Robust Induced Presynapse on Artificial Substrate as a Neural

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Biological and Medical Applications of Materials and Interfaces

Robust Induced Presynapse on Artificial Substrate as a Neural Interfacing Method Joohee Jeon, Min-Ah Oh, Wonkyung Cho, Sun-Heui Yoon, Ji Yong Kim, and Taek Dong Chung ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20405 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 2, 2019

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ACS Applied Materials & Interfaces

Robust Induced Presynapse on Artificial Substrate as a Neural Interfacing Method Joohee Jeon†,‡, Min-Ah Oh†,‡, Wonkyung Cho†, Sun-Heui Yoon†, Ji Yong Kim† and Taek Dong Chung*,† ,§

† Department of Chemistry, Seoul National University, Seoul, 08826, Republic of Korea § Advanced Institutes of Convergence Technology, Suwon-Si, Gyeonggi-do 16229, Republic of Korea ‡ These authors contributed equally to this work.

KEYWORDS: Neural interface, Induced synapse, Protein engineering, Surface modification, Primary cultured neuron, Organotypically cultured brain slice, Synaptic activity

ABSTRACT Over the recent years, the development of neural interface systems has stuck to using electrical cues to stimulate neurons and read out neural signals, although neurons relay signals via chemical release and recognition at synapses. In addition, conventional neural interfaces are vulnerable to cell migration and glial encapsulation due to the absence of connection anchoring the neuron into

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the device, unlike synapses are firmly sustained by protein bonding. In order to close this discrepancy, we conducted intensive investigation into the induced synapse interface by employing engineered synaptic proteins from a neural interface perspective. The strong potential of induced synaptic differentiation as an emerging neural interfacing technique is demonstrated by exploring its structural features, chemical release kinetics, robustness, and scalability to the brain tissue. We show that the exocytosis kinetics of induced synapses is similar to that of endogenous synapses. Moreover, induced synapses show remarkable stability, despite cell migration and growth. The synapse-inducing technique has broad application to cultured hippocampal and cortex tissues and suggests a promising method to integrate neural circuits with digital elements.

Introduction Bolstered by advances in bioengineering1, material science2,3, and neuroscience4, research on neural interfacing has witnessed rapid development throughout the last decade. Previous studies attempting to improve on existing neural interface models share the same fundamental strategy: utilizing electrical cues to stimulate neurons5–7 and read out neural signals7–11. However, neurons relay information in the form of electrical potential strictly for intracellular transduction, and signaling across the extracellular space is executed via chemical release and recognition at synaptic junctions12–14 supported by synaptic protein pairs15–18. Interestingly, research on neuronal development over the past few years has revealed that synapses are not only capable of forming between neurons, but also between non-neuronal substrates and neurons through synapse-inducing proteins.15,19 With bioengineering techniques, those synapse-inducing proteins can be modified into forms that can be immobilized on artificial surfaces using the biotin-streptavidin interaction20, antibodies18, or glycosylphosphatidylinositol16. Some pioneering studies show that surface functionalized with engineered synaptic proteins allows


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neurons to transform the contact area into synapses.16–18,20–22 The induced synaptic junction is formed between the functionalized substrate and the presynapse of neurons. Inspired by these approach, we are convinced that synapse induced interfaces can be applied as neural interfaces. Unique features of the synapse enable surfaces modified with engineered synaptic proteins to establish biochemical interactions with neurons (Scheme 1). First, synaptic junction proteins mediate the highly stable interaction in the induced synaptic interface. Structure of endogenous synapse is maintained with synaptic junction proteins.15–18 At the induced synapse, membrane proteins of neuron and engineered synaptic protein immobilized on the substrate are also tightly bound to each other, resulting in a stable interface. Second, in the induced synapse the synaptic junction spans a length of approximately 20 nm23, which is too narrow to be penetrated by other cells such as microglia24. Efforts to alleviate glial response against external devices exist, such as coating surfaces with proteins25 or polymers26,27. This would address current issues concerning cell migration that renders the neuron-device interface dysfunctional28, and alleviate scar tissue ingrowth29–31. Third, synaptic release of neurotransmitters can be captured with high spatial resolution by the induced synapse interface. Because synapses are junctions specifically made between partner neurons23, the specificity inherent to synapses is also attained through the induced synapse method. Finally, this type of interface can handle more sophisticated signals. Unlike intracellular action potentials, chemicals serve as the messengers at synaptic junctions.32 The amount of neurotransmitters can be measured with single vesicle resolution using electrochemical methods.33,34 Despite its application potential, the synapse-inducing technique has been long used as a subsidiary method in neuroscience to identify protein-protein interactions.15–18 Existing studies on synaptic proteins have never examined the synapse-inducing technique as a method for


