Bulk Electroporation Research Articles

Efficient electroporation in primary cells with PEDOT:PSS electrodes.

Precise and efficient delivery of macromolecules into cells enhances basic biology research and therapeutic applications in cell therapies, drug delivery, and personalized medicine. While pulsed electric field electroporation effectively permeabilizes cell membranes to deliver payloads without the need for toxic chemical or viral transduction agents, conventional bulk electroporation devices face major challenges with cell viability and heterogeneity due to variations in fields generated across cells and electrochemistry at the electrode-electrolyte interface. Here, we introduce the use of microfabricated electrodes based on the conducting polymer poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS), which substantially increases cell viability and transfection efficiency. As a proof of concept, we demonstrate the enhanced delivery of Cas9 protein, guide RNA, and plasmid DNA into cell lines and primary cells. This use of PEDOT:PSS enables rapid modification of difficult-to-transfect cell types to accelerate their study and use as therapeutic platforms.

Automating CAR-T Transfection with Micro and Nano-Technologies.

Cancer poses a significant health challenge, with traditional treatments like surgery, radiotherapy, and chemotherapy often lacking in cell specificity and long-term curative potential. Chimeric antigen receptor T cell (CAR-T) therapy,utilizing genetically engineered T cells to target cancer cells, is a promising alternative. However, its high cost limits widespread application. CAR-T manufacturing process encompasses three stages: cell isolation and activation, transfection, and expansion.While the first and last stages have straightforward, commercially available automation technologies, the transfection stage lags behind. Current automated transfection relies on viral vectors or bulk electroporation, which have drawbacks such as limited cargo capacity and significant cell disturbance. Conversely, micro and nano-tool methods offer higher throughput and cargo flexibility, yet their automation remains underexplored.In this perspective, the progress in micro and nano-engineering tools for CAR-T transfection followed by a discussion to automate them is described. It is anticipated that this work can inspire the community working on micro and nano transfection techniques to examine how their protocols can be automated to align with the growing interest in automating CAR-T manufacturing.

Engineering Efficient Car-T Cells via Electroactive Nanoinjection.

Chimeric antigen receptor (CAR)-T therapy has emerged as a promising cell-based immunotherapy approach for treating blood disorders and cancers, but genetically engineering CAR-T cells is challenging due to primary T cells' sensitivity to conventional gene delivery approaches. The current viral-based method can typically involve significant operating costs and biosafety hurdles, while bulk electroporation can lead to poor cell viability and functionality. Here, we develop a non-viral electroactive nanoinjection (ENI) platform to efficiently negotiate the plasma membrane of primary human T cells via vertically configured electroactive nanotubes, enabling efficient delivery (68.7%) and expression (43.3%) of CAR genes in the T cells, with minimal cellular perturbation (> 90% cell viability). Compared to conventional bulk electroporation (BEP), the ENI platform achieves an almost 3-fold higher CAR transfection efficiency, indicated by the significantly higher reporter GFP expression (43.3% compared to 16.3%). By co-culturing with target lymphoma Raji cells, we prove the ENI-transfected CAR-T cells' ability to effectively suppress lymphoma cell growth (86.9% cytotoxicity). Taken together, the results demonstrate the platform's remarkable capacity to generate functional and effective anti-lymphoma CAR-T cells. Given the growing potential of cell-based immunotherapies, we anticipate that a non-viral and efficient nanoinjection platform like the one described here will offer a promising avenue for ex vivo cell engineering, particularly in the context of CAR-T cell therapy. This article is protected by copyright. All rights reserved.

Electroactive nanoinjection platform for intracellular delivery and gene silencing

