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Title

細胞認識機能を有する神経細胞活動記録に向けた次世代
微小電極技術の開発

Author(s)羽賀, 渉

CitationIssue Date

2024-03

Type

Thesis or Dissertation

Text version

ETD

URL

http://hdl.handle.net/10119/19074

RightsDescription

Supervisor: 筒井 秀和, 先端科学技術研究科, 博士

Japan Advanced Institute of Science and Technology

Doctoral Dissertation

Development of next generation microelectrodetechnique toward neuronal activity recordings
with cell recognition capability
WATARU HAGASupervisor: HIDEKAZU TSUTSUI

Graduate School of Advanced Science and Technology
Japan Advanced Institute of Science and Technology
Materials Science
2024 MarchAbstract
Molecules called synapse organizers are involved in the formation of synapses. Amongthem, clustering of neurexin-1 (b) by interacting with neuroligin-1 is known to cause
synaptogenesis. The ability of Neurexin-1 to induce synaptogenesis in synapse
organizers has been studied from various viewpoints, and although the elucidation ofthis molecular function has been widely attempted, the use of this function as amolecular machine has not been quite common. In this study, I developed an engineered
synapse organizer based on Neurexin-1 and established a method to fabricate a glass
microelectrode in which the main body was insulated by glass and only the tip was
metal (Gold). By combining these, I attempted to induce selective synaptogenesis in theglass microelectrode. The engineered synapse organizer we developed formed synapselike structures on the surfaces of inorganic microbeads and glass microelectrodes byartificial binding, not intrinsic binding. The localization of synapsin, which acts as asynaptic marker molecule, and Rab3 in the introduced marker was consistent. The
engineered synapse organizer we developed can be said to be capable of inducing
synaptogenesis in the glass microelectrode. When the tips of microbeads and glass
microelectrodes bearing Rab3 antibodies were contacted with neurons expressing Rab3,
I confirmed the accumulation of Rab3 despite the binding by antibodies. The method forfabricating the glass microelectrode was developed by preferentially selecting a tool thatis universally used for the patch clamp method, which may be a compatible tool forfields that employ such electrophysiological measurement methods. I employed afocused ion beam (FIB) machine for the fabrication of the tip of the microelectrode, andI was able to perform highly reproducible fabrication. This allowed us to efficiently
fabricate an electrode with an arbitrary tip shape using the FIB, if an electrode with gold
conducting could be fabricated by a micropipette puller. Finally, in a proof-of-principle
experiment using chick forebrain neurons, selective synaptogenesis was induced in theglass microelectrode in a non-CO2-dependent culture medium. Chick neurons are
simple to culture, and unlike rats and other mammals, it is known that removing theforebrain neuron from an egg embryo is unlikely to cause biological problems.
Experiments showed that synaptogenesis by the engineered synapse organizer took atleast 4 hours, but chick forebrain neurons transfected at room temperature and open air
survived for at least 8 hours and formed synapses with antibody-modified microbeads
and glass microelectrodes.
Keywords: Electrophysiology, Neuron, Synapse Organizer, Glass Microelectrode,Protein Adsorption

Table of Contents
Abstract................................................................................................................... 0
Chapter 1 General Introduction .......................................................................... 2
Chapter 2

Development of Engineered Synapse Organizer ............................. 21

Chapter 3

Development of Glass Microelectrodes with the Ability to Induce

Synapse Formation ............................................................................................... 50
Chapter 4 Proof-of-Principle of Glass Microelectrodes with Selective Synaptic
Induction Ability................................................................................................... 90
General Discussion............................................................................................. 110
Acknowledgements ............................................................................................. 117

1

Chapter 1

General Introduction2General purposes of this study⚫ Develop a synapse organizer-modified microelectrode to establish a microelectroderecording method with cell selectivity．

Previous study⚫ Measuring neuronal electrical activity using existing electrophysiological techniques(patch clamp and extracellular electrode methods). “NEHER, E., SAKMANN, B.
(1976)”⚫
⚫

Synapse formation between an endogenous synaptic organizer (rat neuron) and a
microelectrode arrays (MEA). “S. Kim et al. (2023)”
Selective synaptogenic activity between modified microbeads by rat neuron(expressing engineered synapse organizers, NOT peptide-tag). “S.A. Hamid et al.
(2023)”

3

Electrophysiology is a technique that can measure the electrical signals of neurons. By
using this technique, we can understand how neurons and neurons transmit and process
information, which can lead to the development of drugs that are useful in the treatmentof neurological and psychiatric diseases. On the other hand, neurons are connected toeach other by a structure called a synapse, and a signal is sent through the synapse. Eachtechnique including the patch-clamp method, which is one of the electrophysiology
techniques, can observe signals through synapses, but it is difficult to observe a specificsynapse (it is possible to search for a "pair" by generating action potentials at thepresynaptic region and searching for a synapse where the response can be seen, but ittakes a huge amount of time). Therefore, we tried to demonstrate the principle of a newelectrophysiology technique to connect an electrode and an arbitrary neuron by asynapse and directly observe signals from the synapse. The new electrophysiology
technique proposed here uses a special molecule called a synaptic organizer that induces
synaptic differentiation. It is known that this molecule causes synaptic differentiation
when two pairs are connected to each other in the extracellular region, and recent
research has discovered that it causes synapse formation even in inorganic materials
other than neurons. It has also been found that even if the connecting part of thesynaptic organizer (genetically modified Mouse NRXN-1 in previous research) is not4

endogenous, as long as it is connected, synaptic differentiation starts from both ends. By
combining these findings, we created a genetically modified synaptic organizer with apeptide tag in the extracellular region and tried to induce synapse formation on
inorganic materials (microbeads and glass microelectrodes) modified with the ligand.
The purpose of this study is to develop an engineered synaptic organizer-modified
microelectrode and to conduct a proof-of-principle study to establish a microelectroderecording method with cell selectivity.

The following is a description of the terms used in this paper, such as cell biology,
electrophysiology, and molecular biology.

1. Neurons
In animals, including us humans, there are neurons that are responsible for information
processing and transmission; neurons are electrically excitable and can generate action
potentials that can be used as signals to transmit information to other neurons [1].

2. Axons and Dendrites
Neurons consist of a cell body and two types of cytoplasmic processes that extend from5the cell body. One of these structures, the axon, transmits information to other neurons
and muscles, while the other structure, the dendrite, receives information from otherneurons. Although there is usually one axon per neuron, there are multiple dendrites [1,2].

3. Neurons
Neurons transmit electrical pulse signals on their axons that reach presynaptically totrigger the release of neurotransmitters that are received presynaptically to modulate theactivity of ion channels. This neurotransmitter release is Ca2 + - dependent and is
carried out by a molecular machinery centered on SNARE proteins (SNAREs are
responsible for docking and fusion of synaptic vesicles to the active zone and the Ca2 +triggering step itself in combination with the Ca2 + sensor synaptotagmin) [1]. The
electrical signals received by the neurotransmitter are processed and become again
electrical pulse signals, which are transmitted on other nerve cells [1, 3].

4. Synapses
Charles S. Sherrington named the structures created at the junctions between neurons
synapses. Synapses are the junctional structures where neurons communicate. Memory
6and learning in the brain are expressed as changes in signal transmission throughsynapses (synaptic plasticity). There are about 1.000 billion neurons in the human brain,
which are further connected to form a vast network [2]. Some synaptic transmissions
involve the use of electricity and others involve the release of chemical substances;these are called electrical synapses and chemical synapses, respectively. A typicalsynaptic structure is shown below [4] (fig. 1)

7

Fig. 1. A typical transmission electron micrograph of a synapse of a mouse neuron.
Structures such as postsynaptic density (PSD) and synaptic vesicles are visible.

5. Synapse Organizers
Neurons are connected to other cells via synapses. The formation of synapses involves
molecules called synapse organizers. Neuroligin, located in the postsynaptic membrane,
was first described as a synapse organizer [2].
Synapse organizers are known to induce synaptic differentiation [5].

6. Variety of synapse organizers
Synapse organizers are both pre - and postsynaptic and include Leukocyte common
antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs), LRRTMs, CblnGluR δ, NGL-3, PTP δ, Neurexin (NRXN), Neuroligin (NLGN), and PSD-95[6, 7, 8, 9,

8

10, 11, 12, 13].

7. Neurexin and Neuroligin
Neurexins exist in the presynaptic neuronal membrane and are involved in theorganization, and the maintenance of the synapses. The endogenous ligands forneurexins include neuroligins, which exist in the postsynaptic neuronal membrane.
These proteins are essential for synaptic transmission and play an important role in theorganization of brain synapses. They act as cell adhesion molecules and are known toconnect the pre-synapse and postsynapses [6, 7]. NRXN and NLGN couple through atrans-synaptic interaction in which the extracellular domain of NRXN on thepresynaptic neuron binds to the extracellular domain of NLGN on the postsynaptic
neuron, leading to the assembly of pre - and postsynaptic localized molecules such as
synaptic vesicles and receptors required for synaptic function [14]. On the other hand, it
has been suggested that NRXN and NLGN are important for synaptic function but are
not required for synaptogenesis, and that the cytoplasmic tails of Neurexin are notrequired for synaptogenesis [6, 15].

8. Neurexin Family in Mammals
9

Mammals have at least three neurexin genes, which are transcribed by two different
promoters into longer -NRXN or shorter -NRXN isoforms.
Furthermore, - and -NRXN have five and two canonical alternative splice sites,
respectively. These splice variants of NRXN can bind to NLGN but have selectivity
NRXN-binding properties and regulate synaptogenesis in different ways. In addition, and -NRXN have five and two canonical alternative splice sites, respectively, and it isestimated that there are over 1000 variations of NRXN. Similarly, neuroligin is encodedby multiple genes and has alternative splice sites, resulting in diverse isoforms [14,16,
17, 18, 19, 20, 21, 22).

9. Coupling of NRXN and NLGN
It has been shown that NRXN and NLGN signaling for pre-synaptic rigging occurs
because dimerization of neuro-differentiating proteins located post-synapticallypromotes clustering of NRXN located pre-synaptically [23, 24, 25, 26].
In addition, clustering of epitope-tagged NRXNs similarly triggers presynaptic
differentiation, suggesting that clustering in some way is a prerequisite for presynaptic
differentiation [24, 27].

10

10. Induction of Synaptogenesis in Non-biological Materials
According to Yoshida et al., a synapse organizer (IL1RAPL1, PTP δ) can be used toform synaptic structures on magnetic microparticles (particle size ranging from 4.0 to
4.5 [µl] [28]) [. It has also been reported that NRXN  induces presynaptic
differentiation in neurons derived from human induced pluripotent stem cells, and that
NRXN  expressed in non-neuronal cells induces the accumulation of PSD-95, apostsynaptic marker molecule. Furthermore, when NLGN or LRRTM is expressed in
non-neuronal cells, co-cultured neuronal cells form heterogeneous synapses with thecells [14, 15, 17, 29, 30, 31, 32]. Thus, induction of synaptogenesis by some synapse
organizers has been found to be effective against non-neuronal cells expressing synapse
organizers and non-biological materials [21, 22]. This suggests that synapse organizer
molecules can be immobilized on the tip of the glass microelectrode prepared in thisstudy (almost the same as the magnetic microparticle particle size used by Yoshida etal.), thereby sufficiently inducing synaptogenesis.

11. Patch Clamp Technique
The patch-clamp technique is an electrophysiological recording method performed
using glass electrodes. It is a powerful electrophysiological technique that can directly
11measure membrane potential and the amount of current passing through the cell
membrane, and is still a standard technique in electrophysiology and neuroscience [33].
The method was developed by Erwin Neher and Bert Sakmann, who won the Nobel
Prize in Physiology or Medicine in 1991 for achieving precise recordings of theelectrical current flowing through the cell membrane, which is associated with the
opening and closing of a single ion channel [34].

