Subcellular Optogenetic Stimulation for Activity-Dependent

Aug 29, 2016 - activity in vitro for studying axon myelination. KEYWORDS: Optogenetic stimulation, myelination, oligodendrocytes, microfluidics, black...
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Subcellular Optogenetic Stimulation for Activity-Dependent Myelination of Axons in a Novel Microfluidic Compartmentalized Platform Hae Ung Lee, Sudip Nag, Agata Blasiak, Yan Jin, Nitish V. Thakor, and In Hong Yang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00157 • Publication Date (Web): 29 Aug 2016 Downloaded from http://pubs.acs.org on August 31, 2016

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Subcellular Optogenetic Stimulation for Activity-Dependent Myelination of Axons in a Novel

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Microfluidic Compartmentalized Platform

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Hae Ung Lee1, Sudip Nag1, Agata Blasiak1, Yan Jin1, Nitish Thakor1,2, In Hong Yang1,2,*

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Singapore Institute for Neurotechnology, National University of Singapore, Singapore.

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Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, Baltimore,

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USA.

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Abstract

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Myelination is governed by neuron-glia communication, which in turn is modulated by neural

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activity. The exact mechanisms remain elusive. We developed a novel in vitro optogenetic

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stimulation platform that facilitates subcellular activity induction in hundreds of neurons

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simultaneously. The light isolation was achieved by creating a bio-compatible, light-absorbent, black

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microfluidic device integrated with a programmable, high-power LED array. The system was applied

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to a compartmentalized culture of primary neurons whose distal axons were interacting with

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oligodendrocyte precursor cells. Neural activity was induced along whole neurons or was

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constrained to cell bodies with proximal axons or distal axons only. All three modes of stimulation

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promoted oligodendrocyte differentiation and the myelination of axons as evidenced by a decrease

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in the number of oligodendrocyte precursor cells followed by increases in the number of mature

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oligodendrocytes and myelin sheath fragments. These results demonstrated the potential of our

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novel optogenetic stimulation system for the global and focal induction of neural activity in vitro for

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studying axon myelination.

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Keywords: Optogenetic Stimulation, Myelination, Oligodendrocytes, Microfluidics, Black PDMS

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Introduction

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Neuronal activity plays a pivotal role in the survival, proliferation and migration of neurons and glial

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cells, as well as the myelination of axons.1-3 Multiple findings in cell culture systems showed that

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neuronal activation by electrical stimulation (ESTIM) improves myelination.4-7 Accordingly, various in

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vivo models of neuronal activation demonstrated that neural activity influences myelination.8-10

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However, the application of ESTIM via electrodes has considerable limitations. Firstly, it is infeasible

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to simultaneously target multiple neurons with high spatial resolution. Secondly, ESTIM requires

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mechanical proximity and electrical integrity which are laborious and impractical to apply in awake

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and moving animals.11

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Optogenetic stimulation (OSTIM) provides a versatile alternative to ESTIM. A light beam can be

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easily and quickly targeted to stimulate neurons with expression of light-sensitive proteins, such as

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channelrhodopsin 2 (ChR2).11 OSTIM elicits similar levels of activation in neurons as ESTIM does.12

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When ChR2 transfected neurons were exposed to light at 10 - 20 Hz frequency, their electrical

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activity resembled the physiological firing rate of neurons in the brain.13 Even higher frequencies

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can be achieved with modified ultrafast ChR2 (ChETA).14 Recently, Gibson et al. found that neuronal

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activation by OSTIM promotes oligodendrogenesis and myelination in the mouse motor cortex.15

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Their findings demonstrate that OSTIM can be used to study neuronal activity dependent

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myelination mechanism.

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Neurons are highly polarized, as a consequence, multiple cellular processes are region specific.

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Deciphering the role of each subcellular region requires tools that facilitate stimulation and signal

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registration with limited interference from other cellular regions. Traditionally used Campenot

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chamber systems for neuron culture applies a physical constraint and isolates bulky cell bodies from

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thin axons with a set of parallel microgrooves.16 This simple idea has become wide-spread in labs

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around the world with the increased availability of polydimethylsiloxane (PDMS)-based devices. The

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microgrooves have since been replaced with microchannels that connect axonal and somatic

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chambers. The careful balancing of the volumes, and the consequently affected hydrostatic

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pressures, in both chambers combined with the resistance from the microchannels provides fluidic

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isolation. Thus, the axonal and somatic microenvironments can be independently controlled.17, 18

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Another advantage of using PDMS-based microfluidics is easy integration with other techniques. A

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PDMS-based optical system benefits from low fluorescence and high chemical stability. However, its

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application is limited as PDMS causes internal light scattering and reflection generation from the top

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and bottom surfaces, which may affect the input and output light signals. Furthermore, it impedes

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isolated stimulation of a chosen area of the device, as the light is unrestrained and can propagate 2 ACS Paragon Plus Environment

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through PDMS. One of the potential solutions is changing the property of the material and creating

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light absorbent PDMS.19 It is crucial that while blocking the light, the PDMS remains biocompatible –

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the material cannot be toxic and it has to provide sufficient gas permeability to support cellular

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growth.