BackgroundNanoinjection—the process of intracellular delivery using vertically configured nanostructures—is a physical route that efficiently negotiates the plasma membrane, with minimal perturbation and toxicity to the cells. Nanoinjection, as a physical membrane-disruption-mediated approach, overcomes challenges associated with conventional carrier-mediated approaches such as safety issues (with viral carriers), genotoxicity, limited packaging capacity, low levels of endosomal escape, and poor versatility for cell and cargo types. Yet, despite the implementation of nanoinjection tools and their assisted analogues in diverse cellular manipulations, there are still substantial challenges in harnessing these platforms to gain access into cell interiors with much greater precision without damaging the cell’s intricate structure. Here, we propose a non-viral, low-voltage, and reusable electroactive nanoinjection (ENI) platform based on vertically configured conductive nanotubes (NTs) that allows for rapid influx of targeted biomolecular cargos into the intracellular environment, and for successful gene silencing. The localization of electric fields at the tight interface between conductive NTs and the cell membrane drastically lowers the voltage required for cargo delivery into the cells, from kilovolts (for bulk electroporation) to only ≤ 10 V; this enhances the fine control over membrane disruption and mitigates the problem of high cell mortality experienced by conventional electroporation.ResultsThrough both theoretical simulations and experiments, we demonstrate the capability of the ENI platform to locally perforate GPE-86 mouse fibroblast cells and efficiently inject a diverse range of membrane-impermeable biomolecules with efficacy of 62.5% (antibody), 55.5% (mRNA), and 51.8% (plasmid DNA), with minimal impact on cells’ viability post nanoscale-EP (> 90%). We also show gene silencing through the delivery of siRNA that targets TRIOBP, yielding gene knockdown efficiency of 41.3%.ConclusionsWe anticipate that our non-viral and low-voltage ENI platform is set to offer a new safe path to intracellular delivery with broader selection of cargo and cell types, and will open opportunities for advanced ex vivo cell engineering and gene silencing.Graphical abstract

Single cell transcriptomics reveals reduced stress response in stem cells manipulated using localized electric fields.

Membrane disruption using Bulk Electroporation (BEP) is a widely used non-viral method for delivering biomolecules into cells. Recently, its microfluidic counterpart, Localized Electroporation (LEP), has been successfully used for several applications ranging from reprogramming and engineering cells for therapeutic purposes to non-destructive sampling from live cells for temporal analysis. However, the side effects of these processes on gene expression, that can affect the physiology of sensitive stem cells are not well understood. Here, we use single cell RNA sequencing (scRNA-seq) to investigate the effects of BEP and LEP on murine neural stem cell (NSC) gene expression. Our results indicate that unlike BEP, LEP does not lead to extensive cell death or activation of cell stress response pathways that may affect their long-term physiology. Additionally, our demonstrations show that LEP is suitable for multi-day delivery protocols as it enables better preservation of cell viability and integrity as compared to BEP.

Bulk Electroporation for Intracellular Delivery Directly Driven by Mechanical Stimulus.

Electroporation (EP) is an effective and widely accepted intracellular delivery method for fundamental research and medical applications. Existing electroporation methods usually require a commercially available EP system or tailor-made high-voltage (HV, up to kV) power source and are complicated, expensive, harmful to the cells, and even dangerous to the operators. A triboelectric nanogenerator (TENG) is a highly studied device that can generate HV output with limited charges and ultrahigh internal impedance. Here, we developed a Bulk Electroporation System based on TENG (BEST). To maximize the load voltage of the TENG, a flowing EP unit with a capillary was designed as a resistive load to realize impedance matching. A low conductivity buffer was used to further match and assist cell electroporation. Besides, the electrical model and experiments on cells transfected with the BEST showed that the bulk electric field of the cell medium could reach up to 1 kV/cm, therefore resulting in a nearly 30 times increase of trans-membrane potential, thus largely improving transfection efficiency. Finally, using 40 kDa FITC-dextran, we showed that a delivery efficiency above 50% with a cell viability maintained over 90% can be achieved in HeLa cells. This work demonstrated the potential of TENG in the biomedical field as a naturally safe HV power source. It also provided a simple, alternative, and low-cost solution for EP research and related biomedicine applications.

Gene Electrotransfer into Mammalian Cells Using Commercial Cell Culture Inserts with Porous Substrate.

Gene electrotransfer is one of the main non-viral methods for intracellular delivery of plasmid DNA, wherein pulsed electric fields are used to transiently permeabilize the cell membrane, allowing enhanced transmembrane transport. By localizing the electric field over small portions of the cell membrane using nanostructured substrates, it is possible to increase considerably the gene electrotransfer efficiency while preserving cell viability. In this study, we expand the frontier of localized electroporation by designing an electrotransfer approach based on commercially available cell culture inserts with polyethylene-terephthalate (PET) porous substrate. We first use multiscale numerical modeling to determine the pulse parameters, substrate pore size, and other factors that are expected to result in successful gene electrotransfer. Based on the numerical results, we design a simple device combining an insert with substrate containing pores with 0.4 µm or 1.0 µm diameter, a multiwell plate, and a pair of wire electrodes. We test the device in three mammalian cell lines and obtain transfection efficiencies similar to those achieved with conventional bulk electroporation, but at better cell viability and with low-voltage pulses that do not require the use of expensive electroporators. Our combined theoretical and experimental analysis calls for further systematic studies that will investigate the influence of substrate pore size and porosity on gene electrotransfer efficiency and cell viability.