12. Protein Adsorption
Protein adsorption and immobilization on surfaces are influenced by several factors.
These include the properties of the protein itself, the properties of the surface, and theenvironmental conditions in which the interaction occurs. Properties of the protein suchas size, shape, charge, and hydrophobicity affect how the protein interacts with thesurface. For example, a protein with a high surface charge is likely to adsorb to asurface with an opposite charge due to electrostatic attraction, and a protein with ahydrophobic region may preferentially adsorb to a hydrophobic surface [35, 36, 37].
It is also pointed out that the PI (Isoelectric Point) of the protein solution is one of thefactors of adsorption, but it is not dominant, and the effect of water as a medium may belarge [35, 38, 39].
12

13. Protein Adsorption on Metal Surfaces Under the Influence of Voltage/Electric
Field
It has been found that when a voltage is applied to electrodes in a protein solution, theprotein is immobilized on its surface [35, 36, 37, 40, 41].

13

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20

Chapter 2

Development of Engineered Synapse Organizer

21

Purpose of Chapter 2⚫ To develop small, engineered synapse organizers that do not cross-react withendogenous ligands.
⚫ To establish an experimental culture system for proof of principle using
microelectrodes.
Achievement⚫

Using Neurexin-1, synaptogenesis was confirmed by the binding of a small peptide
tag between microbeads and chick or rat neurons.

⚫

Synapse formation was confirmed for chick forebrain neurons within 4 hours at room
temperature and in air.

Remaining Challenges⚫ Search for the minimal functional unit for the active site of chick and mouse NRXN⚫

1.
Confirmation of glutamine release at synapses formed by the developed engineered
NRXN-1 to determine if they are functional synapses.

22

Chapter 2 Key Points⚫

Neurexin-1 (b) without LNS (laminin, neurexin, sex hormone-binding globulin)
domain exerts synaptogenesis via peptide-nanobody interactions⚫

The engineered synapse organizer functions in mammalian and chick forebrainneurons⚫

Chick neurons allow synaptogenesis in CO2-independent medium at room
temperature⚫

Peptide-tag-based multiplexed organizers may be engineered with chick neurons

23Introduction
Membrane potential dynamics in neurons are thought to be fundamental to howneural circuits encode, process, and store information, though the details of thesemechanisms remain one of the unsolved conundrums in science today. Technologicaldevelopment for detecting neuronal electrical activities in more detail is therefore
essential. Despite recent advances in optical imaging approaches, the most stablereadouts of electrical activity have been obtained with microelectrode techniques suchas patch clamp, intracellular recording, and extracellular recording [1-3]. These
techniques were originally suited for recording at a single or a few sites within a circuit,
but recently developed flexible, arrayed electrodes are enabling large-scale parallel
extracellular recording [4]. On the other hand, there are various technical limitations in
the microelectrode-based activity recordings. One of the key limitations is that therecording target is, inherently, not specific to a cell type. The specificity is essential
because neural circuits are composed of different cell types, each of which is believed toplay a significant role in circuit function [5]. Researchers therefore need to identify thetype of the cell under recording through various indirect means, such as visual
identification of marker gene expression, dye injection followed by cell morphology
inspection, waveform analysis, and so on. However, these identifications are not24compatible with large-scale parallel recordings.
We have been seeking to develop a neuron-microelectrode interface with inherent celltype specificity by employing the molecular function of synapse organizers [6]. The
synapse organizers are a series of membrane proteins that play a central role in thegeneration and maintenance of synapses, the junctions between neurons [7-9]. Specificsets of organizers in the axon and dendrite form complexes upon their contact, andtrigger cellular signals to differentiate into pre- and post-synapses. Remarkably, suchfunction of the synapse organizers is generally robust and is exerted even by contact
with non-biological objects whose surfaces are modified with the organizers’ receptor[10, 11]. Then, it is expected that a microelectrode functionalized with the receptor forsynapse organizer is capable of exerting differentiation signals when in contact with atarget neuron expressing that organizer and this leads to the formation of a synapse-likeneuron-microelectrode junction but not with a non-target neuron, thereby establishing acell type-specific interface (Fig. 1). We recently demonstrated that a gold
microelectrode functionalized with Interleukin-1 receptor accessory protein-like 1(IL1RAPL1) exerts such differentiation signals on the cortical neurons contacted [6].
There, IL1RAPL1 is a natural synapse organizer in the postsynaptic membrane andinteracts with PTP, a synapse organizer in the presynaptic membrane [12, 13]. Since25

these natural synapse organizers are pre-existing in neurons, it is necessary to develop
engineered organizers that do not interact with the natural organizers to avoid
interference and achieve a cell-type-specific neuron-microelectrode junction (Fig. 1).
Neurexin-1 (NRXN-1), another natural synapse organizer in the presynaptic
membrane, could serve as a molecular platform for this purpose [14]. The endogenous
receptors for NRXN-1 include neuroligin-1 (Nlgn1) in the postsynaptic membrane.
When NRXN-1 and Nlgn1 come into contact and form a trans-cellular complex, as
Nlgn1 is a dimer-forming protein, clustering of Nrxn1 is promoted and this clustering
then triggers a cellular signal for presynaptic differentiation [10, 15]. Importantly, thesub-region of NRXN-1 recognized by the receptor and that for triggering
differentiation signal is structurally independent [14]. Therefore, a modified NRXN-1,in which the recognition region is replaced by an artificial ligand, is expected to exert
synaptogenic activity following its clustering driven by specific ligand-receptor
interactions. The previous work showed that a variant of green fluorescence proteinfunctions as such an artificial ligand [14]. But the ~27 kD fluorescent protein isrelatively a large ligand. In general, small molecular tools are advantageous from theviewpoint of gene delivery and expression. Also, conditions required for synaptogenic
activities, which provide important information for future prototyping of the synapse
26

organizer-based microelectrode technology, have yet to be fully addressed. We,
therefore, set the purposes of this study as the generation and characterization ofcompact engineered synapse organizers.

Material and Methods
Molecular biology
DNA constructions were performed with the standard PCR-based cloning techniques.
For PCR, ligation, and plasmid purification, we used PrimeSTAR Max DNA
polymerase, DNA Ligation Kit Ver.1, and NucleoBond Xtra Midi Kit, respectively. All
these products are from Takara Bio (Shiga, Japan).

Cell culture
HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (Fujifilm, Tokyo,
Japan) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific,
MA, USA). Primary cortical neurons were prepared from Wister rat embryos onembryonic day 18 as described elsewhere [16, 17], and were cultured in minimum
essential medium (MEM) (Sigma M4655, MO, USA) supplemented with 2% FBS and
1% B27 (Thermo Fisher Scientific, MA, USA). For the preparation of chick forebrain
27

neurons, we established the following procedure which is modified from the previous
reports [18, 19]. Briefly, fertilized chicken eggs purchased from a local distributor wereincubated for 8~9 days at 38 °C. The forebrains were collected in 2 ml ice-cold PBS
without divalent cations. After careful removal of the meninges under a dissecting
microscope, the brain tissue was mechanically chopped with a surgical blade into small
pieces less than 0.5 mm, treated with 0.05% trypsin (Nacalai Tesque, Kyoto, Japan) in
PBS(-) containing 10 unit of DNAaseI (Takara Bio, Shiga, Japan) for 7 minutes,
triturated with a pipette, and filtered through a 100 m mesh cell strainer. Dissociatedneurons were centrifuged at 1000 rpm for 10 minutes, re-suspended with minimum
essential medium (MEM) (Sigma M4655, MO, USA) supplemented with 2 % FBS and
1 % B27 (Thermo Fisher Scientific, MA, USA), and plated onto  12 mm coverslips
coated with a mixture of equal volumes of poly-L-lysine (1.0 mg/ml) and poly-D-lysine(0.1 mg/ml). On the next day, the culture medium was replaced with minimum essential
medium supplemented with 2 % horse serum and 1 % B27. Plasmid transfection to therat hippocampal and chick forebrain neurons was performed at DIV (days in-vitro) 6
and 4, respectively, using Lipofectamine 3000 reagent (Thermo Fisher Scientific, MA,
USA) according to the manufacturer's protocol. All animal experiments followed
ARRIVE guidelines. Thirty-two rat embryos
28

from three pregnant rats and 25 chicken embryos from the fertilized eggs were used forexperiments relevant to this study, including various tests. The sex of embryos was notdistinguished. Experimental animals were used along the institutional guidelines for thecare of animals provided by Japan Advanced Institute of Science and Technology as
well as the National Institutes of Health guide for the care and use of Laboratory
animals.

Evaluation of the synaptogenic activities
The synaptogenic activities of the engineered synapse organizers were evaluated by the
microbeads assay. Synthetic cDNA encoding anti-BC2 nanobody (anti-SpotNb) was
designed (Integrated DNA Technologies, IA, USA), sub-cloned into an Fc fusion
protein expression vector, and transfected into HEK293T cells. The FBS concentrationin the culture medium of HEK cells was reduced to 2%, into which the anti-SpotNb-Fc
fusion protein was secreted. The medium was harvested, passed through a 0.22 m poresyringe filter (Advantech, Tokyo, Japan), and incubated with protein-A magnetic
microbeads ( ~ 4.7 m, PAMS-40-S, Spherotech, IL, USA) in a low protein binding
micro-centrifuge tubes at 4 °C for overnight. Typically, 15 l beads solution was
incubated with ~10 g anti-SpotNb-Fc fusion protein. The protein-conjugated29microbeads were deeply washed with neuron culture medium and transferred onto theneuron culture, typically at the density of 105 beads per a  12 mm coverslip of neuron
culture. Leiboviz L15 based medium was used for the experiments to investigate thetime and CO2 dependence of chick forebrain neurons as described below. After adefined co-culture time (1~20 hours), neurons were fixed and subjected to confocal
microscopy imaging (FV1000D, Olympus, Tokyo, Japan).

Results
The primary structure of NRXN-1 consists of an N-terminal signal peptide (SP), an
LNS (i.e., laminin, neurexin, sex hormone-binding globulin) domain, a transmembrane
domain (TM), and a PDZ domain binding motif at the C-terminal cytoplasmic end (Fig.
2a). The LNS domain is known to constitute the binding interface with Nlgn1 [10]. To
design a compact engineered organizer, I tested whether Spot-tag could act as a ligand
to promote clustering. Spot-tag is a 1.4 kD peptide, PDRVRAVSHWSS in the singleletter code, and is an improved ligand for the anti-BC2 nanobody [20, 21]. To avoid
confusion due to its name, here I refer to anti-BC2 nanobody as anti-SpotNb. We
modified mouse NRXN-1 (Sequence ID: NP_001333889.1) to lack the LNS domain,
have a Spot-tag downstream of the signal peptide and Flag-tag, and also a presynaptic
30

marker, EGFP-Rab3, via a self-cleaving P2A peptide, and named Spot-mNRXN1ECD::P2A-EGFP-Rab3 (Fig. 2a). Flag-tag was not used in the present study. Genetransfer efficiency in primary cultured neurons is generally limited by standard
lipofection methods. Therefore, to observe a sufficient number of contacts between theobjects and the transfected neurons, magnetic microbeads conjugated with protein A(PAMS-40-S, Spherotech, Illinois, USA) were used to emulate microelectrodes (Fig.
2b). Rat cortical neurons were plated onto   mm coverslips, transfected with SpotmNRXN-1ECD::P2A-EGFP-Rab at 6 days in vitro culture, and the next day, antiSpotNb decorated microbeads were added and co-cultured overnight. Neurons
expressing this construct were easily identified by fluorescence derived from EGFPRab3. Accumulations of EGFP-Rab3 signals were often observed at the sites of contact
between the axon and the microbead, which showed synaptogenic activities mediatedthrough the peptide-nanobody interaction (Fig. 2c). In the control experiments in which
the rat cortical neurons were co-cultured with the undecorated microbeads, no suchaccumulations of EGFP-Rab3 were found at the sites of contact (Fig. 2d). Then, I
examined whether Spot-mNRXN-1ECD::P2A-EGFP-Rab3 could exhibit
synaptogenic activity in the chick forebrain neuron culture. This is because primarycultures of chick forebrain neurons growing into typical pyramidal shapes can be31

prepared in a rapid procedure and maintained in a CO2-independent culture medium[18, 19], which should be useful for the future development of the synapse-organizerbased bioelectronic interface. Following co-culture of chick forebrain neurons
expressing Spot-mNRXN-1ECD::P2A-EGFP-Rab3 and the microbeads decorated
with anti-SpotNb, I did observe accumulation of EGFP-Rab3 signals were found at thesites of contact (Fig. 2e). Then, again as a negative control, the cortical neurons
expressing a construct in which Spot-tag has been substituted with a chromophore
deficient variant of fluorescence protein (Venus_Y66G, or YFPnull; [22]) were cocultured with the microbeads. Rab3 signals were not observed (Fig. 2f), which ensured
that the Rab3 signals are not directly caused by the anti-SpotNb decorated microbeads
but reflect the association between the ligand (i.e. Spot-tag) and the receptor (i.e. antiSpotNb). To statistically confirm these trends, I defined an index of Rab3 accumulation
as Ibead/Iaxon-1, where Ibead and Iaxon are the integrals of the EGFP signal in a 50 m2
circular region around the beads and that around the representative axons not in contact
with the beads, respectively, and named as the Rab3 index. The Rab3 index was
analyzed for the laser scanning microscopy images taken under identical imaging
conditions. It was confirmed that the accumulations of Rab3 were statisticallysignificant both to the two negative controls (Fig. 2g).
32