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In this study we present a black PDMS microfluidic device that provides spatial, fluidic, and light

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isolation of the somatic and axonal chambers without compromising biocompatibility. The devices

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were used to apply highly controlled, selective optogenetic stimulation to the chosen neuronal

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subcellular region in a massively parallel fashion and without direct glial cell stimulation. The

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integration of compartmentalized microfluidic co-culture systems with optogenetics allowed testing

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the hypothesis that focal induction of membrane potential shift is sufficient to increase

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oligodendrocyte maturation and myelination of axons.

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Results and Discussion

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OSTIM applied to animals is a promising technique to study neural activity-dependent myelination.

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While in vivo approaches provide invaluable information by incorporating the complexity of the real

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tissue, they suffer from certain disadvantages, e.g. the myelination process might be affected by glial

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scarring after the insertion of light stimulation devices in the brain. Furthermore, the complexity of

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the system with mixed cell types, including multiple neuronal subtypes, different glial cells, etc.,

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impedes deciphering the mechanism of neural activity-induced myelination. Finally, the subcellular

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stimulation of multiple neurons is not feasible. An in vitro model system that facilitates precise

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control over subcellular microenvironments and focal neural activity induction is needed to decipher

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mechanisms behind axon myelination. We aimed to develop such a model by integrating

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neurobiology with microfluidics and optogenetics.

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Spatial control of light exposure

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A compartmentalized PDMS device

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with a pattern outlining the positions of the chambers and microchannels was obtained through soft

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lithography. The pattern was then replicated in a thin layer (approx. 30 μm) of transparent

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polydimethysiloxane (PDMS) by spin-coating and curing the PDMS on top of the wafer. A thick layer

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(approx. 7 mm) of PDMS supplemented with 1% carbon black particles (CB-PDMS) was cured on top

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of the transparent PDMS. Subsequently, the PDMS pad was peeled off, the wells (somatic and axonal

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chambers) were punched out and the pad was bonded to a glass coverslip via oxygen plasma

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activation (Fig. 1A, C). A high-power LED set up was developed to stimulate a chosen chamber of the

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was modified to limit light scattering. Briefly, a silicon wafer

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device: somatic, axonal or both. The device contained an array of blue LEDs that were arranged in a

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2×3 matrix and individually controlled with a programmable controller (Fig 1D). The controller

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received commands from an USB-based computer interface. Received commands were decoded

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and stored inside a non-volatile FLASH memory and utilized for generating real-time stimulation

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sequence (Fig.1B). The system functioned in stand-alone mode if the PC was not present, while

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reading the pre-programmed stimulation parameters. The CB-PDMS device containing up to three

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compartmentalized culture systems was placed on top of the LED array, such that each of the six LED

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lights was under one of the chambers (Fig.1D). The pre-programming assured the small size of the

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set up - the LED system fit into a Ø10 cm Petri dish that could be placed in an incubator - without

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losing versatility of the stimulation modes. The CB-PDMS acted as an optical trap – a 1.5 mm sheet

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blocked more than 99.99% of the light from the LED set up. Additionally, the bottom of the glass

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coverslip underneath the unstimulated chamber was covered with aluminum foil and dark tape to

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prevent the light exposure. As a result, when only one of the chambers was stimulated, the light

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intensity reaching the opposite chamber was limited to < 1.5%.

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The CB-PDMS light trap not only provided isolated LED light exposure of a chosen chamber but also

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limited any internal scattering and reflections from the PDMS. The carbon black particles were of

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sizes < 1μm, and therefore if the CB-PDMS mixture was directly applied at the master, it could

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interfere or even block 3 μm-high microchannels. The thin layer of transparent PDMS allowed

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precise replication of the pattern. The submillimeter thickness of the layer ensured that when

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subsequently covered with the thick layer of CB-PDMS the presence of the transparent PDMS did

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not affect the light isolating properties of the device. The strict cleansing steps facilitated the

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removal of toxic compounds from CB-PDMS and provided its biocompatibility. As such, this material

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can be used for any fluorescent cell assays that benefit from limiting the spatial light exposure and

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limited level of light reflections , e.g. from the top surface of closed PDMS chamber. 21

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Fig. 1. Experimental setup for subcellular OSTIM. A. The preparation of CB-PDMS microfluidic device followed previously published protocols except for addition of CB-PDMS layer on top of the thin layer of transparent PDMS. B. Block diagram of the multichannel optogenetic stimulator. A USB transceiver communicated with the work station computer for incoming commands. The received signal was passed to a low power microcontroller, which decoded and processed the commands. The microcontroller also generated timing and control sequences for six LEDs for optical stimulation. The blue LEDs were interfaced with a driver array that was capable of providing required high current to the LEDs. C. One CB-PDMS device contained up to three sets of independent culture chambers compatible with the alignment of LED array D. The optogenetic device was composed of an operating module and a LED module. E.. The OSTIM platform facilitated light exposure of six CBPDMS chambers (three cultures, each with two chambers) in an independent fashion.