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.

Materials for Improving Immune Cell Transfection.

Chimeric antigen receptor T cell (CAR-T) therapy holds great promise for preventing and treating deadly diseases such as cancer. However, it remains challenging to transfect and engineer primary immune cells for clinical cell manufacturing. Conventional tools using viral vectors and bulk electroporation suffer from low efficiency while posing risks like viral transgene integration and excessive biological perturbations. Emerging techniques using microfluidics, nanoparticles, and high-aspect-ratio nanostructures can overcome these challenges, and on top of that, provide universal and high-throughput cargo delivery. Herein, the strengths and limitations of traditional and emerging materials for immune cell transfection, and commercial development of these tools, are discussed. To enhance the characterization of transfection techniques and uptake by the clinical community, a list of in vitro and in vivo assays to perform, along with relevant protocols, is recommended. The overall aim, herein, is to motivate the development of novel materials to meet rising demand in transfection for clinical CAR-T cell manufacturing.

Non-viral reprogramming of human nucleus pulposus cells with FOXF1 via extracellular vesicle delivery: an in vitro and in vivo study.

Intervertebral disc (IVD) degeneration is characterized by decreased cellularity and proteoglycan synthesis and increased inflammation, catabolism, and neural/vascular ingrowth. Regenerative methods for IVD degeneration are largely cell-therapy-based or involve viral vectors, which are associated with mutagenesis and undesired immune responses. The present study used bulk electroporation and engineered extracellular vesicles (EVs) to deliver forkhead-box F1 (FOXF1) mRNA to degenerate human nucleus pulposus (NP) cells as a minimally invasive therapeutic strategy for IVD regeneration. Bulk electroporation was used to investigate FOXF1 effects on human NP cells during a 4-week culture in 3D agarose constructs. Engineered EV delivery of FOXF1 into human IVD cells in monolayer was determined, with subsequent in vivo validation in a pilot mouse IVD puncture model. FOXF1 transfection significantly altered gene expression by upregulating healthy NP markers [FOXF1, keratin 19 (KRT19)], decreasing inflammatory cytokines [interleukin (IL)-1β, −6], catabolic enzymes [metalloproteinase 13 (MMP13)] and nerve growth factor (NGF), with significant increases in glycosaminoglycan accumulation in human NP cells. Engineered EVs loaded with FOXF1 demonstrated successful encapsulation of FOXF1 cargo and effective uptake by human NP cells cultured in monolayer. Injection of FOXF1-loaded EVs into the mouse IVD in vivo resulted in a significant upregulation of FOXF1 and Brachyury, compared to controls at 7 d post-injection, with no evidence of cytotoxicity. This is the first study to demonstrate non-viral delivery of FOXF1 and reprogramming of human NP cells in vitro and mouse IVD cells in vivo. This strategy represents a non-addictive approach for treating IVD degeneration and associated back pain.

High Throughput and Highly Controllable Methods for In Vitro Intracellular Delivery.

In vitro and ex vivo intracellular delivery methods hold the key for releasing the full potential of tissue engineering, drug development, and many other applications. In recent years, there has been significant progress in the design and implementation of intracellular delivery systems capable of delivery at the same scale as viral transfection and bulk electroporation but offering fewer adverse outcomes. This review strives to examine a variety of methods for in vitro and ex vivo intracellular delivery such as flow-through microfluidics, engineered substrates, and automated probe-based systems from the perspective of throughput and control. Special attention is paid to a particularly promising method of electroporation using micro/nanochannel based porous substrates, which expose small patches of cell membrane to permeabilizing electric field. Porous substrate electroporation parameters discussed include system design, cells and cargos used, transfection efficiency and cell viability, and the electric field and its effects on molecular transport. The review concludes with discussion of potential new innovations which can arise from specific aspects of porous substrate-based electroporation platforms and high throughput, high control methods in general.

Imaging the Dynamic Branching and Synaptic Differentiation of Xenopus Optic Axons In Vivo.