Next, I sought to examine whether the use of NRXN-1 derived from the chick protein
database could result in similar or any further enhancement in the synapse-inducing
activity. After searching the National Center for Biotechnology Information database,
we focused on Gallus gallus NRXN-1 (Sequence ID: NP_001185905.1) and generated
a construct named Spot-cNRXN-1ECD::P2A-EGFP-Rab3 which again lacks theextracellular LNS domain to prevent interaction with the natural ligands (Fig. 3a). The
resulting construct contained 7 amino acid mismatches compared to the mouse NRXN1-based construct (Fig. S1). Synaptogenic activities were evaluated with microbead
assays in rat cortical and chick forebrain neurons. In both types of neurons whencontacted with microbeads decorated with anti-SpotNb, synaptogenic activity similar tothat observed with mouse neurexin-derived sequences was observed (Fig. 3b, c), but notwith the control bare microbeads (Fig. 3d). The trend was also confirmed through theimage analysis (Fig. 3e). Thus, it was found that the mouse and chick Nrxn1-derived
engineered synapse organizers are compatible in their synaptogenic activity in rat
cortical and chick forebrain neurons. In both cortical neurons and chick forebrainneurons, there was no significant difference in the strength of synaptic induction
between Spot-mNRXN-1ECD::P2A-EGFP-Rab3 and Spot-cNRXN-1ECD::P2AEGFP-Rab3.
33

Our particular future direction is to exploit the neuron-microelectrode junction formed
with the engineered synapse organizer as a bioelectronic interface. Knowledge of thetime, temperature, and atmospheric conditions required for synapse induction against
the objects will be essential for future research and development. I addressed theseissues using chick forebrain neurons because of the advantages mentioned above. The
time required for induction was investigated by fixing the chick forebrain neurons
transfected with Spot-cNRXN-1ECD::P2A-EGFP-Rab3 after different times of coculture with the microbeads decorated with anti-SpotNb. The effects of atmosphere andtemperature were investigated by performing the co-culture in the medium based onminimum essential medium (MEM) at 37 °C, Leibovitz's L15 medium at 37 °C, andL15 medium at room temperature (25 °C). Here, MEM is a CO2-dependent culture
medium, used in the presence of 5% CO2, and L15 is a CO2-independent medium, used
under the normal atmospheric condition. All the culture medium was supplemented with
2% horse serum. After 2 hours, Rab3 accumulations were not typically observed,
although were seen infrequently. (Fig. 4a, Fig. S2). After 4, 8, and 12 hours of the coculture, Rab3 accumulations were more common at the sites of contact between axonand microbeads in all of these conditions (Fig. 4a). As confirmed by the analysis of the
Rab3 index (Fig. 4b), the induction efficacy tended to increase with time gradually, but
34there were no significant differences in the Rab3 index between MEM and L15 media
and between 37°C and 25°C.

Discussion
In this study, we developed Spot-tag-based compact engineered synapse organizers
using NRXN-1 sequences derived from mouse or chick genes. Both of theseorganizers showed synaptogenesis in rat cortical neurons and chick forebrain neurons
upon contact with objects decorated with anti-SpotNb. The results suggested threemajor implications.
The first is on the utility of using a peptide tag. Since several different combinations ofshort peptide tags and their specific nanobodies have been developed [23, 24], thesuccessful induction of the presynaptic structure through the interaction of Spot-tag with
its nanobody opens the possibility of further developing multiple engineered synapse
organizers and their specific activators. Such a set of orthogonal molecular tools is
expected to realize a multiplexed molecularly induced bioelectronics interface, as well
as modification of neural circuits at unprecedented levels of detail. Furthermore, owing
to the small ligand size, the total cDNA sequences for the engineered organizer and thedownstream presynapse marker (i.e. EGFP-Rab3) do not exceed 2k base pairs, the35practical limit of cDNA size for packaging into adeno-associated virus (AAV) vectors. It
is expected that the gene transfer efficiency will be improved with the use of AAV
vectors, which is of practical importance in increasing lab work efficiency.
The second is on NRXN-1 itself. There were seven mismatches between the sequencescomprising the transmembrane and cytoplasmic regions of mouse and chick NRXN-1
used in this study (Fig. S1), but no clear differences in the synaptogenic activity of these
proteins were found in rat cortical and chick forebrain neurons, at least by theevaluations with EGFP-Rab3. Thus, the region of NRXN-1 responsible for the
induction signaling of synaptogenesis is conserved and appears to function universallyand even across species. This trend is in sharp contrast to the high specificity withwhich NRXN-1 forms transcellular heterophilic complexes with its ligand in theextracellular space. The observed universality may be explained by the relatively
conserved amino acid sequence of the cytoplasmic region of mouse and chick NRXN1 with the PDZ domain binding motif at its end, but, on the other hand, the previous
study has shown that the cytoplasmic region of NRXN-1 is not necessarily required forexerting the induction signal [25]. Understanding the core region responsible for the
induction as well as the downstream molecular mechanism would be one of theimportant future directions.
36

The third is on the practical advantages of chick forebrain neurons. The results indicatedthat there is a high degree of compatibility between rat cortical neurons and chickforebrain neurons concerning synaptogenic activities by the NRXN-1-basedengineered synapse organizers. This is not simply explained only by the structuralsimilarity of NRXN-1 but suggests the conserved molecular mechanisms that detect its
clustering and activate the downstream signal cascade for differentiation. In addition to
the benefits that the experiments of forming neuron-object junctions can be conductedunder air atmosphere typically in ~4 hours, the culture of chick forebrain neurons has
several practical advantages over mammalian neurons, including a rapid andinexpensive procedure and fewer concerns regarding the use of laboratory animals.
Thus, the chick forebrain neurons expressing the engineered synapse organizers
developed in the present work would serve as a useful cellular model for establishing
the molecularly-inducible neuron-microelectrode interface and also for developing
molecular tools for neural circuit editing.

37

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43

Fig. 1 Role of an engineered synapse organizer for the formation of cell-type-specificneuron-microelectrode junction. The microelectrode functionalized with a receptor
exerts synapse differentiation in contact with a target neuron expressing the engineeredorganizer with an artificial ligand, through the formation of the ligand-receptor complex(left) but not with a non-target neuron (right).

44

Fig. 2 (a) A design of mNRXN-1-based engineered organizer. (b) A scheme showing
microbeads assay of the synaptogenic function of an engineered organizer. (c-f)
Representative results of microbeads assay in the four conditions noted. Cx: corticalneuron, UN: undecorated, CFB: chick forebrain neuron, cdFP: chromophore deficientvariant of a green fluorescence protein. Arrows indicate Rab3 accumulations. Bar = 5
m; applied to all images. (g) Analysis of Rab3 index in four conditions indicated.
Asterisks indicate significance in the Tukey test (*p < 0.1%). The number of samples
was 26, 16, 18, and 24 from left to right.
45

Fig. 3 (a) A design of chick NRXN-1-based engineered organizer. (b-d) Representative
results of microbeads assay in the three conditions noted. Arrows indicate Ra3
accumulations. Bar = 5 m; applied to all images. (e) Analysis of Rab3 index in theconditions indicated. Asterisks indicate significance in the Tukey test (*p < 0.1%). The
number of samples was 28, 14, and 13 from left to right.
46

Fig. 4 (a) Representative results of microbeads assay after co-culture of the different
times as indicated. Some of the Rab3 accumulations are indicated by arrows. Bar = 5
m; applied to all images. (b) Rab3 index versus co-culture time plots. Data in Mem (+CO2, 37 °C), L15 (- CO2, 37 °C), and L15 (- CO2, 25 °C) are plotted in open circle,
open square, and filled square, respectively. The number of samples ranged from 11 to25.
47

Fig. S1 Alignment of amino acid sequences of mNRXN-1 (Seq. ID: NP_001333889.1)
and cNRXN-1 (Seq. ID: NP_001185905.1). Identical residues are boxed. The LNS
domain, transmembrane domain, and the PDZ domain binding motif are shown in blue,
gray, and pink, respectively. The asterisks indicate seven mismatches in the region usedin the constructs. Note that the gap after G201 in the LNS domain of mNRXN-1 is due
to ss4(-) alternative splicing and the coding amino acid sequence in that region is anexact match to cNRXN-1 (see Seq. ID: NM_001346959.2).

48

Fig. S2 Examples of Rab3 accumulations in the microbeads assay in co -culture time of
2 hours. The arrows indicated accumulations. Bar = 5 mm; applied to all images.

49Chapter 3

Development of Glass Microelectrodes with the
Ability to Induce Synapse Formation

50

Purpose of Chapter 3⚫ Establish a method for fabricating glass microelectrodes with synapse organizer
molecules immobilized on the tip.
⚫ Establish a method for immobilizing synapse organizer molecules on glass
microelectrodes and confirm how they are immobilized.
Achievement⚫ A simple method for fabricating glass microelectrodes was established using theelectrode fabrication equipment used in the patch-clamp method.
⚫

The protein was effectively immobilized by applying a pulse voltage to the metal
part at the tip of the electrode in a protein solution. Fluorescent proteins wereindirectly immobilized, and the immobilization was confirmed by observing thefluorescence.

Remaining Challenges⚫ Circuit design and electrode shape optimization for measurement via glass
microelectrodes.

51

Chapter 3 Key Points⚫

Proteins were immobilized by applying a rectangular pulse voltage to the gold
surface at the tip of a glass microelectrode.

⚫

Using a micropipette puller and FIB (Focused Ion Beam), gold was successfully
exposed at the tip of a glass microelectrode.

⚫

A protein called ProteinA was bound to the tip of the fabricated glass microelectrodeto allow binding of proteins with optional Fc regions.

⚫

Protein immobilization experiments using the fabricated glass microelectrodes
suggest that the voltage value of the rectangular pulse and the period of the pulse areimportant.

⚫

The mechanism of protein immobilization and a highly reproducible micropipettepuller processing method remained unresolved.

