Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Multifunctional Fibers as Tools for Neuroscience and Neuroengineering Published as part of the Accounts of Chemical Research special issue “The Interface of Biology with Nanoscience and Electronics”. Andres Canales,†,‡,∇ Seongjun Park,‡,§,∇ Antje Kilias,∥,⊥,#,∇ and Polina Anikeeva*,†,‡ †
Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States § Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States ∥ Bernstein Center Freiburg, University of Freiburg, 79104 Freiburg, Germany ⊥ Biomicrotechnology, Institute for Microsystems Engineering, University of Freiburg, 79110 Freiburg, Germany # Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany ‡
CONSPECTUS: Multifunctional devices for modulation and probing of neuronal activity during free behavior facilitate studies of functions and pathologies of the nervous system. Probes composed of stiff materials, such as metals and semiconductors, exhibit elastic and chemical mismatch with the neural tissue, which is hypothesized to contribute to sustained tissue damage and gliosis. Dense glial scars have been found to encapsulate implanted devices, corrode their surfaces, and often yield poor recording quality in long-term experiments. Motivated by the hypothesis that reducing the mechanical stiffness of implanted probes may improve their long-term reliability, a variety of probes based on soft materials have been developed. In addition to enabling electrical neural recording, these probes have been engineered to take advantage of genetic tools for optical neuromodulation. With the emergence of optogenetics, it became possible to optically excite or inhibit genetically identifiable cell types via expression of light-sensitive opsins. Optogenetics experiments often demand implantable multifunctional devices to optically stimulate, deliver viral vectors and drugs, and simultaneously record electrophysiological signals from the specified cells within the nervous system. Recent advances in microcontact printing and microfabrication techniques have equipped flexible probes with microscale light-emitting diodes (μLEDs), waveguides, and microfluidic channels. Complementary to these approaches, fiber drawing has emerged as a scalable route to integration of multiple functional features within miniature and flexible neural probes. The thermal drawing process relies on the fabrication of macroscale models containing the materials of interest, which are then drawn into microstructured fibers with predefined cross-sectional geometries. We have recently applied this approach to produce fibers integrating conductive electrodes for extracellular recording of singleand multineuron potentials, low-loss optical waveguides for optogenetic neuromodulation, and microfluidic channels for drug and viral vector delivery. These devices allowed dynamic investigation of the time course of opsin expression across multiple brain regions and enabled pairing of optical stimulation with local pharmacological intervention in behaving animals. Neural probes designed to interface with the spinal cord, a viscoelastic tissue undergoing repeated strain during normal movement, rely on the integration of soft and flexible materials to avoid injury and device failure. Employing soft substrates, such as parylene C and poly-(dimethylsiloxane), for electrode and μLED arrays permitted stimulation and recording of neural activity on the surface of the spinal cord. Similarly, thermally drawn flexible and stretchable optoelectronic fibers that resemble the fibrous structure of the spinal cord were implanted without any significant inflammatory reaction in the vicinity of the probes. These fibers enabled simultaneous recording and optogenetic stimulation of neural activity in the spinal cord. In this Account, we review the applications of multifunctional fibers and other integrated devices for optoelectronic probing of neural circuits and discuss engineering directions that may facilitate future studies of nerve repair and accelerate the development of bioelectronic medical devices.
1. INTRODUCTION Understanding physical mechanisms underlying function of the nervous system demands tools capable of sensing and modulating activity of neurons and glia. Decades of neural engineering © XXXX American Chemical Society
Received: November 7, 2017
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DOI: 10.1021/acs.accounts.7b00558 Acc. Chem. Res. XXXX, XXX, XXX−XXX
Article
Accounts of Chemical Research
neural tissues.1 Neural probes are often based on stiff inorganic materials such as silicon, silica, and metals (Young’s moduli in the gigapascal range), while the surrounding tissue is organic and exhibits moduli in the kilo- to megapascal range. The elastic mismatch is particularly relevant when the neural tissue moves relative to the bone-mounted probes.9 These observations have led to the hypothesis that employing materials with mechanics and surface chemistry mimicking those of biological tissues may extend the lifetime and improve the performance of neural interfaces.10 Consequently, a variety of probe architectures based on ultrathin or perforated structures and soft materials such as polymers and composites have been engineered.11,12 In addition to photolithographic and microcontact printing techniques, thermal drawing of multimaterial fibers has emerged as a facile approach to integrate electrical, optical, and microfluidic functional features within microscale
research have delivered a diversity of electrical, optical, chemical, and genetic tools for probing and controlling neural dynamics with ever-increasing temporal and spatial resolution.1 Combined with behavioral studies, these tools have offered insights into the neurobiology of disorders such as addiction, depression, and Parkinson’s disease.2−4 However, the longer-term experiments necessary for understanding progressive diseases and for the design of neuroprosthetic brain−machine interfaces have revealed the reliability challenges of neural probes.5 Implantation of probes into the nervous system causes acute and chronic damage to the surrounding tissue, including neuronal death and glial scarring around the probes.6,7 The foreignbody reaction to neural probes is correlated to their functional failure and has been extensively reviewed elsewhere.8 One of the proposed causes of foreign-body response to neural interfaces is the mechanical and chemical mismatch between devices and
Figure 1. Examples of multimaterial fibers. (A) Thermal drawing of multimaterial fibers. The diameter of the fiber is determined by the ratio of the downfeed and capstan speeds and is monitored continuously by a laser micrometer during drawing. Reprinted with permission from ref 14. Copyright 2017 Nature Publishing Group (NPG). (B) Cross-sectional scanning electron microscopy (SEM) image of a piezoelectric fiber for conformal acoustics. Reprinted with permission from ref 19. Copyright 2012 John Willey and Sons. (C) Cross-sectional SEM image of the structure of the surface-emitting fiber laser cavity (top) and a schematic illustration of the operating principles of a photonic band gap fiber laser (bottom). Reprinted with permission from ref 20. Copyright 2006 OSA Publishing.