In the developing Xenopus tadpole visual system, the targeting and branching of optic axons in the brain is a dynamic process that is closely intertwined with the morphological differentiation and maturation of their postsynaptic neurons and with the formation, stabilization, and elimination of functional synapses. The coordinated addition and retraction of axonal and dendritic branches guides the gradual recognition between pre- and postsynaptic neuronal partners, which subsequently allows synaptic connections to be formed. Axon and dendrite branching and selective synapse formation and stabilization are developmental mechanisms largely orchestrated by an array of signaling molecules that interact in vivo for the proper formation of functional visual circuits. In vivo real-time imaging of individual fluorophore-labeled neurons in living Xenopus tadpoles has allowed investigation of molecular and cellular mechanisms mediating circuit assembly at a cellular level in the intact organism. In this protocol, we describe the use of bulk and single-cell electroporation to rapidly and efficiently transfect individual retinal ganglion cells (RGCs) with different reagents and to simultaneously visualize optic axon arbor morphology and presynaptic sites in real time. Similar techniques for labeling and visualizing RGC axons can be combined with the use of morpholino antisense oligonucleotides, as we describe here, to alter gene expression cell autonomously.

Nanochannel-Based Poration Drives Benign and Effective Nonviral Gene Delivery to Peripheral Nerve Tissue.

While gene and cell therapies have emerged as promising treatment strategies for various neurological conditions, heavy reliance on viral vectors can hamper widespread clinical implementation. Here, the use of tissue nanotransfection as a platform nanotechnology to drive nonviral gene delivery to nerve tissue via nanochannels, in an effective, controlled, and benign manner is explored. TNT facilitates plasmid DNA delivery to the sciatic nerve of mice in a voltage-dependent manner. Compared to standard bulk electroporation (BEP), impairment in toe-spread and pinprick response is not caused by TNT, and has limited to no impact on electrophysiological parameters. BEP, however, induces significant nerve damage and increases macrophage immunoreactivity. TNT is subsequently used to deliver vasculogenic cell therapies to crushed nerves via delivery of reprogramming factor genes Etv2, Foxc2, and Fli1 (EFF). The results indicate the TNT-based delivery of EFF in a sciatic nerve crush model leads to increased vascularity, reduced macrophage infiltration, and improved recovery in electrophysiological parameters compared to crushed nerves that are TNT-treated with sham/empty plasmids. Altogether, the results indicate that TNT can be a powerful platform nanotechnology for localized nonviral gene delivery to nerve tissue, in vivo, and the deployment of reprogramming-based cell therapies for nerve repair/regeneration.

A Novel Electroporation System for Living Cell Staining and Membrane Dynamics Interrogation.

A novel electroporation system was developed to introduce transient membrane pores to cells in a spatially and temporally controlled manner, allowing us to achieve fast electrotransfection and live cell staining as well as to systematically interrogate the dynamics of the cell membrane. Specifically, using this platform, we showed that both reversible and irreversible electroporation could be induced in the cell population, with nano-sized membrane pores in the former case being able to self-reseal in ~10 min. In addition, green fluorescent protein(GFP)-vinculin plasmid and 543 phalloidin have been delivered successively into fibroblast cells, which enables us to monitor the distinct roles of vinculin and F-actin in cell adhesion and migration as well as their possible interplay during these processes. Compared to conventional bulk electroporation and staining methods, the new system offers advantages such as low-voltage operation, cellular level manipulation and testing, fast and adjustable transfection/staining and real-time monitoring; the new system therefore could be useful in different biophysical studies in the future.

A Simple and Efficient Method for Visualizing Individual Cells in vivo by Cre-Mediated Single-Cell Labeling by Electroporation (CREMSCLE).

Efficient methods for visualizing cell morphology in the intact animal are of great benefit to the study of structural development in the nervous system. Quantitative analysis of the complex arborization patterns of brain cells informs cell-type classification, dissection of neuronal circuit wiring, and the elucidation of growth and plasticity mechanisms. Time-lapse single-cell morphological analysis requires labeling and imaging of single cells in situ without contamination from the ramified processes of other nearby cells. Here, using the Xenopus laevis optic tectum as a model system, we describe CRE-Mediated Single-Cell Labeling by Electroporation (CREMSCLE), a technique we developed based on bulk co-electroporation of Cre-dependent inducible expression vectors, together with very low concentrations of plasmid encoding Cre recombinase. This method offers efficient, sparse labeling in any brain area where bulk electroporation is possible. Unlike juxtacellular single-cell electroporation methods, CREMSCLE relies exclusively on the bulk electroporation technique, circumventing the need to precisely position a micropipette next to the target cell. Compared with viral transduction methods, it is fast and safe, generating high levels of expression within 24 h of introducing non-infectious plasmid DNA. In addition to increased efficiency of single-cell labeling, we confirm that CREMSCLE also allows for efficient co-expression of multiple gene products in the same cell. Furthermore, we demonstrate that this method is particularly well-suited for labeling immature neurons to follow their maturation over time. This approach therefore lends itself well to time-lapse morphological studies, particularly in the context of early neuronal development and under conditions that prevent more difficult visualized juxtacellular electroporation.

Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation.

Exosomes are attractive nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and of their ability to penetrate physiological barriers that are impermissible to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs (mRNAs), into cell-secreted exosomes leads to low yields. Here, we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs, and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared to bulk electroporation and to other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and more than a 103-fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic PTEN-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced tumour-growth inhibition, and increased animal survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.

Micromachining of Polymeric Microfluidic Micro/Nanoelectroporation Device.

Micro/nanochannel electroporation can deliver gene/drug into single cell with precise dosage control and much higher cell viability compared to traditional bulk electroporation. However, single cell micro/nanochannel electroporation has the problems of low efficiency and complicated operation. By integrating microfluidic with micro/nanochannel electroporation, a large number of cells can be processed within a short time. In this chapter, we provide a detailed protocol of fabrication microfluidic nanochannel electroporation devices. The fabrication of this microfluidic nanochannel electroporation device integrates soft lithography, DNA combing and imprinting, and micromilling. This device is appropriate for gene/drug delivery to a batch of cells. It has the advantages of both the single cell nanochannel electroporation and microfluidic based cell manipulation. The procedures of device fabrication, holder fabrication, cell trapping, and electroporation are included in this protocol.

Nanostructured Materials for Intracellular Cargo Delivery.

Intracellular cargo delivery is an essential step in many biomedical applications including gene editing and biologics therapy. Examples of cargo include nucleic acids (RNA and DNA), proteins, small biomolecules, and drugs, which can vary substantially in terms of their sizes, charges, solubility, and stability. Viruses have been used traditionally to deliver nucleic acids into cells, but the method suffers from limitations such as small cargo size, safety concerns, and viral genome integration into host cells, all of which complicate therapeutic applications. Commercially available techniques using biochemicals and bulk electroporation are, in general, poorly compatible with primary cells such as human induced pluripotent stem cells and immune cells, which are increasingly important candidates for adoptive cell therapy. Nanostructures, with dimensions ranging from tens of nanometers to a few micrometers, may play a critical role in overcoming cellular manipulation and delivery challenges and provide a powerful alternative to conventional techniques. A critical feature that differentiates nanostructures from viral, biochemical, and bulk electroporation techniques is that they interface with cells at a scale measuring ten to hundreds of nanometers in size. This highly local interaction enables application of stronger and more direct stimuli such as mechanical force, heat, or electric fields than would be possible in a bulk treatment. Compared to popular viral, biochemical, and bulk electroporation methods, nanostructures were found to minimally perturb cells with cells remaining in good health during postdelivery culture. These advantages have enabled nanostructures such as nanowires and nanotubes to successfully interface with a wide variety of cells, including primary immune cells and cardiomyocytes, for in vitro and in vivo applications. This Account is focused on using nanostructures for cargo delivery into biological cells. In this Account, we will first outline the historical developments using nanostructures for interfacing with cells. We will highlight how mechanistic understanding of nano-bio interactions has evolved over the last decade and how this improved knowledge has motivated coupling of electric and magnetic fields to nanostructures to improve delivery outcomes. There will also be an in-depth discussion on the merits of nanostructures in comparison to conventional methods using viruses, biochemicals, and bulk electroporation. Finally, motivated by our observations on the lack of consistency in reporting key metrics such as efficiency in literature, we suggest a set of metrics for documenting experimental results with the aim to promote standardization in reporting and ease in comparing. We suggest the use of more sophisticated tools such as RNA transcriptomics for thorough assessment of cell perturbation attributed to intracellular delivery. We hope that this Account can effectively capture the progress of nanostructure-mediated cargo delivery and encourage new innovations.