52

Introduction
As described in Chapter 2, I performed co-culture of microbeads and Chick, Rat neurontransfected with epitope-tagged NRXN-1, which was developed based on NRXN-1
from mammals and birds, and observed its activity. By combining Anti BC2 Nanobody
and SPOT-tag added to the N-terminus of NRXN-1, I was able to induce synaptogenesis
by promoting the clustering of NRXN-1. Based on this result, I considered that applying
this mechanism to the tip of a glass microelectrode such as used in the patch clamp
method, it would be possible to make synapses between the electrode tip where Anti BC2
Nanobody was immobilized and the fabricated Axon of neuron transfected with NRXN1. In Chapter 2, binding by Anti BC2 Nanobody and SPOT-tag induces synaptogenesisby clustering NRXN-1 at the presynaptic terminal, but I am interested in performing
selective neuronal observation by using different peptide tags.
The patch clamp technique is an electrophysiological technique that can directly measure
the membrane potential and the amount of current flowing across the plasma membrane.
It was developed by Erwin Neher and Bert Sakmann, who was awarded the Nobel Prizein Physiology or Medicine for achieving single-channel recording in 1991 [1]. The patch
clamp technique is an electrophysiological technique that can directly measure themembrane potential and the amount of current flowing across the plasma membrane.
53

Since the patch clamp technique was first developed, various derived techniques havebeen actively developed to this day [2,3,4]. However, this technique also has a problem
that a specific cell cannot be selectively observed. Based on the experience I have gainedso far; I have been able to create a glass microelectrode by an original technique. By
placing the fabricated electrode tip and a silver chloride electrode in a protein solution
and applying a pulsed voltage, the protein in the solution is immobilized on the gold
surface at the tip of the electrode. By this technique, synapse organizer molecules were
immobilized on the electrode tip. Although many have proposed that protein adsorption
on the surface is linked to factors such as the zeta potential and the isoelectric point, there
is still no theory that can provide a unified explanation [5, 6, 7, 8]. There are also studies
that have shown that it is possible to immobilize proteins on electrodes using a direct
current voltage or a pulsed voltage [9, 10, 11].
In order to realize selective cell observation, I developed a prototype microelectrode fornext-generation neural activity recording with cell recognition function using synapticorganizer molecules that induce synapses.
Materials and Methods
In order to make a glass micropipette-type microelectrode with cell-selectivity compatible
with conventional patch-clamp instruments, I designed its overall shape, including the54

back of the electrodes and the lengths of the tubes, so that it was no different from theglass electrodes used for patch-clamp applications. This allowed us to make connections
to manipulators and ensure conduction without any problems. The fabrication method of
the electrodes was based on the work of B. B. Katemann and W. Schuhmann[12].
Platinum-containing microelectrodes and their development method are presented [1]. In
this study, I used borosilicate glass tubes and gold wires to make glass microelectrodes.
The glass microelectrode fabrication method is described separately for each item below.
Seal
I made a SEAL MACHINE (Fig. 1a). I used a Cantal wire heater (PC-10H, NARISHIGE)
as the filament. I vacuumed the glass tube from the left and right sides using a vacuum
pump (DAP-6D, ULVAC KIKO) while heating the glass tube with a gold wire (NILACO,
0.08 [mm]) of about 1 [cm] at the center filament, so that the glass at the filament position
was shrunk and sealed (Fig. 1b). The ultimate pressure of the vacuum pump I used was
6.65 [kPa]. The heating time by the filament was 2 minutes and 20 seconds, and thecurrent and voltage values were set at 12.20 to 13.20 [A] and 3 to 4 [V], respectively. I
performed sealing while observing the gold wire shrinking surrounded by the molten
glass using an optical microscope. I adjusted the voltage and heating time accordingly. I
made a straight gold-sealed glass.
55

Pull
A metal wire was inserted inside a glass tube, sealed, and pulled simultaneously with amicropipette puller (P-1000, SUTTER INSTRUMENT). The metal wire was stretchedthin (as in Kintaroame), while leaving room for glass and gold for later processing. A
glass micropipette containing gold immediately after pulling (Fig. 1c). The P-1000 canbe programmed to adjust the pulling of the glass tube, and a typical glass pipette for patch
clamping can be made with good reproducibility by using the filaments and programs
listed in the recipe book. The programs and filaments for making the microglass tube
electrode used in this process are shown in Table 1. The Ramp test was always executed
before the experiment to confirm the parameters such as Ramp value, temperature, andhumidity before pulling. For the first pull of the experiment day, carefully check [View
Pull Results] in the Menu of the Program. Program adjustment is done mainly bychanging the Heat parameter; changes in Pull and Vel other than Heat have been found tohave a significant effect on electrode shape. By carefully checking the [View Pull Results]
and making adjustments to get close to the last successful [View Pull Results], such asslightly increasing the Heat if the Ramp value is low or slightly decreasing the Heat if thetemperature is high, it is possible to efficiently fabricate an electrode with a good shape56

in which the gold is fully connected inside the glass capillary.
The prepared electrode is examined with an optical microscope near the glass tip, and if
the gold is conductive from the inside of the glass tube to the tip, the tip is processed in
the next step.

FIB Processing
Once the gold-filled glass pipette is prepared, it is processed by a focused ion beammachine (SMI-3050, Hitachi High-Tech Science Corporation). The focused ion beammachine (FIB) is a machine that processes a sample by focusing an ion beam andirradiating it with the focused ion beam. The resolution is 30 kV and 4 nm, and the tip
diameter of the glass microelectrode to be processed this time is 1 to 5 [µm] (Fig. 1d).
Since the gold at the electrode tip cannot be seen during processing unless the gold isgrounded, the FIB machine placed the electrode on a special pedestal and grounded thegold inside the electrode securely. The tip was processed by cutting it with a beam etching
function. The goal was to process the electrode tip into a pencil shape with a sharp point.
By making four cuts at different angles, I finished it into a square pyramid shape. I
adjusted it so that the gold was exposed at the apex. This shape was devised to immobilize
the synapse organizer molecules only at the electrode tip, and the tip diameter of 1 to 557

[µm] should be similar to the particle size of the microbeads used in Chapter 2.

Protein Immobilization
An antibody-binding protein, ProteinA (TCI chemicals, Tokyo, Japan), was immobilizedon the tip of FIB-processed glass microelectrodes using a technique unique to ourlaboratory and described in the introduction (Fig. 1e). I used this "Protein Labeling" toimmobilize ProteinA on the electrode tip and further bound proteins that bind ProteinA,such as Fc Venus (a fluorescent protein) and Fc BC2 Nanobody (a nanobody that binds
to epitope-tagged NRXN-1 created in this study). I unbound the Fc region by washingwith MgCl2 and switched the protein to be immobilized on the surface of the electrodewith ProteinA.
The protein solution used in the protein immobilization experiments was made from a
buffer containing ProteinA. The buffer was 20 mM Hepes (pH 9.0) + 0.05% TritonX
mixed with 5 mM NaCl. 400 [μ l] of the buffer was prepared, and 2 [μ l] of ProteinA wasused in the experiments. Triton-X contained in the buffer was a surfactant and was used
to avoid immobilization of unnecessary proteins on the glass surface of the electrode. A
disk-shaped silicon chamber (outer diameter 1.2 [cm], inner diameter 0.5 [cm], thickness
1 [mm]) was attached to a slide glass with PDMS, and 50 [μ l] of ProteinA solution was
58

placed. A silver chloride electrode and a glass microelectrode were placed in the ProteinAsolution, and a pulse voltage was applied between the microelectrode and silver chloride
using a source meter (2401, Keithley). A pulse voltage was applied by connecting to thesource meter via a breadboard so that the current value could be monitored by the source
meter. The electrode was fixed with a patch clamp manipulator (U-1C, NARISIGE) andthe silver chloride electrode was fixed with a negative electrode. The glass microelectrodewas fixed to a pipette holder and then set on the manipulator. The silver chloride electrodewas fixed to a rod (H-1, NARISIGE) and then set. The pulse voltage was set between 1.7- 2.4 [V], and the width of a rectangular pulse was set between 50 or 100 [ms]. The
magnitude of the voltage was determined by checking the diameter of the electrode tip
and how much current flowed in a test just before the experiment. The number of pulses
was set to be between 100 or 200 times as 1 set, and the voltage and the number of pulses
were adjusted accordingly while observing the current waveform. The most typical
pattern was 2.0 [V], 50 [ms] × 200 pulses, 2 sets.
After the application of the pulse, the electrode was washed with pure water and overnightin the optional Fc protein. After the overnight, the electrode was removed and washedwith PBS (phosphate-buffered saline) before observation, experiment, etc. Since theelectrode can be washed and reused as long as it is not destroyed, the electrode with59

immobilized ProteinA was stored in PBS as much as possible, and when theimmobilization of ProteinA was performed again, all organic matters on the electrode tip
were removed with Piranha solution, and then the experiment was repeated.

Microscopy
When Fc Venus was bound to the tip of the electrode, the presence of ProteinA can be
indirectly confirmed by fluorescence observation because the binding between Fc protein
and ProteinA is almost certain. Fluorescence observation was performed with amicroscope (IX71, Olympus) connected with a xenon lamp (Ushio, Tokyo) and a CCD
camera (Orca Flash 4.0 C11440, HAMAMATSU). Excitation filters from 460 to 480,
emission filters from 495 to 540 (Olympus), and a 490 GFP dichroic mirror were used toobserve Fc Venus. Data were acquired using IDL (Research Systems, USA) and analyzed
with ImageJ.

Results
The results of the protein-immobilization experiments, showing the fluorescence of the
tip of the electrodes (Fig. 2, 3). I used a low voltage in Figure 2 and a high voltage in
Figure 3. The ProteinA solutions used were all of the same composition and were60

previously described in the section on Protein Immobilization. The results of theexperiments showed that ProteinA could be immobilized on the tip of the electrodes whenpulsed voltage was applied to the electrodes in ProteinA solutions to make Fc-Venus
overnight. I used piranha solutions to remove ProteinA. Piranha solutions are strongorganic solvents made by mixing concentrated sulfuric acid with hydrogen peroxide in aratio of 3:1. Because of the risk of explosion, I had to make sure that concentrated sulfuric
acid was placed in the vessel first and hydrogen peroxide was mixed in it. I made piranhasolutions and immediately put the tip of the electrodes in it and washed them. I could
confirm the removal of organic matter by confirming the generation of bubbles, so I
washed them until no bubbles were generated. After washing, I performed fluorescenceobservation again and confirmed that no fluorescent proteins were observed, so I foundthat both electrodes could be washed in about 10-15 seconds. Also, as previously
described, I could only release Fc binding with MgCl2 solutions. I prepared MgCl2
solutions, washed the tip of the electrodes for about 10 seconds, and observed under amicroscope that no fluorescence remained. Then, I could overnight with any Fc proteins(FcBC2 Nanobody, in this study) and switched the proteins immobilized on the tip of theelectrodes.
By observing the fluorescence of Fc Venus and indirectly confirming the immobilization
61

of ProteinA, it can be said that Fc BC2 Nanobody could be immobilized in a similarmanner. The diameter of the electrode tip was almost the same as the microbeads used in
the experiments in Chapter 2, and the situation in which Fc BC2 Nanobody isimmobilized by Fc-binding in it can also be said to be the same. Therefore, it is expectedthat the results of the experiments on inducing synaptogenesis in microbeads in Chapter
2 can be obtained by bringing the glass microelectrode prepared here into contact with
Neuron.

Notes on Experiments
The rough flow of the experiment has already been explained in Materials and Methods,
but the knowledge obtained by actually preparing the electrode is shared below. The
precautions in the preparation of the glass microelectrode are explained item-by-item
below.

