Gene-Embedded Nanostructural BioticâAbiotic Optoelectrode Arrays

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Biological and Medical Applications of Materials and Interfaces

Gene-Embedded Nanostructural Biotic-Abiotic Optoelectrode Arrays Applied for Synchronous Brain Optogenetics and Neural Signal Recording Wei-Chen Huang, Hui-Shang Chi, Yi-Chao Lee, Yu-Chun Lo, Ta-Chung Liu, Min-Yu Chiang, Hsu-Yan Chen, Ssu-Ju Li, You-Yin Chen, and San-Yuan Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03264 • Publication Date (Web): 07 Mar 2019 Downloaded from http://pubs.acs.org on March 10, 2019

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ACS Applied Materials & Interfaces

Gene-Embedded Nanostructural Biotic-Abiotic Optoelectrode Arrays Applied for Synchronous Brain Optogenetics and Neural Signal Recording Wei-Chen Huang a, Hui-Shang Chi b, Yi-Chao Lee c, Yu-Chun Lo c, Ta-Chung Liu b, Min-Yu Chiangb, Hsu-Yan Chen d, Ssu-Ju Li d, You-Yin Chen c, d, *, and San-Yuan Chen b,e,*

a Graduate

Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, No.

250 Wu-Xing St., Taipei 11031, Taiwan, R.O.C. b

Department of Materials Science and Engineering, National Chiao Tung University, No. 1001,

Ta-Hsueh Rd., Hsinchu, Taiwan 30010, R.O.C. c

The Ph.D. Program for Neural Regenerative Medicine, Taipei Medical University, No. 250

Wu-Xing St., Taipei 11031, Taiwan, R.O.C. d Department

of Biomedical Engineering, National Yang Ming University, No.155, Sec.2, Linong St.,

Taipei, Taiwan 11221, R.O.C. e

Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua

University, No. 101, Sec. 2, Kuang-Fu Rd., Hsinchu 30013, Taiwan, R.O.C.

*Correspondence

should be addressed to either of the following:

San-Yuan Chen, Department of Materials Science and Engineering, National Chiao Tung University, No. 1001, Ta-Hsueh Rd., Hsinchu, Taiwan 30010, R.O.C. Email: [email protected]; You-Yin Chen, Department of Biomedical Engineering, National Yang Ming University, No.155, Sec.2, Linong St., Taipei, Taiwan 11221, R.O.C. E-mail: [email protected]

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ABSTRACT Optogenetics is a recently established neuromodulation technique in which photostimulation is used to manipulate neurons with high temporal and spatial precision. However, sequential genetic and optical insertion with double brain implantation tends to cause excessive tissue damage. In addition, the incorporation of light-sensitive genes requires the utilization of viral vectors, which remains a safety concern. Here, combining device fabrication design, nanotechnology, and cell targeting technology, we developed a new gene-embedded optoelectrode array for neural implantation to enable spatiotemporal electroporation (EP) for gene delivery/transfection, photomodulation, and synchronous electrical monitoring of neural signals in the brain via one-time implantation. A biotic-abiotic neural interface (called PG) composed of reduced graphene oxide (rGO) and conductive polyelectrolyte, 3, 4-ethylenedioxythiophene-modified amphiphilic chitosan (PMSDT) was developed to form a nanostructural hydrogel with assembled nanodomains for encapsulating non-viral gene vectors (called PEI-NT-pDNA) formulated by neurotensin (NT) and polyethylenimine (PEI)-coupled DNA. The PG can maintain high charge storage ability to respond to a minimal current of 125 μA for controllable gene delivery. The in vitro analysis of PG-PEI-NT-pDNA on the microelectrode array (MEA) chip showed that the microelectrodes provided electrically inductive electro-permeabilization, which permitted gene transfection into localized rat adrenal pheochromocytoma (PC-12) cells with a strong GFP expression that was up to 8-fold higher than that in non-treated cells. Furthermore, the in vivo implantation enabled on-demand spatiotemporal gene transfection to neurons with 10-fold enhancement of targeting ability compared with astrocytes. Finally, using the real optogenetic opsin, Channelrhodopsin-2 (ChR2), the flexible neural probe incorporated with an optical waveguide fiber displayed photo-evoked extracellular spikes in the thalamic ventrobasal region after focal EP for only 7 days, which provided a proof of concept for the use of photomodulation to facilitate neural therapies.