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Scheme 1. Conceptual Illustration of the Induced Presynapse Interfacea

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With protein engineering techniques, synaptic proteins can be immobilized on the artificial substrate. When a neuron meets the functionalized substrate, partner synaptic proteins of the neuron are recruited to the contact site, and presynaptic differentiation is triggered. In this interface, the functionalized substrate directly faces the induced synapse with robust protein binding.

neural interfacing concerning kinetics and stability. Here, we investigate the feasibility of using the synapse inducing technique to fabricate neural interfaces. The most important features of the neural interface are structural stability, signaling activity of neurons at the interface and scalability to brain tissues. Using microbeads and engineered neuroligin1 (NLG1), a well-known synapse-inducing protein35–39, we examine the structural and functional features of the induced synapse by immunostaining and membrane recycling analysis using N-(3-Triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM 1-43). Stability of the induced synapse interface is examined over several days. Additionally, we confirm the scalability of induced synapse interface by inducing synaptogenesis in brain slices. We, therefore, firmly believe that the present study has enabled a better understanding of the induced synapse interface, opening up new avenues for application in interfacing neural systems and manufactured devices.


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Experimental Section Protein production and immobilization on microbeads eNLG1 was produced and isolated from HEK293 cells transfected with ‘His6 - ectodomain of NLG1 – TagRFP - Avi tag – IRES - BirA’ in pDisplay vector (Figure S1 of the Supporting Information). IRES (internal ribosome entry site) is a RNA sequence which links two open reading frame sequences in a single vector and allow the translation of both proteins40. The HEK293 cell was chosen for complete glycosylation. Biotinylated protein complex was purified using Ni-Co resin (TALON®, Metal Affinity Resin). 0.6 μg/μL eNLG1 solution 200 uL was mixed with 300,000 streptavidin-coated polystyrene (PS) microbeads, 2.8 μm in diameter (Invitrogen, Dynabead® M-280 Streptavidin) or amine group coated microbead (Invitrogen, Dynabead® M-270 Amine) as a control group. The beads were washed with Dulbecco’s phosphate-buffered saline (DPBS) 3 times, and then put into 10 DIV (days in vitro) primary cultured neuron or 13 DIV brain slice for 2-3 days at a density of 25,000 beads/mm2. In the case of BSA beads, 0.6 μg/μL biotinylated BSA (Thermo Scientific) solution was used instead of eNLG1. Configuration analysis of immobilized eNLG1 To investigate the arrangement of eNLG1 assembled on the microbead surface, 4 mM bis(sulfosuccinimidyl) suberate (BS3, Thermo Scientific) solution of varying concentrations were added to 300,000 eNLG1 beads diluted in 60 μL DPBS, and the mixtures were allowed to react in RT for 30 min. The reaction was quenched by addition of 1 M Tris-HCl (pH 8.0) to a final concentration of 200 mM. To separate the crosslinked eNLG1 from the bead surface, they were mixed with 30 μL 0.1% SDS solution and heated at 120 °C for 5 min. Detached protein solution was centrifuged and the supernatant 16 μL was taken. Same method was applied for eNLG1 solution to examine the multimerization of eNLG1. The composition of the protein complexes was