Seal
The transition temperature of borosilicate glass is 525 ℃, and the filament is heated toapproximate this temperature. At this temperature, the gold does not change and is only
enclosed and enclosed in the glass. To prevent static electricity from causing the thin gold62to stick to the glass, rubber gloves are not worn when gold is placed in the glass tube. By
picking up the gold at the end of the glass tube, the gold can be placed in the glass tube
without bending the gold line. It has been found that stretching the gold as much as
possible does not adversely affect the subsequent pull, so the bent gold is used as muchas possible. It can be straightened by rolling the gold, but it is difficult to remove the strain
once it has been applied to other than 0.08 [mm], for example, 0.10 [mm], and it isdifficult to handle 0.05 [mm] for the same reason. Since the change in the diameter of thegold line also requires adjustments in the program used for the subsequent pull, theprocessing is performed under unified conditions if possible. Although it is not anessential element, it is important for the subsequent pull process that neither the glass tube
nor the gold is subjected to excessive force as much as possible, and the change isminimized.
The sealing machine consists of a heating mechanism by a filament and a glass tube fixing
part as shown in Figure 1a. The heating mechanism is made by cutting an aluminum rod
and carving a screw hole and a groove to guide the filament. The glass tube fixing part isalso made by carving a screw hole and a groove, and the same four aluminum parts areused in total. It is good if the temperature at which the filament can output is high enoughto melt the electrode. However, if the current value is too high or the glass tube is in63contact with the filament, the glass tube will melt away. It is carefully observed under the
microscope for changes in the glass through the filament. When the preparation of thedevice is completed, before heating the glass tube, a vacuum pump is connected to bothsides of the electrode by a tube and air is pulled. After sealing, the encapsulation of theelectrode is confirmed by attaching a silicon tube of about 1.0 [mm] to the disposable
syringe and connecting it to the sealed glass tube. If the electrode is encapsulated aroundthe center, it can be confirmed immediately because the syringe tries to return to its
original position. To prevent the influence of environmental changes, a certain number ofthem are sealed together and used for Pull.

Pull
It was processed using a micropipette puller (P-1000, SUTTER). At present, I use theprogram shown in Table1, but it is highly dependent on the state of the filament. In
addition, I have confirmed that changes in temperature and humidity affect the Pull result.
Therefore, I clarify here that basically the same program does not guarantee the same
result. During the search for a suitable program, I changed the type of filament and theprogram to match it, and finally I was able to create a glass electrode in which the gold
was continuous inside.
64

As already mentioned, the parameter adjustment is mainly carried out by changing only
the Heat. The guideline for the Heat parameter is the Ramp value + 10. Pull and Vel are
greatly related to the shape of the electrode, and frequent changes in the electrode shape
are not desirable in this study in which the state of the electrode tip is important. In orderto stabilize the experimental results, the Ramp value was measured after the start of theexperiment, and the program was run as it was if the value, room temperature, andhumidity did not change greatly from the previous experiment. If there was a change in
the Ramp value, the Heat was adjusted accordingly. The Ramp value in significant
environmental changes, such as the Ramp value in summer and winter, changed greatly,
but the Ramp value usually did not change greatly to 10 or 20 in a month. If there was achange, the change or breakage of the filament and the state of the machine had to bechecked and adjusted again. For the reasons mentioned above, it was important to keep
the room temperature constant, so in this experiment, the experiment was carried out by
fixing the room temperature to 23-24 ℃. The humidity varied from 30% to 70%, but if
the humidity was higher than 50%, the Heat was also adjusted up, and on the contrary, if
the humidity was lower than 50%, the Heat was adjusted down. The adjustment range ofthe Heat was basically 5. The most important thing in the Pull is the adjustment of the
program, and it is the key to finish the adjustment early on the day of the experiment.
65When the adjustment of the program is successful and the electrode with a good shape iscompleted, as many electrodes as possible are pulled and stocked. When the experimentis carried out on an annual basis, it is convenient to record the good programs roughly by
seasons.
After completion of Pull, the electrodes were stored on a plastic Petri dish with thickdouble-sided tape placed on it.
FIB Processing
Various preparations are required before FIB processing. In particular, it is important toprotect both the equipment and electrodes when using the FIB equipment. Since the mainchamber where the FIB equipment is processed must be kept in a vacuum state, theelectrodes, and the FIB stage where the electrodes are installed must be cleaned inadvance to eliminate organic contamination. A pointed contact probe (C-NP20, MISUMI)
was packed into the electrode to ensure the electrical connection of the electrode.
In some electrodes, the gold is not exposed at the tip. In that case, the glass tube isremoved until the gold is exposed and prepared for FIB processing. A small scissor or
tweezers is used for processing. The glass at the tip of the electrode is in the form of astring, and that portion is cut off and the gold is connected to the tip of the glass tube.
When protein immobilization experiments are performed, attention is paid to the crack of66

the glass because proteins may enter the inside of the glass tube through the crack of theglass. Such pre-processing of the tip shape is only an aid for FIB processing, and it is not
for re-processing a relatively thick electrode or a destroyed electrode to finish it into athin electrode by FIB processing. It is performed only when it is necessary as spare
processing. It is difficult to reuse an electrode that failed in Pull (thick tip, gold is notconnected inside, etc.).
A special stage was fabricated for FIB processing (Fig. 4a). A conductive tape was used
to secure the electrode to the stage, and a contact probe (CP) was inserted inside. The CP
was pressed from the inside to the tip of the electrode to measure the conduction (Fig.
4b). It was confirmed microscopically that the gold was touching the CP. Since the CP
alone was not long enough to protrude outside the glass tube, a metal wire (0.40 [mm],brass, Dydo Hunt) of about 1 [cm] was pressed from the back of the CP to push in the CP,
and the metal wire was pressed and fixed by a metal plate. Since the CP was fixed while
being pressed by the metal wire, the CP was always in a pressed state, which was anindicator of good electrical conduction.
Next, I explain machining with the FIB device. As previously described, the FIB device
is used to sharpen the tips of the electrodes in a pencil-like shape, and the electrodes areshaved with a laser so that the gold is exposed. The following detailed explanation is67based on the FIB device SMI-3050. The electrodes installed in the FIB device are oriented
so that the tips of the electrodes are oriented in the downward direction (180 °) on theStage Controller screen of the FIB. During FIB operation, the tips of the electrodes canbe oriented upward on the screen by setting the Scan Rotation to 180 ° in advance. This
makes it easier to see when the electrodes are tilted. The stage is attached to the holder
specified for the FIB device with conductive tape. It is attached to the rod and the stage
is transferred to the Main Chamber. After the prescribed Daily Adjust, the process moves
to the machining screen. The flow of machining (Fig. 4). Eucentric the electrodes with an
Image Scale of 40 [µm] or finer. The height (Z) after the Eucentric is recorded, and theheight can be set without Eucentric again by using it after the Stage Rotation. Note that
the Eucentric is required every time the Image Scale is changed. After the Eucentric, theelectrodes are machined by adjusting the Scan Rotation to an arbitrary angle, mainly at
an oblique angle (the image is adjusted at an oblique angle instead of the machining frame
at an oblique angle). The machining frame is created after the image is captured with the
Scan Speed set to 2, and the Beam type is set to URough (Rough is recommended for
careful machining) and the material is set to SiO2 by opening the properties of themachining frame. First, the tips are cut horizontally little by little to identify the positionof the gold on the tips of the electrodes. If the gold is already visible before machining,68this machining is not necessary. Once the gold is identified, the electrodes are cut on theleft and right sides at an arbitrary angle. After the cutting is completed twice, tilt is
performed, and the gold is confirmed to be on the "ridge" created on the tips of theelectrodes. The machining is adjusted by repeating machining a little, if necessary, butevery time the machining is repeated, the electrodes become thicker, so I try to minimize
the machining. After the left and right sides of the electrodes are cut, the electrodes arerotated by 90 ° in the Stage rotation (if the Scan rotation is also set to 90 °, the Scan
rotation before the Stage Rotation is the same as that at 180 °). The tips of the electrodes
are projected on the screen and tilted (50 ° - 60 °. For SMI-3050, the upper limit is 60 °).
After tilting, the same as before. If the tip of the electrodes is less than 5 [µm], the Image
Scale should be smaller than [µm] and the Rough beam should be used. In order to prevent
unnecessary irradiation of the beam during machining, the machining frame duringmachining should be observed and the machining should be stopped by STOP when themachining is finished. The position and location of the machining frame can also be
adjusted in the ST postoperative direction. If the electrodes can be formed into the target
shape, the next step is taken.

Protein Immobilization
69

The protein is immobilized on the gold exposed at the tip of the fabricated glass tube
microelectrode. In order to apply the pulsed voltage, conduction is ensured by CP as in
the preparation for FIB processing. Using a glass micropipette holder, the positions of the
microelectrode and the silver chloride electrode were fixed, and the aforementioned
protein solution was placed in a silicon chamber. I have already described the method of
the experiment, so I will omit it. The experimental results show that it is possible toestimate to some extent whether the immobilization of the protein is successful by looking
at the current flow during the experiment. Specifically, the probability of immobilization
is high when the current waveform falls like a function with the inverse of a natural
number (Fig. 3). In particular, the success rate of immobilization increased when thevoltage value was adjusted so that the spike immediately after the pulsed voltage wasapplied was 0.1 [µA] or more. The number of pulsed voltage applications is set to 2 sets
as already described, but in the case of most electrodes, it was found that the current value
decreases with each repetition of the set of pulsed voltage. In this case, the immobilization
probability increases more by repeating the application of pulsed voltage until the current
value becomes about 1/10. However, I cannot deny from our experience that too highvoltage application and excessive number of experiments do not increase theimmobilization probability.
70

I describe how to perform overnight after the experiment. I used a 96-well microplate forovernight. I placed approximately 200 [µl] of an arbitrary Fc protein solution in the wellplate, and then placed the electrode tip in the well. I used a rubber band and a wooden
board with a groove so that the electrode tip could be fixed in the Fc protein solution
while protecting it. The wooden board was carved with a groove of about 1 [mm] at theposition where the outer circumference and the electrode were fixed. The electrode was
fixed along the outer circumference of the wooden board with the rubber band, and ascrew was attached to prevent the wooden board from moving from the well plate (Fig.
5). Wet tissues for moisture retention and the well plate were placed in an airtightcontainer and overnight at 4 ℃.

Alternative Methods
So far, I have described the electrode fabrication process that I mainly use at present. If I
can switch methods and make appropriate electrodes according to the purpose andsituation, I will be able to easily consider new electrodes using these electrodes andcombinations with unprecedented methods. To create the possibility of efficiently solving
future problems, I will describe different methods that I established in the past.

71

Seal
In the past, I established a method to establish electrode conduction by spot-welding agold wire and a stainless-steel wire. This method differs from the current method ofprocessing with contact probes in that a dedicated terminal needs to be connected to bringthe conduction to the outside of the glass tube. Therefore, I need to think about which
conduction method to use the electrode before sealing it. I spot-weld a 0.10 [mm] stainlesssteel wire (NILACO) and a gold wire with a spot-welding machine (KTH-MWST, Kondo
Tech). I cut the stainless-steel wire to a length of about 10 [cm], cross the gold and thestainless-steel wire, and spot-weld them. I place them in the glass tube, place the gold in
the center, and seal them while vacuuming. I spot-weld the stainless-steel wire and thegold in only one place, but I can also do it in two places by sandwiching the gold. In thatcase, the two glass tubes created later by Pull may both be processed as microelectrodes
if the gold is connected inside.