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KEYWORDS: Neural interface, Optogenetics, Electroporation, Non-viral gene delivery, Nanotechnology, Graphene

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INTRODUCTION Over the past decades, electrical neuromodulation using implantable devices has been recognized as a therapeutic option for neurodegenerative disorders

1-6.

Through simultaneous probing and

manipulation of neuronal activity using electrical currents from microminiaturized electrode sites, it has been shown that deep brain stimulation (DBS) can be successful in treating Parkinson’s diseases. However, the electrical current is electrophysiologically and biologically non-specific, which tends to cause unpredictable activation of normal neurons. Recently, an invasive neurostimulation technique, known as optogenetics, was developed to permit cell-specific regulation 7-9. Optogenetics enables depolarization of gene-transfected neurons immediately under exposure to light of a specific wavelength to allow for rapid and reversible control over behaviors specific to the targeted cells 10-13. Optogenetic operation typically requires sequential craniotomy for repeated brain implantation including gene injection followed by optic incorporation. Unfortunately, penetration through the superficial brain structures to reach more ventral brain regions results in damage to superficial brain areas, highlighting the challenge in precisely positioning the optical fiber locally. In addition, the present system of optogenetics had detrimental effects on the viability of neurons in the vicinity of the target. Much progress has been made in integrating neural implants with functional surface modification to enhance neural recording and modulation using electrical, chemical, and/or optical modalities, thus supporting the promise of therapeutic perspectives for neuroprosthetic treatments. Particularly, inspired by nanotechnology, electrodes coated with conductive polymer can soften and roughen their corresponding surface to enhance mechanical, electrical, and biological congruence for neural interfacing

14-16.

The nanostructural surface provides high surface-to-volume area to

encapsulate bioactive molecules such as drugs, growth factors, imaging agents, or genes that can be protected and transported toward the target cells via the shortest route

17-21.

In addition, compared

with integrated microfluidic channels, the nanostructural surface can permit seamless contact 4


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between the implant and brain tissues without increasing the footprint of the device, and the implant has a higher biocompatibility for prolonged utilization. Implantable neural electrode arrays integrated with nanoscale vehicles can facilitate site-specific neural stimulating/monitoring and spatiotemporal electrically-controlled delivery

14, 22-23,

beyond which electrical pulses also enabled

electroporation (EP) to facilitate cellular uptake of the bioactive molecules

24-26.

Accordingly, we

presented nanoscale vehicles-coupled implantable optoelectrode probe with a flexible and amenable fabrication technique for use in the enhancement on optogenetic neural interfacing, which provides a new optogenetic stimulating system for electrically controlled gene delivery/transfection, optogenetic stimulation, and simultaneous acquisition of synchronous electrical monitoring of neural signals via one-time implantation. The internalization of naked genes into neurons is difficult and creates challenges in the subsequent degradation in lysosomal compartments. Ideal gene vehicles need to provide protection for nucleic acid cargos, as well as carry bio-specificity for neurons or neuronal subpopulations. To facilitate gene delivery and transfection efficiency, we also focused on creating non-viral gene nano-vehicles with the following dual functions: (1) response to EP in the enhanced gene transfection and (2) strengthening recognition of targeted neurons

27-29.

Graphene was attractive in

the development of gene vectors, bioelectrodes, and biosensors due to its facile synthesis process 30-34,

the high specific surface area of the nanostructure, excellent optical and electrical properties

and easily tunable surface functionalization

36,37.

35

Graphene oxides (GO) has been shown to respond

to external physical stimuli in the enhanced gene transfection 38. On the other hand, chitosan was a soft tissue-mimic polymer that is good for electrically controlled delivery based on the sensitively switchable swelling/deswelling ability

39-41.

Amphiphilic chitosan also self-assembled into nanogel

structures to provide a high surface-to-volume ratio beneficial for neural interfacing

42.

Bioactive

conductive polymer composed of conductive polymers and natural biocompatible macromolecules such as proteins or polysaccharides presented a tissue-mimicked three-dimensional environment that 5



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not only facilitated the interconnection with surrounding cells, but also enhanced the transduction of electrical and chemical signals 43-46. Here, we presented a new implantable optic-driven neural probe that was equipped with gene vehicle-embedded bioelectrodes to enable precise optogenetics via targeted optogenetic gene delivery as shown in Scheme 1. The biotic-abiotic conjugates composed of

reduced

graphene

oxide

(rGO)

and

conductive

polyelectrolyte,

3,

4-ethylenedioxythiophene-modified amphiphilic chitosan (PMSDT) at thermodynamic equilibrium self-assembled into a nanogel-based architecture with tissue-mimicking properties, ionic/electrical conductivity, and biocompatibility

47,48.