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analyzed using SDS-PAGE. The samples were heated in 3X loading buffer, containing Tris-HCl, glycerol, SDS, bromophenol blue, and β-mercaptoethanol at 98 °C for 5 min before electrophoresis. 5% stacking gel and 8% resolving SDS-PAGE gel were used for electrophoresis, followed by western blot staining using 0.05% SAV-HRP (Invitrogen). Primary neural cell culture and organotypic brain slice culture All animal studies and experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Seoul National University. Primary rat hippocampus was isolated from Sprague-Dawley rat embryos at day 18 of gestation (E18). Hippocampal tissues were separated into single cells with papain (Worthington), and then the dissociated hippocampal neurons were plated on poly-D-lysine (Sigma-Aldrich, MW 30,000-70,000) coated glass coverslips (200 cells/mm2). Neurons were cultured in Neurobasal medium (Gibco) supplemented with GlutaMaxTM (Gibco), B-27 (Gibco), and penicillin-streptomycin (Welgene), in an incubator maintained at 37 °C and 5% CO2. The culture medium was changed every other day. For organotypic brain slice culture, the hippocampus and cerebral cortex were extracted from the brain of E18 rat embryo. Then, tissues were transferred to 4% agarose (Fisher Scientific, low melting) in ACSF. Upon rapid gelation in ice bath, the agarose casts were carefully cut and glued onto the metal chuck of ice-cooled vibratome (Leica VT 1200S). Tissues were cut into 300-μmthick slices in ice-cold ACSF solution. The brain slices were placed onto poly-D-lysine coated membrane inserts (Millipore, PICMORG50), which then were transferred into 6-well plate filled with basal medium eagle (Gibco) supplemented with N-2 supplement (Gibco). Cultures were kept in an incubator atmosphere of 37 °C, gassed with 5% CO2. The medium was renewed every other day (Figure S2). FE-SEM imaging


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Cultured neurons were fixed with 2.5% glutaldehyde in 0.05 M PBS for 2 h, followed by washing with 0.05 M PBS. It was additionally fixed with 1% osmium tetroxide in 0.05 M PBS for 2 h, and then washed with water briefly. The sample was dehydrated by immersing in a graded series of ethanol (25, 50, 70, 90, 100, 100%, 10 min each), followed by gradual replacement of solution with hexamethyldisilazane (Sigma-Aldrich). Finally, it was dried in vaccum chamber. For clear imaging, platinum was sputtered on the sample. Sputtered Pt thickness was approximately 4 nm. Samples were then examined using Carl Zeiss SIGMA field-emission scanning electron microscope. Immunocytochemistry Sample was fixed with 4% paraformaldehyde in PBS for 25 min, and rinsed with PBS. After fixation, it was incubated in 4% normal donkey serum (Jackson ImmunoResearch Inc.) and 0.1% Triton X-100 in TBS to block non-specific antibody adhesion and assist membrane penetration. 30 min later, the sample was replaced with primary antibody solution and kept overnight at 4 °C. Then, the sample was washed with TBS and incubated in secondary antibody solution for 1 h at RT, followed by TBS washing. In the case of brain slices, 5% NDS and 0.3% Triton X-100 in PBS was used as blocking solution. Fixation and blocking steps were carried out for 1 h each. Subsequent steps were same as for the primary culture samples. Solutions of primary and secondary antibodies were prepared by dilution of stock with blocking solution. Mouse anti-Synapsin1 monoclonal antibody (Synaptic systems), guinea pig antivesicular glutamate transporter polyclonal antibody (Millipore), rabbit anti-vesicular glutamate transporter polyclonal antibody (Synaptic systems), mouse anti-neurexin1β antibody (NeuroMab), rabbit anti-synaptophysin antibody (abcam), mouse anti-bassoon antibody (abcam), mouse antiPSD antibody (Invitrogen), rabbit anti-gephyrin antibody (Synaptic systems), rabbit anti GluN2


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antibody (Synaptic systems) rabbit anti-microtuble-associated protein 2 antibody (abcam), and mouse anti-glial fibrillary acidic protein antibody (Invitrogen) were used as the primary antibody. Corresponding secondary antibodies linked with alexa-488 or alexa-633 (Invitrogen) were used. Chemical release-kinetics analysis using FM 1-43 Neurons were immersed in 5 μg/mL FM 1-43 (Invitrogen) in stimulation solution (120 mM NaCl, 22 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 1.5 mM KH2PO4, 8.1 mM Na2HPO4) for 2 min to enable FM 1-43 loading into synaptic vesicles. Solutions were then perfused with PBS without Ca2+ and Mg2+ for 10 min to clear away extracellular FM 1-43. Then, neurons were re-stimulated with a secondary stimulation solution (70 mM NaCl, 80 mM KCl, 1.2 mM CaCl2, 0.5 mM MgCl2, 1.5 mM KH2PO4, 8.1 mM Na2HPO4). During exocytosis, real-time images were acquired every 2 s during 200 s around the stimulation with Zeiss LSM880 confocal laser scanning microscope equipped with ZEN black (2009) software. ROI was set on beads or native synapses, and normalized intensity of fluorescence was plotted over time. For the analysis on native synapse, endogenous synapses were determined using two criteria. First, intersections between two neurons that showed bright FM 1-43 fluorescence were identified with light and fluorescent image. Second, of these regions those that decreased FM 1-43 fluorescence upon stimulation were selected. ROI was drawn up on the assigned region, and intensity decay was plotted over time. Image acquisition and data analysis were executed in the same way with the induced synapse on eNLG1 beads. Chemical release studies were conducted in 10 times independently. Fluorescence Imaging and analysis. Fluorescent measurement of modified beads and eNLG1-Nrx1β binding assay were carried out with Nikon eclipse Ti fluorescent microscope equipped with NIS-elements AR 4.2 software. For immunostained protein analysis and real-time monitoring of FM 1-43 assay we used Zeiss