Combining two photon microscopy and CMOS MEA to uncover the mechanism of aberrant activity in blind retina

Event Abstract Back to Event Combining two photon microscopy and CMOS MEA to uncover the mechanism of aberrant activity in blind retina Meng-Jung Lee1, 2, 3*, Prerna Srivastava3, 4, 5, Luke Rogerson3, 4, 5, Philipp Berens4, 5, 6, Thomas Euler4, 5, 6, Timm Schubert4, 5 and Günther Zeck1 1 Natural and Medical Sciences Institute, Neurophysics, Germany 2 Multi Channel Systems MCS GmbH, Germany 3 Centre de formation des diplômés en neuroscience, Universität Tübingen, Germany 4 Werner Reichardt Centrum für Integrative Neurowissenschaften (CIN), Universität Tübingen, Germany 5 Institut für Ophthalmologische Forschung, Universität Tübingen, Germany 6 Bernstein Zentrum für Computational Neuroscience Tübingen, Germany Motivation Increasing spontaneous activity and intrinsic oscillations have been reported in different cell layers in photoreceptor degenerated retina [1, 2]. Understanding the biophysical characteristics and the cellular origin of these oscillations may facilitate the development of strategies to treat diseases like Retinitis Pigmentosa and age-related macular degeneration. Here, we present the simultaneous recording of somatic calcium/synaptic glutamate signals in the inner and outer retina using two photon imaging and of action potentials of retinal ganglion cells (RGCs) in the ganglion cell layer using high-density CMOS based microelectrode arrays (MEAs). Material and Methods In this study, retinas from adult photoreceptor-degenerated rd10 mice were used. In the calcium imaging experiments, bipolar cells and amacrine cells in the retinal whole mount were labeled with Oregon Green BAPTA-1 using bulk electroporation [3]. For the glutamate imaging experiments, the AAV-encoded iGluSnFR biosensor was injected intravitreally into the mouse eye 3-4 weeks prior to the dissection of the retina [4].The labeled retina was placed RGCs down onto a poly-lysine coated CMOS-based MEA (CMOS 5000, Multi Channel Systems MCS) with a 1 mm2 sensitive area. The two photon recording was performed in a local area of 71.5 x 26.8 µm xy-scans for calcium imaging and 47.6 x 11.9 µm for glutamate imaging at different planes using an excitation laser tuned to 927 nm (Mai Tai DeepSee HP; Spectra Psychics). Data acquired from two photon imaging was analyzed with IGOR Pro, while spikes from RGCs were first sorted with CMOS-MEA-Tools and then further analyzed with Matlab. Results The main challenge of simultaneous two-photon imaging and extracellular recording using MEAs is the electrical artifact caused by the scanning laser beam in the sensitive electronic structures. This artifact occurred only in the MEA electrodes underneath the scanning laser. It could not be removed simply by applying filters to the signals as in previous work [5] . Instead, we applied a reset paradigm where the floating gate voltages of all sensing transistors were briefly assigned to one common voltage using internal switches. The reset parameters (duration and frequency) are defined by the user. Using this paradigm, continuous simultaneous two photon imaging and electrophysiological recordings with only 20 ms data loss during the resets were possible, yielding simultaneously calcium or glutamate signals in the outer and inner retina as well as spikes from the RGCs. Thus, combining two photon imaging and MEA recordings enabled us monitoring the activity in most retinal cell classes (remnant cones, bipolar cells, amacrine cells and RGCs) simultaneously. Conclusion Our results demonstrate that two photon imaging can be performed together with CMOS-MEA based recordings to study vertical signaling in the degeneratedive retina. This combination balances strengths and weaknesses of both recording techniques, offering a more complete and comprehensive way to study neuronal networks. Acknowledgements This work was supported by the Marie Curie Program switchBoard to M-J.L , P.S , T.S. and T.E. switchBoard receives funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 674901. In addition, this work was funded by the Funding from the BMBF (FKZ031L0059A to G.Z. and FKZ 01GQ1601 to P.B.) is acknowledged by G.Z, the BW-Stiftung (NEU013) and the Deutsche Forschungsgemeinschaft (DFG, SFB 1233; BE5601/4-1).

Dye Electroporation and Imaging of Calcium Signaling in Xenopus Nervous System.

Electroporation is an efficient method of transferring charged macromolecules into living cells in order to study their morphology, function, and connectivity within neuronal networks. Labeling cells with fluorophore-coupled macromolecules can be used to trace projections of whole neuronal ensembles, as well as the fine morphology of single cells. Here, we present a protocol to visualize pre- and postsynaptic components of a sensory relay synapse in the brain, using the olfactory system of Xenopus laevis tadpoles as a model. We apply bulk electroporation to trace projections of receptor neurons from the nose to the brain, and single cell electroporation to visualize the morphology of their synaptic target cells, the mitral-tufted cells. Labeling the receptor neurons with a calcium-sensitive dye allows us to record stimulus-induced presynaptic input to the dendrites of the postsynaptic cells via functional calcium imaging.