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Primary neuron/glia co-culture in compartmentalized CB-PDMS platform for optogenetic

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stimulation

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Primary dorsal root ganglion (DRG) neurons from embryonic day 13 mice were plated in the somatic

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chamber of the CB-PDMS devices (Fig. 2A,B) andtransfected with light-sensitive ChR2-EYFP probe

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under the control of the CaMKII promoter.11 After 6-7 days in vitro (DIV), axons sent by the cell

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bodies crossed through the 500μm-long microchannels to the axonal chamber and extensively

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elongated. At 10 DIV the axonal chamber was filled in with axons (Fig.2C). The neuronal growth

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pattern was not significantly different from that observed in the transparent PDMS devices. Neuron

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morphology was regular as visualized with the neurofilament stain. The expression of ChR2-EYFP

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was confirmed along the whole cell as the EYFP signal overlapped with the neurofilament stain (Fig.2 5 ACS Paragon Plus Environment

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D-G). Most of the axons in the axonal chamber expressed ChR2-EYFP indicating that the transfection

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did not affect axonal growth. Oligodendrocyte precursor cells (OPCs) were sourced from postnatal

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day 1 mouse pups and were purified by a magnetic separation system. The purity of separated OPCs

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was 85-90% as verified with anti-O4-PE. OPCs seeded in the axonal chamber of CB-PDMS with 10

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DIV-old axons survived in a co-culture for at least 14 DIV. LED optical pulses were used to stimulate

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ChR2-expressing DRG neurons along their whole length (WholeSTIM), in the axonal chamber

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(AxonSTIM) and in the somatic chamber (SomaSTIM) (Fig.2 H-J).

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Fig.2. The optogenetic platform was compatible with primary neuron/glia co-cultures and allowed for neuron subcellular stimulation in three different modes. A. Schematic of the compartmentalized culture system. Somatic and axonal chambers were connected with 5 μm-wide and 500 μm-long microchannels. Cells were plated in the somatic chamber and axons, but not cell bodies, crossed to the axonal chamber. B. Primary DRG neurons transfected with the ChR2-EYFP probe were plated in the somatic chamber of the CB-PDMS device. There was no effect of the CB-PDMS on the neurite growth or morphology as visualized with anti-neurofilament stain. C. At 10 DIV axons passed through the microchannels and extensively invaded the axonal chamber. D. Transfected primary neurons expressed ChR2-EYFP in cell bodies and proximal axons in the somatic chamber as visualized in the YFP channel. E. Axons that crossed to the axonal chamber expressed ChR2-EYFP. F. The merged image indicated that almost all neurons in the somatic chamber (cell bodies and proximal axons) expressed ChR2. G. Almost all axons that crossed to the axonal chamber expressed ChR2 as demonstrated by the co-localization of YFP and neurofilament signals. H-J. Schematics of the stimulation modes: whole cell stimulation (H; WholeSTIM) where both chambers were illuminated 6 ACS Paragon Plus Environment

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with pulses from blue LEDs; axonal chamber stimulation (I; AxonSTIM) and somatic chamber stimulation (J; SomaSTIM), where blue LEDs only stimulated a single, chosen chamber.

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The compartmentalized system provided spatial and fluidic isolation of distal axons and cell bodies.

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OPCs were exclusively interacting with the axons in the axonal chamber, such that the presence of

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cell bodies did not interfere with the myelination process. This arrangement reflects the in vivo

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condition that distal regions of DRG axons reaching towards the target tissue experience different

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microenvironments than cell bodies clustered in a posterior root of a spinal nerve. Accordingly, axon

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myelination in vivo can occur hundreds of micrometers away from the cell bodies, so the cell bodies

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can affect this process only by changing the state of their axons, e.g., inducing anterograde

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propagation of an electrical signal.

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CB-PDMS devices contained up to three individual, compartmentalized cultures (a somatic and an

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axonal chamber for each culture; Fig1. C, D and E). The LED array with independent control of each

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light source allowed stimulation of all three cultures simultaneously (Fig1. D and E). This

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arrangement not only increased the replicates of the experiments, but also allowed a direct

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comparison of the effects of the three modes of the stimulation (WholeSTIM vs. SomaSTIM vs.

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AxonSTIM) as they could have been applied simultaneously for reducing experimental variations (e.g.

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culturing conditions).

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Several methods have been designed to obtain subcellular optogenetic stimulation, including

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subcellular channel-rhodopsin-assisted circuit mapping (sCRAM)22 and the two photon activation

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approach.23 These systems facilitate high spatial resolution of stimulation (around 50 μm for sCRAM

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and