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LSM880 confocal laser scanning microscope equipped with ZEN black (2009) software. Regions of interest were defined and intensity profile obtained using corresponding software. Statistics. Error bars indicates the uncertainty range of one standard deviation. Statistical significance was evaluated using the one-way analysis of variance method. Statistical significance is indicated by n.s. for p > 0.05, * for 0.05 > p > 0.01, ** for 0.01 > p > 0.001 and *** for p > 0.001. All statistical analysis was performed using Microsoft Excel 2013.

Result and Discussion Synapse-inducing recombinant protein - eNLG1 The key constituent of our novel neural interface is the genetically engineered recombinant protein based on NLG1 (Figure 1a). Neuroligin (NLG) is one of the most widely studied postsynaptic transmembrane protein families.35–39 It is known to trigger presynapse formation autonomously, without association with auxiliary proteins.16,20 NLG1 specifically binds to neurexin1β (Nrx1β)41,42 located in the outer membrane of neighboring neurons43, then the assembly of Nrx1β mediates presynaptic differentiation15,19 through direct binding of intracellular synaptic proteins like CASK, Mints, and synaptotagmin44–46. The design of the engineered protein was adopted from our previous report that demonstrated capability of synapse induction20. Briefly, only the extracellular domain of NLG1, which contains the site of neurexin1β interaction41,47, was used for high production efficiency48. TagRFP, a red fluorescent protein with high light efficiency49,50, was attached facilitate quantification of the recombinant protein. Finally, an Avi-tag was placed at the C-terminus. Avi-tag is a special peptide sequence to which biotin is attached post-translation by in vivo enzymatic processes.51,52 With


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Figure 1. Synapse-inducing protein, eNLG1, and its immobilization on microbeads. (a) Structure of eNLG1. The structure of eNLG1 was reconstructed after NLG1 (PDB ID, 3BIW)41 and TagRFP (PDB ID, 3NED)50. (b) TagRFP fluorescent image of beads modified with varying concentrations of eNLG1 solution (upper row). DIC image (lower left). TagRFP fluorescence of eNLG1 modified amine beads (as opposed to streptavidin beads) (lower right). Scale bar: 1 μm. (c) FM-SEM image of eNLG1 beads, showing the nano-groove structure of beads. Scale bar: 1 μm, 200 nm, respectively. (d) Plot of fluorescent intensity against increasing concentration of eNLG1 solution. (55 > n > 130 for each condition) (e) SDS-PAGE gel image of eNLG1 stripped from beads, and stained with streptavidin-HRP. Two bands indicating monomeric eNLG1 (black arrow), and multimeric eNLG1 complex (white arrow) are shown.

biotinylation, eNLG1 readily fixed to the artificial substrate through biotin–avidin interaction53, known as one of the strongest non-covalent interactions (Kd ≈ 10−14 M)54. To validate that the purified eNLG1 could be immobilized on substrates by biotin-avidin interaction, streptavidin-coated polystyrene microbeads with nano-grooves were incubated in eNLG1 solution and fluorescence from the TagRFP was observed (Figure 1b, c). The choice of