Moreover, in order to increase the efficiency of gene

transfection, a new non-viral gene vector consisting of neurotensin (NT) and polyethylenimine (PEI)-coupled DNA (called PEI-NT-pDNA) was developed to enhance the targeting ability toward neurons. By taking advantage of device fabrication in combination with nanomaterials and biological technology, the new neural optoelectrode probe embedded with both genetics and optics were expected to achieve electroporation

49

for gene transfection and spatiotemporal control of opsin

distribution locally. We demonstrated the use of targeted optical modulation and signal monitoring on specific cell types within intact tissue, which confirms the feasibility of targeted photomodulation through one-time implantation.

EXPERIMENTAL SECTION Synthesis of rGO/amphiphilic polysaccharide-modified PEDOT (PG) conductive hydrogels Polydimethylsiloxane-modified chitosan (PMSC) copolymers were prepared by two-step synthesis: carboxymethylation and esterification. In the first step, N,O-carboxymethyl chitosan (NOCC) was synthesized by grafting carboxymethyl groups onto the amino site (N-site) and the hydroxyl site (O-site) of pristine chitosan

47, 48.

Briefly, 5-g chitosan was suspended in 2-propanol

(50 ml) at room temperature for 30 min. The resulting suspension was gently mixed with 12.5 ml NaOH solution. A mixture containing NaOH of 13.3N was mixed with 25 g of chloroacetic acid to 6


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prepare carboxymethyl chitosan. After NOCC was obtained, 2 g of dried sample was dissolved in distilled water (50 ml) and stirred for 24 h. The resulting solutions were mixed with methanol (50 ml), and then stirred vigorously for 4 h, followed by the addition of bis(hydroxyalkyl) terminated polydimethylsiloxane, which was dispersed in 2-propanol as a solution (50% v/v) in advance. Following catalysis with sulfuric acid, the mixture was maintained and reacted at 60 oC for 24 h to be esterified, the resulting solution was dialyzed (Mw cut-off 12,400 g/mol, average flat width 33 mm) with ethanol/ diethyl ether (9 : 1 v/v) for 1 day. After repeating dialysis 3 times, the purified product was obtained after drying at 50 oC overnight. For the doping with 3, 4-ethylenedioxythiophene (EDOT), the 1-g dried PMSC sample was re-suspended in 50 ml deionized water and stirred for 24 h. Then, FeCl3‧6H2O (FeCl3/EDOT = 3/1 mole/mole) was added and the mixture was stirred for another hour. The EDOT monomer of 11.7 ml was added to solutions for 24 h to carry out the oxidation polymerization at room temperature

50.

The product was obtained after washing with

ethanol three times and dried at 60oC overnight. To synthesize the rGO/amphiphilic polysaccharide-modified PEDOT, called PG, rGO was incorporated into 2% PMSDT water solution at solid weight ratios of PMSDT: rGO at 1 : 0.25, 1 : 1, 1 : 3, and 1 : 4 for PG1, PG2, PG3, and PG4, respectively. The rGO was prepared according to the Hammers method, as described by Zhijun Zhang et al. 51. The resultant solution of PG was obtained following ultrasonic homogenization for 30 sec.

Characterization of rGO/amphiphilic polysaccharide-modified PEDOT (PG) conductive hydrogels The intermolecular interaction was investigated by Fourier transform infrared spectroscopy (FTIR, Spectrum 100 FTIR, Perkin Elmer Inc., Waltham, MA, USA) analysis. Dried sample (0.2 mg) was mixed with 200 mg dry KBr and pressed into a pellet using a macro KBr die kit. The FTIR spectrum ranging from 450 to 4,000 cm-1 was obtained after 50 scans at 4 cm-1 resolution. The surface charge was obtained by electrophoretic light scattering to analyze zeta potentials. The PG 7



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substrate was obtained through casting the solution. The morphology of the substrates was investigated by scanning electron microscopy (SEM, JEOL 6700, Tokyo, Japan) and atomic force microscopy (AFM, Bruker Innova, Billerica, MA, USA). A tapping mode of AFM coupled with a scanning probe microscope (PPP-NCSTR-20, nanosensersTM, Neuchâtel, Switzerland) was used to investigate the surface morphology. The scan rate was 0.5 Hz, and the scanning dimension was set to 2  2 μm2 for each sample. The equilibrium water content (EWC) of the hydrogel substrates was obtained by measuring the weight before and after immersion in phosphate-buffered saline (PBS, pH = 7), which was determined by Eq. 1:

EWC  100 (%) 

mw  md mw

(1)

Where mw is the weight of the wet hydrogel and md is the weight of the dry hydrogel. The stiffness, i.e., the displacement in response to force, was obtained by nanoindentation test using a dynamic model (MTS-Nano Instruments, Oak Ridge, TN, USA). During the indentation experiment, the indenter tip was pressed into the gel surface at a constant rate of 0.5 nm s−1. Once the maximum penetration depth of 2,000 nm was reached, the indenter was held for 10 sec before being withdrawn to allow time for stress relaxation of plastic materials. All measurements were performed at 25 °C and the Poisson ratios were assigned a value of 0.5. Electrochemical properties of the hydrogel substrates were analyzed based on cyclic voltammetry (CV) and AC impedance using the CHI 614C electrochemical workstation (CHI Instruments, Austin, TX, USA). The three-electrode system cell configuration was used and 0.1 M phosphate-buffered saline solution (PBS, pH = 7) was used as the electrolyte. The signal was controlled by the Ag/AgCl reference electrode and returned via platinum (Pt) counter electrode, while the working electrode was a material-coated neural electrode. For electrochemical measurement, the scan rate was set as 0.05 V/sec with a potential range from -0.5 V to 0.5 V. The impedance response of the hybrids was assessed by application of amplitude sinusoids of 5 mV across the frequency range of 1 – 1M Hz. The mean impedance magnitude was presented on 8


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a Nyquist plot.

In vitro biocompatibility study The rat adrenal pheochromocytoma cell line (PC-12), which was kindly donated by Dr. Chia-Hsin Liao (Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan), was used in this study. The PC-12 cells were cultured in DMEM medium with 10% horse serum (HS), 10% fetal bovine serum (FBS), 100 μg/ml penicillin and 100 μU/ml streptomycin, and were maintained in a 5% CO2 humidified atmosphere at 37 oC. For cell differentiation, the media was supplemented with 5% HS, 100 μg/ml penicillin, 100 μU/ml streptomycin, and 100-ng/ml nerve growth factor (NGF, Sigma-Aldrich, St. Louis, MO, USA). Cell viability and proliferation were evaluated using MTT assay (Catalog # V13154, Thermo Fisher Scientific Inc., Waltham, MA, USA). Cells were seeded onto 24-well plates at a density of 5 × 104 cells per well, followed by the incorporation of sample solutions. After 24 h, the media was replaced with MTT reagent-incorporated fresh medium for an additional 4 h incubation to form purple formazan crystals. After the purple formazan crystals were dissolved by DMSO, the cell viability was obtained by measuring the optical density (OD value) at 492 nm with the enzyme-linked immunosorbent assay (ELISA) reader (Sunrise™, Tecan Group Ltd., Männedorf, Switzerland). The glass substrate was used as a control to define the 100% cell viability. For the PC-12 cell adhesion and differentiation assay, cells were seeded in 24-well coated plates with PG, PMSDT, and rGO, and culture media containing 5% HS and 100 ng/ml NGF to induce cell differentiation. After 7 days of differentiation, β- tubulin (III) was stained to observe cell differentiation and measure the neurite outgrowth. A confocal microscopy system (Eclipse C1 Plus, Nikon Instruments Inc., Melville, NY, USA) was used to obtain cell fluorescent images. For the quantification, the neurite length of the cells was measured with Image J software (ver 1.47, National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov/ij/). Length was defined as the distance from the tip of the neurite to the 9



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junction between the cell body and neurite. Neurite length was obtained by measuring the average in three random images.

Synthesis and characterization of gene vectors Gene vectors were synthesized through the incorporation of 25k-branch polyethylenimine (PEI) and neurotensin (NT)

38, 52.