Pull
As already mentioned, the production of glass pipettes by micropipette pullers is notstable and requires a lot of adjustments due to the continuity of the gold inside the tube.
For reference, the program used in the past is shown in Table 2.
72

I describe the processing of spot-welding stainless steel wire and gold. A terminal was
attached to the back end of a glass tube electrode to ensure continuity for use in FIB
processing and later protein immobilization experiments. A single-row IC socket(XR2C0008A, OMRON) was used. The terminal was separated into one terminal each.
The terminal was of a standard that can be inserted into itself, and the terminal at the backend of the electrode and the terminal for ensuring continuity to be bonded to it were thesame. All of the resin on the exterior of the IC socket was removed. The pin portion of
the terminal was ground small so that it could be inserted into the glass tube. A
microgrinder (UC100, URAWA) was used to polish the metal portion. After the pin waspolished small, a small amount of solder was applied in advance. The back portion was
cut to adjust the length so that the glass pipette could be placed on the FIB stage. The
stainless-steel wire inside the glass tube was exposed when the glass tube was shortened,
so it was removed leaving only 2-3 [mm] from the back end of the tube. The prepared
terminal was inserted into the back end of the electrode while carefully securing the glass
tube with a tool clipper or double-sided tape, taking care not to break the glass tip. The
side of the terminal and the stainless-steel wire were soldered. The direction of theterminal was made as parallel as possible to the direction of the glass tube, so that theholder and the electrode could be made parallel during the experiment. After the terminal73

was soldered, the stainless-steel wire and the terminal were reinforced with epoxy resin,
etc. If the terminal was ready to be connected with the terminal for continuity, it could be
used for FIB and experiments. The terminal for continuity could be used for spot welding
and experiments by soldering it to a covered wire, etc. and connecting it to the terminalat the back end of the electrode. This method is easy to ensure continuity and can be usedwithout repair in most cases. Continuity by CP cannot be broken when the CP pushedinside can no longer make contact with the gold, but processes such as spot welding andsoldering are easy to check because of strong bonding. Two of the disadvantages are thehigh processing difficulty and the lack of flexibility in the processing method. It isdifficult to attach the terminal so as not to break the tip of a pulled glass pipette, and theglass electrode after spot welding and attaching the terminal portion cannot be further
processed unless part of it is broken. In particular, the rear portion of the glass electrodeis a terminal portion, which makes it impossible to attach it to a patch clamp manipulator.
This was the biggest reason for not choosing this continuity method in our experiments.
I next describe the use of gallium to ensure conduction. When gallium, indium, and tin
are mixed at a ratio of 68.5%, 21.5%, and 10.0% while heating (at approximately 40 ℃),
gallium is converted to Galinstan ™, a room-temperature liquid metal (this alloy ishereafter referred to as gallium). Gallium in liquid form is injected into the inside of the74

electrode to ensure conduction. A heated and stretched disposable syringe was used to
inject gallium. This method is superior to other methods in the ease of conduction, and it
can also be used for protein immobilization experiments by simply attaching it to thepipette holder. The major drawbacks are the extremely high wettability of the glass andthe adhesion of gallium to other devices. Even if gallium is injected into the inside of theglass tube using a syringe, if it is not injected near the gold near the tip, it will touch theglass first. Once it touches the glass, it will continue to spread through the glass wall andhardly touch the gold. It was difficult to remove gallium, and in particular, the connection
with the pipette holder was noticeably contaminated and damaged by gallium. Although
it is characterized by the ability to ensure the conduction of the glass microelectrode later,
it was difficult to handle due to the liquid.

Protein Immobilization
Streptavidin-Alexa 488 (Thermo Fisher Scientific, USA) or ProteinA-Alexa could be
immobilized on the electrode tip in the same way as the immobilization of ProteinA.
However, these proteins had weaker fluorescence than ProteinA and were more easily
immobilized on the glass part. The advantage of immobilizing these proteins on theelectrode tip is that the fluorescence can be observed directly without Fc Venus overnight.
75

Although they may be useful when searching for protein immobilization conditions, sincedifferent proteins are expected to have different optimal immobilization conditions, these
proteins were not used in this experiment because I aimed to produce a microelectrode,which was the original purpose.

Discussion
In this chapter, I recorded in detail the technique that realized the immobilization ofproteins on the gold surfaces at the tips of the developed glass microelectrodes. The
fabrication method of the electrodes was based on the conventional patch-clamp method
and the technique developed by B. B. Katemann et al. [12]. Although I was able toimmobilize ProteinA on the tip of the fabricated microglass tube electrode, themechanism of protein adsorption, which is the core of this technology, is not deeply
understood. There is an opinion that two large forces, ionic amine-silanol bond andcooperative aggregation force between protein and glass, are involved in protein
adsorption. Usually, proteins are adsorbed more easily on the glass surface, but ourexperimental results show that they are rather adsorbed more strongly on the gold tip thanon the glass. Although pH is considered to be an important factor related to protein
adsorption, it is also suggested that the state of water as a medium has a greater effect [8,769, 13, 14]. However, in this study, when the electrode tip was immersed in the protein
solution, almost no active immobilization of proteins on the glass part was observed in awide area near the tip, which is probably because the number of proteins immobilized on
the gold surface of the electrode tip was larger than that of proteins (ProteinA, Fc Proteins)that might have adhered to the glass. Although I did not attempt to change the pH or
immobilize proteins other than ProteinA in the present experiments, it is possible thatinduction by electrical force, as suggested by many literatures [11, 16, 20], acted on theproteins to immobilize them efficiently on the tip of the electrode area.
Protein adsorption on the metal surfaces was effectively performed using a pulsed voltage.
Comparing the results of Figure 2 and Figure 3, it is considered that the voltage of about
2.0 [V], the pulse width of 50 [ms], and the number of times of 200 are more likely toimmobilize the protein more efficiently. Since the results of the experiments with dc
voltage are not available, it is not clear which is easier to immobilize the protein comparedwith pulsed voltage. However, the experiments performed with dc, ac, and pulsed
voltages all confirmed the immobilization of the protein [6, 10, 19], so the method of thisexperiment is considered to be sufficiently reproducible for the experiment, while themore efficient method remains unknown. Additionally, some studies suggest that theimmobilization of proteins on electrodes is related to the magnitude of the external
77electric field, and at least the results of this experiment are not inconsistent with them [5,10]. In order to establish an efficient protein immobilization method and to prevent
unnecessary protein immobilization on the electrode, clarification of such protein
adsorption phenomena is a future issue. Once protein adsorption technology is established,
it is expected to become a technology that spans both the biological and physical fields,
and it is expected to be the basis of various new technologies. In addition, protein
desorption phenomena on metal surfaces are also expected to be elucidated. For implant
devices with metal-based or glass surfaces that operate in vivo, protein adsorption must
be controlled [14, 17, 18]. Elucidation of the mechanism of protein adsorption anddesorption will be important for such areas as well. Elucidation of the mechanism will
require real time observation of adsorption phenomena, detailed analysis of adsorption
surfaces, and combinations of different types of metals and proteins.
I am interested in selective neuronal observation and elucidation of the mechanism ofprotein adsorption on metals by fabricated electrodes. The former allows evaluation of
the performance of glass microelectrodes by making contact with Neuron and acquiring
data as in the patch clamp method, while the latter considers it important to expand theknowledge on protein adsorption and acquire and analyze quantitative data.

78

References[1] NEHER, E., SAKMANN, B. Single-channel currents recorded from membrane ofdenervated frog muscle fibres. Nature 260, 799–802 (1976).
[2] Hamill, O P et al: Improved patch-clamp techniques for high-resolution current
recording from cells and cell-free membrane patches. Pflugers Archiv : European
journal of physiology vol. 391,2 (1981): 85-100. doi:10.1007/BF00656997[3] Kodandaramaiah SB, Franzesi GT, Chow BY, Boyden ES, Forest CR (2012):
Automated whole-cell patch-clamp electrophysiology of neurons in vivo. Nat
Methods 9: 585–587.
[4] Schroeder K, Neagle B, Trezise DJ, Worley J (2003): IonWorksTM HT: A new
high-throughput electrophysiology measurement platform. J Biomol Screen 8: 50–
64.
[5] Htwe EE, Nakama Y, Yamamoto Y, Tanaka H, Imanaka H, Ishida N, Imamura K(2018): Adsorption characteristics of various proteins on a metal surface in thepresence of an external electric potential. Colloids Surf B Biointerfaces 166: 262–
268.
[6] Wang Z, Yan Y, Qiao L (2017): Protein adsorption on implant metals with various
deformed surfaces. Colloids Surf B Biointerfaces 156: 62–70.
79

[7] Lapizco-Encinas BH, Ozuna-Chacón S, Rito-Palomares M (2008): Protein
manipulation with insulator-based dielectrophoresis and direct current electricfields. J Chromatogr A 1206: 45–51.
[8] M. Rabe, D. Verdes, S. Seeger, Understanding protein adsorption phenomena atsolid surfaces, Adv. Colloid Interface Sci. 162 (2011) 87–106.
https://doi.org/10.1016/j.cis.2010.12.007.
[9] [1] H. Noh, S.T. Yohe, E.A. Vogler, Volumetric interpretation of protein adsorption:
Ion-exchange adsorbent capacity, protein pI, and interaction energetics,
Biomaterials. 29 (2008) 2033–2048.
https://doi.org/10.1016/j.biomaterials.2008.01.017.
[10] J. Barberi, S. Spriano, Titanium and protein adsorption: An overview of
mechanisms and effects of surface features, Materials (Basel). 14 (2021).
https://doi.org/10.3390/ma14071590.
[11] S. Takahashi, K. Kishi, R. Hiraga, K. Hayashi, Y. Mamada, M. Oshige, S. Katsura,
A new method for immobilization of his-tagged proteins with the application of
low-frequency AC electric field, Sensors (Switzerland). 18 (2018).
https://doi.org/10.3390/s18030784.
[12] B.B. Katemann, W. Schuhmann, Fabrication and characterization of needle-type Pt80

disk nanoelectrodes, Electroanalysis. 14 (2002) 22–28.
https://doi.org/10.1002/1521-4109(200201)14:1<22::AID-ELAN22>3.0.CO;2-F.
[13] Suvajyoti Guha, Joshua R. Wayment, Mingdong Li, Michael J. Tarlov, and Michael
R. Zachariah Langmuir, Characterizing the Adsorption of Proteins on Glass
Capillary Surfaces Using Electrospray-Differential Mobility Analysis 2011 27 (21),13008-13014 DOI: 10.1021/la202792g[14] Mizutani T, Mizutani A. Estimation of adsorption of drugs and proteins on glass
surfaces with controlled pore glass as a reference. J Pharm Sci. 1978
Aug;67(8):1102-5. https://doi.org/10.1002/jps.2600670820.
[15] Silva-Bermudez, P., & Rodil, S. E. (2013). An overview of protein adsorption on
metal oxide coatings for biomedical implants. In Surface and Coatings Technology(Vol. 233, pp. 147–158).
[16] P. Cha, A. Krishnan, V.F. Fiore, et al., Interfacial energetics of protein adsorption
from aqueous buffer to surfaces with varying hydrophilicity, Langmuir. 24 (2008)
2553–2563. https://doi.org/10.1021/la703310k.
[17] J. anne N. Oliver, Y. Su, X. Lu, P.H. Kuo, J. Du, D. Zhu, Bioactive glass coatings
on metallic implants for biomedical applications, Bioact. Mater. 4 (2019) 261–270.
https://doi.org/10.1016/j.bioactmat.2019.09.002.
81

[18] J. Barberi, S. Spriano, Titanium and protein adsorption: An overview of
mechanisms and effects of surface features, Materials (Basel). 14 (2021).
https://doi.org/10.3390/ma14071590.
[19] Visalakshan, Rahul Madathiparambil et al., Biomaterial Surface Hydrophobicity
Mediated Serum Protein Adsorption and Immune Responses, ACS Applied
Materials & Interfaces 2019 11 (31), 27615-27623 DOI: 10.1021/acsami.9b09900[20] M.A. Brusatori, Y. Tie, P.R. Van Tassel, Protein adsorption kinetics under anapplied electric field: An optical waveguide lightmode spectroscopy study,Langmuir. 19 (2003) 5089–5097. https://doi.org/10.1021/la0269558.

82

Fig. 1. Fabrication Process of Glass Microelectrodes. (a) Heated by the filament of thesealing machine, the gold is sealed inside the glass tube. (b) Sealed glass tube. (c) Glasstube immediately after pulling with a micropipette puller. (d) Tip of a glass tube
electrode processed by the FIB system. Scale is 10[µm]. (e) Schematic diagram ofprotein immobilization experiment.