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microbeads as the interfacing substrate was primarily due to the similarities in size (diameter ~2.8 μm) with neurites, which makes it suitable for studies on surface interaction with neurons. In addition, it can be readily and reproducibly modified, and offers a large number of samples from each batch. This was crucial to obtain reliable statistical data. After incubation, fluorescence was detected from the surface of the microbeads, but not from the amine-coated beads despite the same PS core. Result showed that eNLG1 was immobilized on the substrate by biotin-streptavidin specific interactions. Because biotin is located at the Cterminus of eNLG1, it is expected that the ectodomain of NLG1 is directed away from the surface to the external environment. As the concentration of eNLG1 solution is increased, the fluorescent intensity rapidly rose (Figure 1d). At a protein concentration above 0.6 μg/μL, the fluorescent intensity showed saturation, which means every available binding sites is filled by eNLG1. The immobilization of eNLG1 did not affect the overall structure of the microbead (Figure 1c and figure S3). Subsequent experiments used microbeads saturated with eNLG1. Because density of the synaptic protein is critical factor for synapse formation55, close packing of the eNLG1 is a basic requirement for synapse induction. To investigate the configuration of eNLG1 assembled on the microbeads’ surface, eNLG1 beads were treated with bis(sulfosuccinimidyl) suberate (BS3) which makes amine-to-amine crosslinks between closely packed proteins, followed by biotin-streptavidin dissociation using SDS. Size composition of the protein complex stripped from the microbeads was analyzed by western blot (Figure 1e). Results indicate a protein mixture of different molecular mass. Also, heavier protein complex is formed with eNL1. Since the length of spacer in BS3 is about 11.8 Å, heavier complex must be derived from multimeric or very closely packed forms of eNLG1.


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Constituents of the induced synapse at eNLG1 bead To demonstrate that eNLG1 establishes neural interfaces by mediating presynaptic differentiation, NLG1 bead were incubated with primary cultured hippocampal neurons (10 DIV) for 2-3 days. Morphology of the neuron-eNLG1 interface was observed using FE-SEM. Acquired images clearly show that neurites meet eNLG1 beads, follow the bead surface and develop a wide contact region (Figure 2a, left column). Neurites’ tendency to favor eNLG1 beads were reproducibly observed. When neurites were placed between two eNLG1 beads, the direction of growth was orthogonal to the beads and fine structures were generated at both beads to make further contacts (Figure 2a, right column). Notably, the neural interface that results is also in part due to the neurons actively undertaking changes in its morphology to cover the eNLG1immobilized substrate. In contrast, bare or BSA beads were not recognized by axons, which merely passed by without interaction (Figure 2b). This is consistent with previous reports of neurons adjusting their growth direction toward surfaces immobilized with synCAM (another type of synapse-inducing protein18,56), and changing their morphology during synaptogenesis57. Presynaptic differentiation was confirmed with immunocytochemistry, a method in which fluorescence markers are bound to specific protein for identification and analysis15,19. Accumulation of key pre- and post-synaptic proteins that is at the neuron-bead interface were examined. Proteins exclusively located at the pre- or post-synapse at nerve terminal were selected as the marker proteins. In accordance with results obtained with HEK293 cell (Figure S4), ICC imaging shows that Nrx1β in primary neurons had assembled on the eNLG1 beads (Figure 2c, d). In addition, synapsin1 (Syn1), a representative presynaptic protein in the cytoplasmic surface of synaptic vesicle58, was recruited and co-located with Nrx1β showing characteristic puncta geometry. Other intracellular presynaptic proteins such as synaptophysin (a glycoprotein that is


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Figure 2. Morphology and protein constitution changes in primary hippocampal neurons induced by eNLG1 beads. Representative FE-SEM images of the interface between hippocampal neuron and eNLG1 beads (a) or control groups (b). Scale bar: 500 nm (upper row of (a) and (b)), 2 μm (lower row of (a)). (c) Representative images of immunostained Nrx1β (red) and synapsin1 (green) in neurons that are in contact with eNLG1 beads. Scale bar: 5 μm. (d) Fluorescent analysis of preand post-synaptic proteins in each bead condition. Intensities are compared between each bead condition. (37 < n < 68 for each condition. For Syn1 and Vglut1, 7 times of independent experiments are conducted. There were 1~4 replicates for other synaptic proteins) Statistical significance is indicated by n.s. for p > 0.05 and *** for p > 0.001.