Briefly, NT (10 mg/ml) was activated with crosslinker succinimidyl

3-(2-pyridyldithio)propionate (SPDP, 22 mM) in H2O for 30 min at room temperature, while PEI was mixed with another crosslinker, Traut’s reagent (2-iminothiolane) (100 mM) in H2O. These two solutions were mixed for 8 h to get PEI-NT. The reaction was monitored by measuring the absorbance of displaced pyridine-2-thion at 343 nm. Subsequently, the PEI-NT solution was mixed with different amount of GFP-conjugated plasmid DNA (pDNA). For the in vitro study, the pDNA was derived from pmWasabi-NT (ABP-FP-WNNCS, Protech Technology Enterprise Co., Ltd., Taipei, Taiwan). The n/p ratios were 1 k, 0.1 k, 0.05 k, and 0.01 k (molars of nitrogen atoms (n) of PEI-NT to the molars of phosphate groups (p) of pDNA). Gel electrophoresis was used to define the final conjugation degree of the PEI-NT-pDNA composites. The complexes were loaded on 1% (w/v) agarose gel containing 0.025% dye in TAE (Tris-acetate-EDTA) buffer at 120 V (MP-100, Major Science, Saratoga, CA, USA) for 2 h. The band for pDNA was visualized on the UV transilluminator (MUV21-254/365, Major Science, Saratoga, CA, USA). For in vivo study, channelrhodopsin-2 (ChR2) opsin plasmids (AAV-CAG-ChR2-GFP, plasmid #26929, Addgene, Cambridge, MA, USA) were used instead to apply real optogenetics. The final composite, called PEI-NT-ChR2 was fabricated as described above and used to perform electrically induced optogenetics.

Controlled delivery of gene-incorporated PG electrodes The PEI-NT-pDNA was incorporated into PG at different volume ratios (PG: PEI-NT-pDNA, 2 : 1, 4 : 1, and 10 : 1) to obtain the final product called PG-PEI-NT-pDNA. To investigate DNA 10


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release under EP, an electrical pulse under current-control was applied. Three different currents, 125 μA, 250 μA, and 500 μA, were used, while the frequency was set as 1 Hz and the application duration was 250 ms for 2 h per day. The DNA released into PBS was labeled by the intercalating fluorescent dye, i.e., PicoGreen dsDNA Reagent and Kits (Thermo Fisher Scientific Inc., Waltham, MA, USA). The fluorescence intensity was measured with a fluorescence spectrophotometer (CLARIOstar®, BMG Labtech, Ortenberg, Germany) at excitation and emission wavelengths of 480 nm and 525 nm, respectively. All experiments were performed three times, and values are expressed as mean ± standard deviation (SD).

Electrically induced gene transfection from PG in vitro PC-12 cells (5 × 105) were seeded on laboratory-designed microelectrode array (MEA) chips 53 that were pre-coated with a film of PG-PEI-NT-pDNA by dipping. Cells were cultured at 37 °C under 5% CO2 for 24 h to allow complete cell adhesion. After 24 h incubation, EP was performed on a selected channel site of the MEA chip with an isolated pulse stimulator (Model 2100, AM-systems, Carlsborg, WA, USA). Electrical pulses under 250 μA at the frequency of 1 Hz and pulse width of 250 ms were applied for 5 cycles of 10-min stimulation followed by 10-min rest. The result of transfection was measured by observing expressed GFP using fluorescence microscopy (Eclipse C1 Plus, Nikon Instruments Inc., Melville, NY, USA). Image J was used to quantify the GFP intensity of four zones of equal dimensions that intersected at the center of the MEA. The integral intensity of each zone was quantified on days 0, 3, and 6. All of the data were reported as the mean ± SD obtained by three similar tests.

Implantable optoelectrode probe Our multi-functional neural probes were designed for electroporation of opsin gene in the adult mouse brain, which combined with optogenetic stimulation and neural recording. Their specification 11



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and fabrication was depicted in Table S1 and Figure S2, respectively, in the Supporting Information Note 1. To fabricate optical waveguides onto the flat shaft of the laboratory-designed neural probe, the subsequent fabrication steps were schematically shown in Figure 1. The geometrical pattern of the mold for the optical waveguide was designed using computer-aided design software Autodesk (AutoCAD 2017, Autodesk, Inc., San Rafael, CA, USA). The convex of the pattern was milled on a Poly (methyl methacrylate) (PMMA) sheet (Figure 1a) using a computerized numerical control (CNC) mini-engraving machine (Everprecision Tech Co., New Taipei City, Taiwan) with an end-mill (ϕ = 0.1 mm, double-edged, tungsten steel) for the positive master as shown in Figure 1b. The positive master mold was cleaned with mild detergents, and washed in pure water for 15 min with ultrasonic, and then flushed dry. Liquid Polydimethylsiloxane (PDMS, SYLGARD™ 184, Dow Corning, Auburn, MI, USA) mixture (prepolymer: curing agent = 10 : 1 w/w,) was poured into the cleaned positive master with the convex pattern top-down to the predefined depth, then degassed for ~10 min, and cured for 2 h at 75 °C in an oven. After curing, the positive PMMA master mold was removed, leaving the solidified PDMS mold with the 100-m deep negative pattern of the optical waveguide (Figure 1c). We trimmed and stripped the graded-index multimode fiber optic cable (GIF625, Thorlabs Inc., Newton, NJ, USA) to expose the cladding (125 µm) and core (62.5 µm), which acted as the interconnect between the optical waveguide and laser light source, and then was placed onto the tail segment of the concave pattern of the optical waveguide of the PDMS mold Figure 1d. After injection of the precure ultraviolet (UV) polymer (OG146, Epoxy Technology Inc., Billerica, MA, USA), which had a relatively low viscosity (82 cps), into the concave pattern of the PDMS mold using a fine tip syringe as shown in Figure 1e, the liquid solution automatically spread and filled up the concave pattern due to capillary effects. The neural probe subsequently was aligned the optical waveguide pattern and then attached onto the PDMS mold with the top side. The UV polymer was crosslinked by UV radiation at an intensity of 100 mW/cm2 for 30 sec to form the optical waveguide. 12