Figure 2. Fabrication of fiber-based microelectrode arrays using a two-step TDP. (A−C) (top) Photographs of multielectrode probe preforms and (bottom) SEM cross-sectional images of the fiber during (A) the first step and (B, C) the second step of TDP. (D) SEM images of probes incorporating (top) nine electrodes around a hollow channel and (bottom) a linear array of tin electrodes. (A−C) and the top panel in (D) are reprinted with permission from ref 13. Copyright 2015 NPG. B
DOI: 10.1021/acs.accounts.7b00558 Acc. Chem. Res. XXXX, XXX, XXX−XXX
Article
Accounts of Chemical Research
materials and features of the desired probe. Then, by the application of heat and tension, the preform is drawn into a fiber, maintaining the cross-sectional geometry but reducing the linear dimensions of the preform by 1−2 orders of magnitude. At the same time, the length of the resulting fiber increases by 2−4 orders of magnitude, making TDP a scalable method for the fabrication of neural probes (Figure 1A). While versatile, TDP poses constraints on the materials constituting the preform, as all of the components must exhibit similar viscosities at the drawing temperature.21 This can be achieved by selecting materials with
probes with elastic moduli ranging from megapascals to a few gigapascals.13−17
2. FIBER DRAWING OF NEURAL PROBES Although traditionally applied to glasses to fabricate optical telecommunications fibers, the thermal drawing process (TDP) is compatible with a wide range of geometries and materials.18 Advances in multimaterial fiber technology have resulted in a variety of fiber-based devices19,20 (Figure 1). TDP begins with the construction of a macroscopic preform that incorporates the
Figure 3. Devices combining optical stimulation and electrophysiology. (A) Tetrode microwire bundles arranged around an optical fiber and integrated with a microdrive. Reprinted with permission from ref 41. Copyright 2011 NPG. (B) Utah array with one recording electrode replaced by a gold-coated silica fiber. Reprinted with permission from ref 42. Copyright 2012 IOP Publishing. (C) Michigan probe equipped with an optical waveguide. Reprinted with permission from ref 43. Copyright 2010 John Wiley and Sons. (D, E) Thermally drawn fibers combining a hollow core for patch recordings with a waveguide for simultaneous light delivery (D) and a metallic coating for LFP recordings (E). Reprinted with permission from (D) ref 44 and (E) ref 45. Copyright 2011 NPG and 2013 PLoS, respectively. (F) Monolithically integrated μLEDs within silicon neural probes. Reprinted with permission from ref 46. Copyright 2015 Cell Press. (G) Transparent square zinc oxide pillar array. Reprinted with permission from ref 47. Copyright 2015 NPG. C
DOI: 10.1021/acs.accounts.7b00558 Acc. Chem. Res. XXXX, XXX, XXX−XXX
Article
Accounts of Chemical Research
processing techniques has enabled the fabrication of silicon-based arrays carrying more than 1000 electrodes.31,32 Although microfabricated silicon probes offer an unprecedented volume of neural data,33 the foreign-body response to these probes motivated the development of ultrathin carbon-based electrodes,34 recording arrays on flexible and stretchable substrates,35 and injectable electrode meshes.36 To enable high-resolution recordings while minimizing the impact on the surrounding tissue, TDP has been applied to tin embedded in poly-(etherimide) (PEI) to produce flexible arrays containing 7−36 electrodes13 (Figure 2). In two TDP steps, the electrode dimensions were scaled down to 5−8 μm (Figure 2A−C). The resulting recording probes exhibited impedances of 570− 1200 kΩ at 1 kHz and permitted recording of isolated spikes with a signal-to-noise ratio of 13 ± 6. As TDP offers versatility in the cross-sectional geometry of the preform, the probe geometry and electrode arrangement can be straightforwardly tailored to a specific neurobiological question (Figure 2B−D). We note that probes currently produced by TDP offer electrode−tissue interfaces only at the fiber tip. Future advances in fiber processing may allow for the development of thermally drawn probes with multiple functional interfaces along the fiber length.
similar glass transition (Tg) and melting (Tm) temperatures. The functional properties of the constituent materials, such as conductivity or optical transparency, can range widely, however, allowing for straightforward integration of multiple functions into miniature probes. Consequently, controlling the geometry of the preform on the macroscale enables fiber function on the microscale (Figure 1B,C). Thus, TDP can integrate more features per device at a lower cost than is achievable with traditional microfabrication.22,23 Below we review multifunctional probes tailored to specific applications in neuroscience and neural engineering.