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abundant in the synaptic vesicle59,60) bassoon (a zinc-finger protein selectively localized at the cytomatrix at active zone61, and is essential for regulated neurotransmitter release62) and vesicular glutamate transporter1 (Vglut1, a Na+-dependent Pi cotransporter associated with the membrane of synaptic vesicle and transporting glutamate into the vesicle63) had also assembled. Presynaptic protein accumulation was significantly higher for eNLG1 beads for than bare and BSA beads (p < 0.01). On the other hand, the amount of postsynaptic proteins such as postsynaptic density protein 95(PSD95, a membrane-associated guanylate kinase exclusively located in the postsynaptic density13,64), gephyrin (a component of the postsynaptic protein density in inhibitory postsynapse, anchoring receptors to the cytoskeleton65), GluN2 (one of the subunits of NMDA receptor, which is a glutamate receptor and positive ion channel) remained at a similar level with both controls, which is a strong indication of surface contact with endogenous presynapse in the absence of endogenous postsynapse at the eNLG1 beads. This confirms that fluorescent signals representing presynaptic proteins do not come from arbitrary overlap of the beads and native synapse, but from the presynapse induced by eNLG1-modified substrate. In conclusion, surfaces modified with eNLG1 initiates presynapse differentiation with neurons, presumably through the Nrx1βdependent pathway, resulting in the formation of a presynapse-interface.

Kinetics of exocytosis at the induced synapse We previously showed that presynapses induced by engineered postsynaptic protein immobilized on artificial substrates can conduct chemical signaling via neurotransmitter release.20 However, chemical release kinetics of the neurotransmitter release has not been verified. Kinetics of induced synapse, especially relative to native synapses is a critical factor for application on the neural interface. To examine the exocytotic process, assay using FM 1-43, a fluorescent dye widely used


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Figure 3. Kinetics of chemical release in the induced synapse. (a) Representative time-lapse image of the fluorescence from FM 1-43 after stimulating an induced synapse (right), and DIC image of the stimulation site (left). Scale bar: 5 μm. (b) Fluorescence intensity of the line profile bisecting the bead over time. Scale bar: 5 μm for inset. (c) Comparison of the normalized fluorescence decay using representative data from induced synapse, endogenous synapse, and neurite near BSA.

for visualizing membrane recycling66,67, was performed. FM 1-43 has a positively charged amine group that prevents diffusion across the cell membrane. Therefore, FM 1-43 can enter or exit from cells exclusively through endocytosis and exocytosis, respectively. FM 1-43 was loaded into vesicles at synaptic boutons via endocytosis by triggering membrane recycling using 20 mM potassium solution. After washing with DPBS for 10 min, neurons were stimulated using 80 mM potassium solution, and release of the FM 1-43 was real-time monitored.


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As a result, intense fluorescence from FM 1-43 within synaptic vesicles was observed at the contact between neuron-eNLG1 bead after the dye loading, indicating that synaptic vesicle formation via membrane recycling is active at the induced presynapse (Figure 3a). When exocytosis was triggered, the fluorescent intensity exponentially decreased (Figure 3b), which means FM 1-43 was released from the synaptic vesicle to the synaptic gap and diffused out to the bulk extracellular space66. The fluorescence intensity of FM 1-43 surrounding the eNLG1 beads decreased immediately after applying stimulating solution, and the reduction of FM 1-43 intensity at endogenous synapses occurred at the same time point. On the other hand, the neurons at BSA beads showed no initial staining and subsequent destaining upon stimulation. It means the decrease in FM 1-43 intensity was localized event. FM 1-43 is a membrane impermeable dye that can only move into a cell through membrane recycling. Taken together, it supports that the change in fluorescence intensity at the eNLG1 beads is a result of synaptic vesicle exocytosis at induced synapses. Next, the rate of the fluorescence decay was compared between the endogenous (Figure S5) and induced synapse. The time constants obtained from single exponential fitting are similar in order (37.1 s for endogenous synapse and 49.7 s for induced synapse, respectively) (Figure 3c). This strongly indicates that the presynapse induced by eNLG1 is functional, and its secretion behavior is similar to the endogenous synapse.

Stability of induced synapse-interface Most neural interfaces suffer from a loss of neural contact caused by movement and growth of neurons. Those interfacing methods depend on passive contact without any means to conjugate a neuron and substrate. On the other hand, induced synapse-interface is supported by tight protein-


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Figure 4. Stability of the induced synapse-interface. (a) Relative change in position of soma and eNLG1 bead during 4 days. Inset: Fluorescent image of membrane recycling site assay using FM 1-43 at the eNLG1 bead. While the neuron migrate and grow over time, the synapse-interface is stably preserved. Scale bar: 20 μm and 2 μm for inset. (b) Fluorescence intensity of the line profile bisecting the eNLG1 bead over days.

protein interaction. To demonstrate stability of induced synapse-interface, an eNLG1 bead incubated with 10 DIV primary neurons were observed for severe days. Neurons kept on migrating and growing, changing its location relative to the eNLG1 bead (Figure 4a). Nevertheless, induced synapses formed on the eNLG1 beads were stably preserved (Insets of figure 4a and figure 4b). There was considerable position change especially from 6 to 8 day after the bead seeding, but the configuration of the induced synapses were successfully retained. This result showed the synapseinterface is durable against interface breaking unlike conventional methods.