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Meanwhile, the neural probe was bonded onto the cured optical waveguide as shown in Figure 1f. After the UV polymer was fully cured, the probe with the optical waveguide was easily peeled off from the mold to the optoelectrode probe (Figure 1g).

Animal surgery and gene transfection by focal EP from PG in vivo All in vivo experiments were performed using three healthy adult male C57BL/6 mice (BioLASCO Taiwan Co., Ltd., Taipei, Taiwan) that were 3.5 months old on average (n = 3). All mice were housed in identical housing circumstances in a constant temperature environment (22 ± 3 °C), with a normal 12 h light-dark cycle (lights on at 7 AM). All experimental procedures were approved by the Institutional Animal Care and Use Committees (IACUC) at National Chiao Tung University and National Yang Ming University. Mice were deeply anesthetized using isoflurane (Sigma-Aldrich, St. Louis, MO, USA) mixed with air (induction with 4% isoflurane, maintenance with 2% isoflurane). Anesthetized mice were secured in a stereotactic frame (Stoelting, Wood Dale, IL, USA) on a 37 °C heating plate for surgery. A small craniotomy was performed with a dental drill above the targeted coordinate. After drilling a small hole above the ventrobasal (VB) complex nucleus, we slowly advanced a laboratory-designed 16-channel optoelectrode probe with pre-coating PG-PEI-NT-ChR2 via dipping into the right VB region (AP: −1.7 mm, ML: −1.8 mm, DV: −3.4 mm) and secured it to the skull with stainless steel screws and dental cement. After curing the dental cement, the focal EP was performed using a pair of microelectrodes on the optoelectrode probe (anode: Ch #1 and cathode: Ch #8), which was connected to an isolated pulse stimulator (Model 2100. AM-systems, Carlsborg, WA, USA). The parameters of focal EP in the adult mouse brain were as follows: pulse width = 125 ms, current = 250 μA, frequency = 1 Hz, proceeding with 3 cycles with the unipolar square pulses for 5 sec followed by the rest for 10 sec 54. After EP, the mice were allowed to fully recover from surgery for a week before electrophysiological recording was started. 13



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Recording Thalamic Photo-evoked Neural Activities in Vivo The implanted mice were anesthetized with an intraperitoneal injection of a ketamine cocktail (ketamine 70 mg∕kg, xylazine 10 mg∕kg) and placed in a temperature-controlled stereotaxic frame to combine in vivo optogenetics with electrophysiology. The implanted laboratory-designed optoelectrode probe in the VB thalamic nucleus was connected to a 473 nm laser source (CNI Optoelectronics Tech. Co., Changchun, China) with an output power level of 3 mW via a 120-μm diameter optical waveguide. The photo-evoked neural activities were conducted at select sites of the laboratory-designed optoelectrode probe modulated by light stimulation at 12 Hz with 30% duty cycle. The neural signals from each microelectrode were differentially filtered to obtain spike data (200 Hz - 8 kHz). Data were amplified at 20 ×, digitized, further digitally amplified at 20-100 ×, and recorded using the Plexon Multichannel Acquisition Processor (MAP, Plexon Inc., Dallas, TX, USA). Spikes in our digitized data were first detected with the median method and then imported into our unsupervised spike-sorting software

55

for discrimination of single populations of action potentials

by principal component analysis. Spike times and interspike intervals (ISIs) were determined simultaneously. In this study, the Wilcoxon signed-rank test with a significance level at p

Gene-Embedded Nanostructural BioticâAbiotic Optoelectrode Arrays

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