83

Table 1. Program and Filament for Glass microelectrode Fabrication.
Line

Heat

Pull

Vel

Time

Pressure: 500

1x4

510

50

5

0

Preheat: ON

2x2

510

40

5

0

Filament: 220B

3x1

505

0

8

0

Room Temp: 24℃

4x1

505

10

5

0

Humidity: 60%

For Time/Delay setting, Time was selected.
The preheat function is enabled by opening Menu in any program window and checking
the [Preheat Jaws 70°C] checkbox. The program will then confirm whether to preheat
when it is opened. This function allows the starting temperature to be constant.
Delay Mode is not enabled.
A typical example of an electrode pulled by this program is shown below (Fig S1).

84

Fig. 2. Proteins were immobilized at the tip of the electrode by applying a pulsed

voltage. The two experiments differ in pulse interval and frequency. Overnight with FcVenus after immobilization of ProteinA (4℃, dark). 20220831-2 is immobilized at thetip, and 20220504-2 is immobilized near the tip, down to the glass area.

85

Fig. 3. Proteins were immobilized at the tip of the electrode by applying a pulsed
voltage. The two experiments differ in voltage. Overnight with Fc-Venus after
immobilization of ProteinA (4℃, dark). 20230911-2 is immobilized at the tip, and20230831-4 is immobilized near the tip, down to the glass area.

86Fig. 4. Preparation for FIB processing and FIB processing. (a) 30[mm] square size stage
for FIB processing. (b) Side view of the stage (upper). Red lines indicate conduction
paths (lower). (c) FIB process flow. (1) Cut horizontally to expose the gold. (2) Cut theleft and right sides. (3) Cut again twice at different angles. The pyramidal shape isobtained.

87Fig. 5. Fixation method for safe oversight of the electrode tip. Electrodes are grabbed
with tweezers and secured through rubber bands. Multiple electrodes can be attached at
the same time. (a) A wood plate with electrodes fixed with rubber bands and a 96 wellplate. By placing two screws in each well and fixing them in place, the entire woodplate is stuck and can be stored for a long time without the tip of the electrode touching
the walls or bottom of the well. In use (left) and disassembled (right). (b) Well andwooden board in use (Side view). Electrodes are always pressed by the rubber band.

88Fig. S1. A typical example of an electrode pulled by micropipette puller (Program isshown Table 1). The scale shows 1 [mm]89Chapter 4

Proof-of-Principle of Glass Microelectrodes
with Selective Synaptic Induction Ability

90

Purpose of Chapter 4⚫ To induce selective synapse formation using the developed engineered synapticorganizer and glass microelectrodes.
Achievement⚫ Using microglass electrodes on neurons expressing engineered synaptic organizers,
the formation of synapses between them was confirmed.
Remaining Challenges⚫

Simultaneous use of several different peptide tags to measure the activity of eachgenetically different neuron in a neural circuit.

91

Chapter 4 Key Points⚫
⚫

⚫

The fabricated glass microelectrodes formed synapse-like structures with axons ofneurons expressing engineered synapse organizers.
At least temperature conditions are not essential for culturing chick forebrain neurons in
L15, and chick neurons themselves are also easy to culture, making them easier to use inexperiments than rat neurons and others.

⚫

Chick forebrain neurons could also be cultured in open air, at room temperature (21 ℃),
and without a perfusion device, but some necrosis was observed.
The optimal conditions for the tip shape of the fabricated glass microelectrodes were notknown. However, ProteinA was also immobilized on the glass part of the tip, and Rab3
accumulated on the glass part.

⚫

Selective synaptogenesis could be realized by combining the fabricated engineered
synapse organizers with glass microelectrodes if experimental conditions were suitable,
but the reproducibility was low, leaving room for improvement in many steps.

92Introduction
The patch-clamp technique developed by Erwin Neher and Bert Sakmann has made it
possible to observe ion channel currents and neuronal activities, including detailed dataacquisition of synaptic currents using in vivo patch-clamp recording [1]. Prior to thistechnique, intracellular electrode techniques were mainly used to obtain current data ofcells larger than the electrode tip [1]. After the advent of the patch-clamp technique, cellrecording of various techniques has become possible using this technique with greater
convenience. This technique has greatly influenced the development of molecular andcell biology and has become a powerful technique in physiology and pharmacology.
However, although the patch-clamp technique is a powerful tool that can observe ionchannel currents of various cells, it has some problems such as non-destructive
observation and low throughput. In addition, the inability to observe networks
consisting of multiple cells cannot be ignored today, when detailed data are required forfurther advanced research.
In Chapter 2, I introduced an engineered synapse organizer consisting of a part of mouseor chicken neurexin-1

sequence and an N-terminal peptide (SPOT-tag) epitope into Rat

neuron and Chick neuron and described the observation of synaptic inductive activity
induced from the binding of peptide tag and Nanobody (immobilized on the surface of93

microbeads). The formation of synapse, the junction between neurons, was induced in an
artificial object, and the inductive activity of synapse by SPOT-tag was observed,
indicating that preparation of tags with specific binding (HAT-tag, His-tag, FLAG-tag,etc.) and their antibodies enabled selective synaptic formation with multiple neurons
simultaneously [2].
Chapter 3 describes the fabrication of glass microelectrodes with gold cores and theimmobilization of functional proteins (mainly ProteinA) on the electrodes. On the other
hand, synaptic formation can be induced on the electrode surface by preparing a large
number of substrate-type electrodes by lithographic techniques and immobilizing
endogenous synapse organizers on the electrode surface, followed by culturing neurons
on them. [3] There are also mesh electrodes [4], etc. By using these and engineered
synapse organizers, it is possible to perform efficient selective measurement of neurons.
The glass microelectrodes with selective synaptic induction ability fabricated in thisstudy can be differentiated from the multiple electrode-type ones described above in thefollowing two ways. One is that the axon of any cell can be approached. The other isthat the related equipment (micropipette pullers, manipulators, electrode holders, etc.)
used for the existing patch-clamp method can be reused as it is. The first one is different
from the existing neuronal measurement methods and multiple substrate-type electrodes94

in that it is quite easy to handle. Although the throughput is not expected to change
compared with the patch-clamp method, it can be said that it has different advantages
considering the possibility of performing measurement with selective neurons via
synapses. The substrate-type electrode with multiple electrodes basically has theelectrode part fixed, so it cannot be moved, and synaptogenesis is possible only when
the corresponding axon approaches the electrode. The second point, that the existing
equipment and equipment can be reused, is relatively important for conducting
experiments. The glass microelectrodes presented in Chapter 3 are also performed inperipheral equipment used in basic electrophysiology except for tip processing. The factthat the existing equipment can be used from the fabrication to the utilization of theglass microelectrodes means that laboratories using electrophysiological methods cansmoothly try these techniques when they can obtain more versatility and stability. If I
can provide a new potential method without major changes compared with the existing
method, the pace of feedback will be faster, so I expect to further improve the method
proposed in this study.
In this chapter, I conduct proof-of-principle experiments on a glass microelectrode withsuch a background and show by proof-of-principle experiments that the developed glass
microelectrode with an engineered synapse organizer immobilized has selective95

synaptic induction ability.

Materials and Methods
To demonstrate the principle, I basically used the materials and methods used in
Chapters 2 and 3. First, I prepared chick neurons cultured in L15 (CO2-independent) at
37 ℃. Chick forebrain neurons were prepared by culturing fertilized eggs purchasedfrom vendors at 38 ℃ for 8-9 days. Since the culture experiments and preparation
experiments using L15 conducted in Chapter 2 showed that the culture in open air andat room temperature was possible for at least 24 hours, I conducted the experimentusing the same L15 culture as presented in Chapter 2 (L15, 2% HS or FBS). The otherculture conditions were the same as those presented in Chapter 2, but the glass for
culturing neurons was changed to one with a size of 18 mm (No. 1. Matsunami), it wascoated with a mixture of 1. 0mg/ml PLL and 0.1mg/ml PDL, and Chick forebrainneurons were cultured by the method described in Chapter 2. Then, the neurons weretransfected with the engineered synapse organizer produced and evaluated in Chapter 2
on the 4th day from the start-up and observed by fluorescence 1 day later. The neurons
thus prepared were transferred under a microscope (IX71) and observed by fluorescencein L15 culture as previously described.
96

The microscope and CCD camera, etc. are basically the same as in Chapter 3. The
appearance of the experimental environment (Fig. 1). The glass microelectrode withsynaptic inductive ability produced in Chapter 3 was approached to the axon of the neurontransfected the engineered synapse organizer we developed. The necessity of theperfusion equipment and the temperature conditions were examined using the perfusion
equipment and the temperature controller (TC-324B, Warner Instruments) so that theneuron would not be deactivated during observation for more than 8 hours. Since the only
difference compared with the experiment using microbeads used in Chapter 2 is that the
microelectrode was cultured in open air and at room temperature, basically the same data
would be expected to be obtained. Therefore, I confirmed that the same synaptogenesis
could be seen by experimenting with the microbead used in Chapter 2 in co-culture at thesame time as the microelectrode produced.
The data obtained were compared and examined. The combination of fluorescence filters
used was the same as in Chapter 3. Excitation and emission filters were selected according
to the fluorescent proteins (EGFP, mcherry) attached to Rab3 at the N-terminus of theengineered synapse organizer. The data were acquired using IDL (Research Systems,
USA) and analyzed using ImageJ.

97

Results
First, I performed an experiment using a perfusion apparatus in which the temperature ofcultured neurons under the microscope was kept constant at 31.6 ℃. The culture medium
was L15, HS2%, and chick forebrain neurons could be cultured using this culture medium
in the incubator without any problems (Fig. 2). Therefore, the temperature was lower thanthe preset temperature of the incubator (37 ℃). Compared with the previous L15 culture,
the open-air culture was the most different. However, not all neurons remained active
after culturing at room temperature (about 21 ℃) without the heater, and some cells were
found dead after culturing for at least 9 h (Fig. 3). However, even under the conditions of
L15 HS2%, room temperature (about 21 ℃) without the perfusion apparatus,
fluorescence representing the accumulation of Rab3 was confirmed on the microbeads onwhich the antibodies of the engineered synapse organizers were modified (Fig. 4). In
order to perform the experiment more stably, I decided to perform the L15, FBS2%, room
temperature (about 21 ℃) with the perfusion apparatus. As a result of the previous roomtemperature co-culture experiment of L15 and microbeads, the accumulation of EGFPRab3 on the microbeads was confirmed as in other experiments (Fig S1). In the systemusing L15, FBS2%, and the perfusion apparatus, the cells were able to be active for atleast 8 h, and the accumulation of Rab3 on the tips of the electrodes was confirmed. The
98

axon and dendrite rupture, which are common in neurons just before collapse, were notobserved (Fig. 5). Based on the above experimental results, I can say that theFcBC2Nanobody immobilized on the tips of the electrodes combined with the engineered
synapse organizers with the SPOT tag, and the synapse-like structure could be inducedby the accumulation of Rab3 and the confirmation of Synapsin localization performed in
Chapter 2. However, due to the characteristics of the experiment, I must also mention an
example of failure. Here, I introduce a typical experimental failure that occurred during
an experiment using glass microelectrodes (Fig. 6). In general, the successful fabricationand processing of glass microelectrodes and the successful culture experiment using chickneurons can provide various data, but it cannot be denied that there are many problems tobe solved in the future, such as the destruction of the electrodes and the failure of culture.