Induced presynapse in hippocampal slices on eNLG1 beads. The ultimate goal of neural interface systems is to make links between the nervous system and external device. Therefore, applicability of the interfacing technique to neurons in vivo is of utmost


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Figure 5. eNLG1 bead induces synapse formation in cultured hippocampal brain slice. Representative image of immunostaining on synapsin1 (green) and Vglut1 (red) in hippocampal slice contact with eNLG1 beads (a) and a image covered a wide scope (c). Scale bar: 5 μm for (a) and 20 μm for (c). (b) Comparison of the fluorescent intensity of the presynaptic proteins in eNLG1 beads and bare beads. Intensities are compared between each bead condition. (n > 78 for each condition from 7 replicates) Statistical significance is indicated by *** for p > 0.001.

importance. To confirm the scalability of the induced synapse-substrate interfacing technique, we co-cultured brain slices with eNLG1 beads. The organotypic brain slice culture68 preserves intrinsic cellular and connective organization69,70 and synaptogenetic activity71,72. Cultured hippocampal slices were incubated with eNLG1 beads for 3 days, followed by immunostaining targeting Syn1 and Vglut1 as representative presynaptic proteins. Results show that the presynapse were formed on the eNLG1 beads in cultured brain tissue. Fluorescence intensity is three times greater than that of control (Figure 5a, b). There were distinct


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synaptic puncta, and the location of Syn1 and Vglut1 clearly overlapped. These results demonstrated that eNLG1-functionalized substrate could trigger presynaptic development in cultured hippocampal brain slice. Similarly, presynapse formation evoked by eNLG1 was observed at the cultured cortex brain slices (Figure S6). The probability of successful presynapse induction at the hippocampal brain slice was about 58%, which is lower than that of primary cultured neurons (about 80%) (Figure 5c). This may be due to the irreversible damage incurred during slicing73 and lower probability of neuron-bead contact in the brain slice due to greater glia to neuron ratio than dissociated neurons70,74.

Conclusion Here, we explored the potential of the synapse-inducing technique as a neural interfacing method. Critical features for neural interface were assessed, including structural features, chemical release kinetics, stability, and scalability to the brain tissue. FE-SEM images show that neurons prefer contacting with eNLG1-immobilzed surfaces, and actively covers the substrate. From the protein analysis, it was verified that eNLG1 beads trigger synaptogenesis of primary neurons, consequentially forming the synapse-interface. Membrane recycling kinetics measurement using FM 1-43 showed that induced synapses also released chemicals in the synaptic vesicles, and their kinetics was similar to that of endogenous synapses. Strikingly, synaptic connection was robust against migration and growth of neuron over several days. The synapse-inducing technique was successfully applied to not only primary cultured neurons but also brain tissues isolated from rat. Our findings provide numerous insight. In the synapse-interface, the substrate and the neuron are firmly fixed to each other by protein-protein binding, and chemicals are secreted directly to the substrate. Though microbeads were the surface of choice in the present study, beads can be readily


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substituted by any avidin-coated substance. Methods have been established for immobilizing streptavidin on electrode materials such as silicon75, gold76,77, and carbon nanotubes78 using chemical conjugation. If a conductive electrode is used as substrate and modified with eNLG1, it would be possible to construct a synapse-electrode interface, and detect the chemical signal electrochemically79. On the other hand, the plasticity of induced synapse is still unknown. Memory relies on synaptic plasticity80,81, which is modulated by the expression of synaptic proteins. If induced synapse possesses plasticity like native synapse, synapse-interface could be used as an adaptive neural interface, changing its synaptic strength depending on the stimulation history. With further studies, the synapse-interfacing technique is groundbreaking in the effort to integrate neural circuits and artificial devices.

