as well as spine formation, were inhibited by GluA2 knockdown in Purkinje cells. GluA2 knockdown augmented the elevation of intracellular Ca2+ concentrations and a higher incidence of oscillation of intracellular Ca2+ concentrations in response to glutamate. These findings suggest that excessive elevation of intracellular Ca2+ concentrations has a negative effect on the dendritic differentiation of Purkinje cells and that the expression of GluA2 inhibits this negative effect in the development of Purkinje cells.

N-Cadherin is Involved in Neuronal Activity-Dependent Regulation of Myelinating Capacity of Zebrafish Individual Oligodendrocytes In Vivo.

Stimulating neuronal activity increases myelin sheath formation by individual oligodendrocytes, but how myelination is regulated by neuronal activity in vivo is still not fully understood. While in vitro studies have revealed the important role of N-cadherin in myelination, our understanding in vivo remains quite limited. To obtain the role of N-cadherin during activity-dependent regulation of myelinating capacity of individual oligodendrocytes, we successfully built an in vivo dynamic imaging model of the Mauthner cell at the subcellular structure level in the zebrafish central nervous system. Enhanced green fluorescent protein (EGFP)-tagged N-cadherin was used to visualize the stable accumulations and mobile transports of N-cadherin by single-cell electroporation at the single-cell level. We found that pentylenetetrazol (PTZ) significantly enhanced the accumulation of N-cadherin in Mauthner axons, a response that was paralleled by enhanced sheath number per oligodendrocytes. By offsetting this phenotype using oligopeptide (AHAVD) which blocks the function of N-cadherin, we showed that PTZ regulates myelination in an N-cadherin-dependent manner. What is more, we further suggested that PTZ influences N-cadherin and myelination via a cAMP pathway. Consequently, our data indicated that N-cadherin is involved in neuronal activity-dependent regulation of myelinating capacity of zebrafish individual oligodendrocytes in vivo.

A modified single-cell electroporation method for molecule delivery into a motile protist, Euglena gracilis.

Single-cell transfection is a powerful technique for delivering chemicals, drugs, or probes into arbitrary, specific single cells. This technique is especially important when the analysis of molecular function and cellular behavior in individual microscopic organisms such as protists requires the precise identification of the target cell, as fluorescence labeling of bulk populations makes tracking of individual motile protists virtually impossible. Herein, we have modified current single-cell electroporation techniques for delivering fluorescent markers into single Euglena gracilis, a motile photosynthetic microalga. Single-cell electroporation introduced molecules into individual living E. gracilis cells after a negative pressure was applied through a syringe connected to the micropipette to the target cell. The new method achieves high transfection efficiency and viability after electroporation. With the new technique, we successfully introduced a variety of molecules such as GFP, Alexa Fluor 488, and exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) RNA probes into individual motile E. gracilis cells. We demonstrate imaging of endogenous mRNA in living E. gracilis without interfering with their physiological functions, such as swimming or division, over an extended period of time. Thus the modified single-cell electroporation technique is suitable for delivering versatile functional molecules into individual motile protists.

Corrigendum: Single cell electroporation for longitudinal imaging of synaptic structure and function in the adult mouse neocortex in vivo.

[This corrects the article on p. 36 in vol. 9, PMID: 25904849.].

Single cell electroporation for longitudinal imaging of synaptic structure and function in the adult mouse neocortex in vivo.

Longitudinal imaging studies of neuronal structures in vivo have revealed rich dynamics in dendritic spines and axonal boutons. Spines and boutons are considered to be proxies for synapses. This implies that synapses display similar dynamics. However, spines and boutons do not always bear synapses, some may contain more than one, and dendritic shaft synapses have no clear structural proxies. In addition, synaptic strength is not always accurately revealed by just the size of these structures. Structural and functional dynamics of synapses could be studied more reliably using fluorescent synaptic proteins as markers for size and function. These proteins are often large and possibly interfere with circuit development, which renders them less suitable for conventional transfection or transgenesis methods such as viral vectors, in utero electroporation, and germline transgenesis. Single cell electroporation (SCE) has been shown to be a potential alternative for transfection of recombinant fluorescent proteins in adult cortical neurons. Here we provide proof of principle for the use of SCE to express and subsequently image fluorescently tagged synaptic proteins over days to weeks in vivo.

A Nonlinear Size-Dependent Equivalent Circuit Model for Single-Cell Electroporation on Microfluidic Chips.

Electroporation (EP) is a process of applying a pulsed intense electric field on the cell membrane to temporarily induce nanoscale electropores on the plasma membrane of biological cells. A nonlinear size-dependent equivalent circuit model of a single-cell electroporation system is proposed to investigate dynamic electromechanical behavior of cells on microfluidic chips during EP. This model consists of size-dependent electromechanical components of a cell, electrical components of poration media, and a microfluidic chip. A single-cell microfluidic EP chip with 3D microelectrode arrays along a microchannel is designed and fabricated to experimentally analyze the permeabilization of a cell. Predicted electrical current responses of the model are in good agreement (average error of 6%) with that of single-cell EP. The proposed model can successfully predict the time responses of transmembrane voltage, pore diameter, and pore density at four different stages of permeabilization. These stages are categorized based on electromechanical changes of the lipid membrane. The current-voltage characteristic curve of the cell membrane during EP is also investigated at different EP stages in detail. The model can precisely predict the electric breakdown of different cell lines at a specific critical cell membrane voltage of the target cell lines.