Discussion
In this chapter, I found that engineered synapse organizers were able to bind to the tips ofProteinA-mediated Anti-SpotNb-immobilized glass microelectrodes and induce theformation of synapses on the tips of the electrodes. This finding allowed us to find thatmeasuring with the fabricated glass microelectrodes can be a method for measuring
electrophysiology through synapses and that the fabricated engineered synapse organizers
99

work with chick neurons, making it possible to measure with the combination of multiple
independent synapse organizers [5]. Looking at Figure 6, I found that Rab3 wasassembled in the glass part at the tip of the electrodes, but this could be improved byincreasing the surface area of the gold at the tip. In addition, although the addition of FBS
to the culture medium and the use of a perfusion device could relatively improve theculture condition of neurons, culturing chick forebrain neurons on a microelectrode arrayand co-culturing with glial cells could potentially lengthen the neuron cells [6].
On the other hand, to control the immobilization of ProteinA, which is a problem withglass microelectrodes, it seems to be more effective to use a method in which the exposedarea of gold is adjusted by plating gold on the electrode tip than in the previous study [3].
In addition, it is likely that electrode tip breakage and electrode shaking, which were
problems in the experiments conducted in this chapter, can be dealt with by improving
the environment around the manipulator and the microscope. In general, although highly
reproducible results were not obtained, the fabricated engineered synapse organizers andglass microelectrodes were able to contact cultured neurons and form synapse-likestructures. Thus, it can be said that the technology that will be the basis of the
microelectrode technology for neuronal activity recording for the next generation withcell recognition function has been established. In the future, I will analyze the quantitative100

data of the engineered synapse organizers discussed in Chapter 2, elucidate the molecularmechanism on synapse induction, and improve the glass microelectrode fabrication
method presented in Chapter 3, in order to establish cell activity measurement via
synapses and selective multiple simultaneous measurement of neurons.

101References[1] T. Ishikawa, M. Shimuta, M. Häuser, Multimodal sensory integration in single
cerebellar granule cells in vivo, Elife. 4 (2015) 1–10.
https://doi.org/10.7554/eLife.12916.
[2] K. Terpe, Overview of tag protein fusions: From molecular and biochemical
fundamentals to commercial systems, Appl. Microbiol. Biotechnol. 60 (2003) 523–
533. https://doi.org/10.1007/s00253-002-1158-6.
[3] S. Kim, M. Imayasu, T. Yoshida, H. Tsutsui, Formation of neuron-microelectrodejunction mediated by a synapse organizer, Appl. Phys. Express. 16 (2023).
https://doi.org/10.35848/1882-0786/acd166.
[4] T.M. Fu, G. Hong, R.D. Viveros, et al., Highly scalable multichannel meshelectronics for stable chronic brain electrophysiology, Proc. Natl. Acad. Sci. U. S.
A. 114 (2017) E10046–E10055. https://doi.org/10.1073/pnas.1717695114.
[5] S.A. Hamid, M. Imayasu, T. Yoshida, et al., Epitope-tag-mediated synaptogenic
activity in an engineered neurexin-1 lacking the binding interface withneuroligin-1, Biochem. Biophys. Res. Commun. 658 (2023) 141–147.
https://doi.org/10.1016/j.bbrc.2023.03.063.
[6] S.Y. Kuang, Z. Wang, T. Huang, et al., Prolonging life in chick forebrain-neuron
culture and acquiring spontaneous spiking activity on a microelectrode array,
Biotechnol. Lett. 37 (2015) 499–509. https://doi.org/10.1007/s10529-014-1704-1.

102

Fig. 1. Appearance of the experimental environment. (a) The area around themicroscope, including the irrigation system, manipulators for electrode manipulation,
and wiring for heaters. (b) A glass plate containing a Chick neuron culture is placed
above the microscope lens. An electrode is approaching from the right. (c) L15 culture
medium being pumped by the perfusion system. (d) Typical neuron in this environment.
The electrode is in contact with the axon.

103

Fig. 2. L15, HS2%, 31.6℃ and with perfusion. Chick neuron cultured under amicroscope. (a) Photos taken every hour for about 9 hours of incubation. (b)Comparison between immediately after the start of cultured and 9 hours later.

104

Fig. 3. L15, HS2%, 21℃ (no heater) and without perfusion. Chick neuron cultured
under a microscope. Neuron incubated in L15, HS2% for 480 minutes were
photographed every 10 minutes. (a) Photos were taken every 10 minutes from the startof incubation. (b) Immediately after the start of the post-incubation imaging and 8 hrslater.

105

Fig. 4. Chick neurons were cultured in L15 (HS 2%) in room temperature (21℃),
without perfusion observed fluorescently. (a) Glass microelectrodes were contacted withaxons of neurons expressing engineered synapse organizers. The accumulation of
EGFP-Rab3 was confirmed at the tip of the electrode after 8 hours at the latest. (b)Chick neurons and Nanobody-immobilized microbeads were co-cultured for 8 h. The
accumulation of EGFP-Rab3 was confirmed in surface of the microbeads (except forthe culture environment, the conditions were the same as those shown in Chapter 2, Fig
4). (c) Control beads were used under the same conditions as in (a) and (b), but no
EGFP-Rab3 was accumulated on the microbeads. Bars = 10 µm.
106Fig. 5. Chick neurons could be successfully cultured in L15, FBS 2% using a perfusion
device (co-cultured with bare-microbeads). (a) Immediately after the start of culture, thetip of the electrode was contacted with axon. (b) After 8 hours, Rab3 accumulation wasobserved at the electrode tip. (c) Fluorescence showing the accumulation of Rab3 at the
beginning of the observation (red) and 8 hours later (green).
Bars = 5 µm.

107

Fig. 6. Typical failure of an experiment using glass microelectrodes. (a) Rab3 (modified
with fluorescent protein) accumulated on the non-gold tip of the electrode. (b) The tip ofthe electrode touched the bottom and was broken. (c) The slight movement of theelectrode during 8 hours of incubation prevents it from contacting the axon (out offocus). Bars = 10 µm.

108Fig. S1. Microbeads modified with a tag that binds to the engineered synapse organizer
were co-cultured with transfected chick neurons. The neurons were co-incubated forapproximately 5 and 7 hours in room temperature (21℃), respectively. EGFP-Rab3(green fluorescence, arrow) accumulates on the surface of microbeads. Bars = 10 µm.

109General Discussion110Achievement⚫ By using microelectrodes with specially processed tips, synaptogenesis could be
induced in specific neurons expressing engineered synapse organizers.
⚫ Remaining Challenges
Improving efficiency of synapse formation experiments with engineered synapse
organizers and the microelectrodes.

111

In this study, I developed and quantitatively evaluated an engineered synaptic organizer
based on Mouse, Chick NRXN-1, and developed glass microelectrodes to approach theaxons of neurons. Proof-of-principle experiments were conducted using a combination ofthese techniques.
Innumerable neurons in the brain are connected by synapses to form a huge network for
information transmission and computation. Information transmission and processing bycomplex neural circuits by normally differentiated and active neurons generate various
behaviors in living organisms. In general, synapses are required to be connected bysynapses to other neurons and muscles, and neurons grow to transmit information toother cells while extending axons. Neurons that reach their target cells and initiate
synaptic differentiation receive information from dendrites themselves and participate in
the development and maintenance of the normal network, becoming one element of theneural circuit architecture.
It has recently become clear that proteins called synapse organizers are involved in theformation of synapses, and research has been actively conducted to elucidate themechanism. Many synapse organizers have a huge number of subfamilies formed byalternative splicing, and it is thought that the realization of innumerable modes ofconnection enables control of normal synapse formation and maintenance. Neurexin
112

(NRXN) and Neuroligin (NLGN), a pair of synapse organizers, have been known for along time, and they are located in the presynaptic and postsynaptic regions, respectively.
It is thought that their intercellular connection promotes the dimerization of NLGN andthe clustering of NRXN, thereby initiating presynaptic differentiation.
Based on this idea, this study attempted to develop and apply a device using anengineered synapse organizer that lacks the LNS of NRXN-1 and has a Spot-tag at the
N-terminus. LNS is the extracellular domain of NRXN and has been shown to bind
NLGN with nanomolar affinity. Prior studies have shown that synaptogenesis can be
induced by artificial binding even in the absence of this endogenous binding site.
Therefore, the application of such molecular tools to glass microelectrodes opens up the
possibility of measuring neuronal activity, which was not possible before. Selective
measurement of neuronal activity is not possible with patch clamp or extracellularelectrode methods, and synapse-mediated measurement is an unprecedented method.
Selective measurement of neuronal activity allows us to obtain data focused on specificneuronal activity within a neural circuit, from which a new understanding of neuralcircuits is likely to emerge. Therefore, we developed a glass microelectrode withsynaptic induction capability using the engineered synaptic organizer we developed, andconducted a proof-of-principle experiment. Glass microelectrodes are microelectrodes113with gold sealed inside and insulated with glass, similar to those used in the patchclamp method. Although the performance of the fabricated electrodes has not been
evaluated, it is known from the experiments described in Chapter 3 that current canflow. Experiments using microbeads and engineered synaptic organizers showed thatsynapse-like structures can be selectively formed. However, it is not clear whether
synapses formed by this mechanism have normal function or excitatory and inhibitory
properties. When microbeads and the tips of glass microelectrodes modified with Spottag and Nanobody were attached to engineered synaptic organizers, both showed
accumulation of Rab3 (a synaptic marker).
Future perspectives include the measurement of signals from synapses created on the tipof glass microelectrodes. To achieve this, the following items must be achieved.
The first is the characterization of electrodes. It has been found that the developed
electrodes carry a current of at most several hundred nanoampere when 1.7 to 2.0 [V] isapplied to them. It is essential to investigate the characteristics of the electrodes in orderto create a dedicated amplifier and to clearly read the signals.
The second is the development of more efficient electrodes and the probability ofsynaptic-guided experiments. The development of electrodes was made efficientthrough the processing of universal machines, but this was not the case with tip
114

processing. In order to repeat experiments using electrodes as described in Chapter 4
and to perform experiments with high efficiency, the electrode processing process andthe method of contacting axons must be revised.
The third is the probability of synaptic-mediated measurement methods. As shown inprevious studies (S. Kim et al., 2023), two types of measurement methods can beconsidered for the measurement of created synapses. One is through capacitive
coupling, and the other is by electrochemically sensing and reading the release of
neurotransmitters.
The first described method is ideal, and if the signal can be detected through directelectrical coupling between a neuron and a microelectrode, it may be possible tomeasure using an amplifier similar to the patch-clamp method. However, as previously
described, the amplifier must be adjusted to the characteristics of the electrodes.
If these are achieved and selective synaptogenesis induction and neuronal signal
acquisition can be achieved, the signal of a specific neuron in a neuronal circuit can betracked, which will lead to the elucidation of the mechanism of a neuronal circuit. In thebrain, neurons are thought to be indispensable for complex activities and behavioral
decisions due to their diversity, and a myriad of neuronal circuit assemblies constructhigher-order functions. Therefore, the elucidation of the function of a neuron in a115

neuronal circuit is thought to benefit the elucidation at the functional unit level of thebrain. The elucidation of the mechanism of the brain is an imperative proposition, andthe unknown function elucidation of neurons, synapses, and neuronal circuits obtainedin the process will surely become important information to be utilized in a wide range offields such as medical treatment and brain science.

116

Acknowledgements
I would like to express my sincere gratitude to my supervisor Professor Dr. Hidekazu
Tsutsui, Area of Bioscience and Biotechnology (JAIST) for all the guidance he gave mein conducting this research. I would also like to express my gratitude to my associate
supervisor, Mr. Goro Mizutani, for his constant support in important situations. I sincerely
appreciate Dr. Yuichi Hiratsuka's generous support and advice as an advisor for Minor
research and for his technical assistance. I would like to thank Ms. Mieko Imayasu forher many experimental aids. I am deeply grateful to the Japan Advanced Institute of
Science and Technology (JAIST) for funding a Doctoral research Fellowship (DRF). I
am grateful to the members of our laboratory and the staff of JAIST's Nanomaterials
Technology Center, especially Ms. Ito, for their support.
Lastly, I give my heartfelt thanks to my family and my grandparents.

117