ASSOCIATED CONTENT The following files are available free of charge. Vector design of eNLG1, Images of neurons and glial cells in organotypically cultured hippocampal slice, FE-SEM image of bare beads, Light and fluorescence image of Nrx1β-GFP transfected HEK cells incubated with eNLG1 beads, FM 1-43 assay image of the endogenous synapse, Induced presynapse in cultured cortex brain slice (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ORCID


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Taek Dong Chung: 0000-0003-1092-8550

ACKNOWLEDGMENT This work was supported by Samsung Science and Technology Foundation under Project Number SSTF-BA1502-09. We thank Jin Sook Kim, Moon Hwa Choi, and Dr. Eun Joong Kim for their technical assistance. We thank Sung-Yon Kim and researchers in his group for their kind assistance with their tissue slicing instrument.

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Figure 1. Synapse-inducing protein, eNLG1, and its immobilization on microbeads. (a) Structure of eNLG1. The structure of eNLG1 was reconstructed after NLG1 (PDB ID, 3BIW) and TagRFP (PDB ID, 3NED). (b) TagRFP fluorescent image of beads modified with varying concentrations of eNLG1 solution (upper row). DIC image (lower left). TagRFP fluorescence of eNLG1 modified amine beads (as opposed to streptavidin beads) (lower right). Scale bar: 1 μm. (c) FM-SEM image of eNLG1 beads, showing the nano-groove structure of beads. Scale bar: 1 μm, 200 nm, respectively. (d) Plot of fluorescent intensity against increasing concentration of eNLG1 solution. (55 > n > 130 for each condition) (e) SDS-PAGE gel image of eNLG1 stripped from beads, and stained with streptavidin-HRP. Two bands indicating monomeric eNLG1 (black arrow), and multimeric eNLG1 complex (white arrow) are shown. 174x70mm (300 x 300 DPI)


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Figure 2. Morphology and protein constitution changes in primary hippocampal neurons induced by eNLG1 beads. Representative FE-SEM images of the interface between hippocampal neuron and eNLG1 beads (a) or control groups (b). Scale bar: 500 nm (upper row of (a) and (b)), 2 μm (lower row of (a)). (c) Representative images of immunostained Nrx1β (red) and synapsin1 (green) in neurons that are in contact with eNLG1 beads. Scale bar: 5 μm. (d) Fluorescent analysis of pre- and post-synaptic proteins in each bead condition. Intensities are compared between each bead condition. (37 < n < 68 for each condition) Statistical significance is indicated by n.s. for p > 0.05 and *** for p > 0.001. 119x159mm (300 x 300 DPI)



Figure 3. Kinetics of chemical release in the induced synapse. (a) Representative time-lapse image of the fluorescence from FM 1-43 after stimulating an induced synapse (right), and DIC image of the stimulation site (left). Scale bar: 5 μm. (b) Fluorescence intensity of the line profile bisecting the bead over time. Scale bar: 5 μm for inset. (c) Comparison of the normalized fluorescence decay using representative data from induced synapse, endogenous synapse, and neurite near BSA. 150x102mm (300 x 300 DPI)


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Figure 4. Stability of the induced synapse-interface. (a) Relative change in position of soma and eNLG1 bead during 4 days. Inset: Fluorescent image of membrane recycling site assay using FM 1-43 at the eNLG1 bead. While the neuron migrate and grow over time, the synapse-interface is stably preserved. Scale bar: 20 μm and 2 μm for inset. (b) Fluorescence intensity of the line profile bisecting the eNLG1 bead over days. 168x65mm (300 x 300 DPI)



Figure 5. eNLG1 bead induces synapse formation in cultured hippocampal brain slice. Representative image of immunostaining on synapsin1 (green) and Vglut1 (red) in hippocampal slice contact with eNLG1 beads (a) and a image covered a wide scope (c). Scale bar: 5 μm for (a) and 20 μm for (c). (b) Comparison of the fluorescent intensity of the presynaptic proteins in eNLG1 beads and bare beads. Intensities are compared between each bead condition. (n > 78 for each condition) Statistical significance is indicated by *** for p > 0.001. 122x89mm (300 x 300 DPI)


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Scheme 1. With protein engineering techniques, synaptic proteins can be immobilized on the abiotic substrate. When a neuron meets the functionalized substrate, partner synaptic proteins of the neuron are recruited to the contact site, and presynaptic differentiation is triggered. In this interface, the functionalized substrate directly faces the induced synapse with robust protein binding. 83x47mm (300 x 300 DPI)



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Robust Induced Presynapse on Artificial Substrate as a Neural

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