3. ELECTRICAL PROBING OF NEURAL ACTIVITY The spatial and temporal precision of recorded neuronal activity is determined by the properties of the electrodes and their proximity to the neural sources.24 Physiological parameters of individual neurons, such as membrane potential and firing threshold, can be measured by intra- or juxtacellular recordings using patchclamp techniques,25 sharp electrodes,26 and recently developed nanostructures.27,28 Low-impedance (1−7 mW/mm2)39 and simultaneously perform electrophysiology40 in deep brain regions. Initial studies relied on silica waveguides adhered to existing recording probes41−43 (Figure 3A−C). Other approaches, inspired by patch-clamp techniques, incorporated metal electrodes and optical waveguides into glass probes, facilitating electrical recording from optically identified neurons during light stimulation or calcium-dependent fluorophore imaging44,45 (Figure 3D,E). Recently, advanced microfabrication approaches have enabled monolithic integration of μLEDs onto silicon-based electrode arrays46 (Figure 3F) as well as the development of transparent arrays of conductive zinc oxide pillars47 (Figure 3G). The ubiquitous use of commercial silica fibers in optogenetics studies motivated the application of TDP to produce probes suitable for simultaneous optical stimulation and electrophysiology.44,45 These pioneering fiber-based probes included silicacore waveguides for optical stimulation and photometric readout of calcium indicator fluorescence. Electrical recording was accomplished either through integration of metal wires45 or through hollow channels filled with saline solution akin to patch-clamp electrodes.44 In addition to their stiffness, the application of these
Figure 5. Carbon-composite electrodes within fiber-based probes. (A) Preform (left) and cross section of a multifunctional fiber probe (right). (B) Electrophysiological recording with a multifunctional fiber probe before, during, and after injection of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) during optogenetic stimulation. (A) and (B) are reprinted with permission from ref 13. Copyright 2015 NPG. (C) Carbon nanofibers within the CPE electrodes align during TDP. Reprinted from ref 52. Copyright 2017 American Chemical Society. (D) Cross section of a fiber probe for “onestep optogenetics”. (E) SEM image of a gCPE electrode. (F) Single-unit activity recorded by the fiber probe in (D). (D−F) are reprinted with permission from ref 14. Copyright 2017 NPG. E
DOI: 10.1021/acs.accounts.7b00558 Acc. Chem. Res. XXXX, XXX, XXX−XXX
Article
Accounts of Chemical Research
Figure 6. (A) Fiber probes were implanted in the BLA and vHPC, and AAV5-CaMKIIα::ChR2-eYFP was delivered through the probe in the BLA, resulting in ChR2-eYFP expression in BLA axons targeting vHPC (right panel, 6 weeks after injection). (B) Optical stimulation through both fibers, in BLA and vHPC, evoked responses 11 days after the virus infusion. (C, D) Optical stimulation of BLA terminals in the vHPC resulted in reduced exploration of the center of an open field (C). This optically induced anxiety-like phenotype was abolished by the infusion of CNQX through the fiber probes implanted in the vHPC (D). Reprinted with permission from ref 14. Copyright 2017 NPG.
with low absorption and autofluorescence to constitute the waveguide core and cladding. Furthermore, to minimize losses in the waveguide and maximize the numerical aperture, the refractive index of the core must exceed that of the cladding. Finally, TDP requires the materials constituting the preform to have similar viscosities at the drawing temperature. A combination of a transparent polycarbonate (PC) core and cyclic olefin copolymer (COC) cladding addressed all three requirements. However, the relatively low glass transition temperatures of these polymers (150 and 158 °C, respectively) made integration of metallic electrodes challenging, and light transmission losses still exceeded those of conventional silica fibers. While recent work indicates the potential of indium as a suitable electrode material,51 conductive carbon composites similarly offer a chemically inert and inexpensive platform for integration of recording capabilities into PC-based probes. Carbon-loaded conductive polyethylene (CPE) has been used as the electrode material, allowing the production of trifunctional fiber-based probes with different geometries and cross-sectional dimensions as small as 180 μm14 (Figure 5). The multifunctional probes with PC/COC optical waveguides, CPE electrodes, and hollow microchannels enabled simultaneous recording and optical stimulation of neural activity in transgenic Thy1-ChR2-YFP mice broadly expressing the bluelight-sensitive cation channel channelrhodopsin 2 (ChR2) (Figure 5A). Infusion of a glutamate receptor blocker through the integrated microchannels reversibly suppressed the optically evoked potential (Figure 5B). The high sheet resistance of commercially available CPE, however, made recordings of isolated single-neuron spikes challenging with conventional electrophysiology setups intended for
electrodes with impedance values of
