Letter Cite This: Nano Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/NanoLett
Stretchable Transparent Electrode Arrays for Simultaneous Electrical and Optical Interrogation of Neural Circuits in Vivo Jing Zhang,†,‡,§,∥ Xiaojun Liu,†,∥ Wenjing Xu,⊥ Wenhan Luo,‡,§,∇ Ming Li,‡,§,# Fangbing Chu,†,∥ Lu Xu,⊥ Anyuan Cao,⊥ Jisong Guan,∇,○ Shiming Tang,‡,§,# and Xiaojie Duan*,†,§,∥ †
Department of Biomedical Engineering, College of Engineering, ‡Peking-Tsinghua Center for Life Sciences, §Academy for Advanced Interdisciplinary Studies, ∥Center for Nanochemistry, Beijing Science and Engineering Center for Nanocarbons, ⊥Department of Materials Science and Engineering, College of Engineering, and #School of Life Sciences, Peking University, Beijing 100871, China ∇ School of Life Sciences, Tsinghua University, Beijing 100084, China ○ School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China S Supporting Information *
ABSTRACT: Recent developments of transparent electrode arrays provide a unique capability for simultaneous optical and electrical interrogation of neural circuits in the brain. However, none of these electrode arrays possess the stretchability highly desired for interfacing with mechanically active neural systems, such as the brain under injury, the spinal cord, and the peripheral nervous system (PNS). Here, we report a stretchable transparent electrode array from carbon nanotube (CNT) web-like thin films that retains excellent electrochemical performance and broadband optical transparency under stretching and is highly durable under cyclic stretching deformation. We show that the CNT electrodes record well-defined neuronal response signals with negligible light-induced artifacts from cortical surfaces under optogenetic stimulation. Simultaneous two-photon calcium imaging through the transparent CNT electrodes from cortical surfaces of GCaMP-expressing mice with epilepsy shows individual activated neurons in brain regions from which the concurrent electrical recording is taken, thus providing complementary cellular information in addition to the high-temporal-resolution electrical recording. Notably, the studies on rats show that the CNT electrodes remain operational during and after brain contusion that involves the rapid deformation of both the electrode array and brain tissue. This enables real-time, continuous electrophysiological monitoring of cortical activity under traumatic brain injury. These results highlight the potential application of the stretchable transparent CNT electrode arrays in combining electrical and optical modalities to study neural circuits, especially under mechanically active conditions, which could potentially provide important new insights into the local circuit dynamics of the spinal cord and PNS as well as the mechanism underlying traumatic injuries of the nervous system. KEYWORDS: Stretchable electronics, transparent electrode, neural electrode array, brain activity mapping, optogenetics, calcium imaging, traumatic brain injury, nanobio interface, carbon nanotube
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optimization of these stimulation strategies and revealing of the underlying mechanism. Conventional neural surface electrode arrays using opaque metal conductors are not suitable for use in simultaneous electrical and optical neural interfacing because they block the field of view and are prone to producing lightinduced artifacts in the electrical recordings.16 Recent efforts of creating transparent electrode arrays based on graphene and indium tin oxide (ITO) films, etc., enabled efficient light delivery through the electrodes and recording of electrophysiological signals concurrently with optical stimulation and imaging.17−19 The flexible nature of graphene-based micro-
ombining optical and electrical modalities in neural interfacing is important for studying the connectivity and function of neural circuits.1−5 Optogenetic stimulation with simultaneous multichannel electrophysiological readout enables the mapping of the dynamics of perturbed neural populations at the network level,5−9 while simultaneous and co-localized electrophysiology measurement and optical imaging of neural structure and function could leverage the spatial and temporal resolution advantages of both techniques, providing new insights into how neural circuits process information.10−12 Recent progress on direct optical stimulation of unmodified neurons provided an alternative to optogenetics and, potentially, for therapies involving neuronal photostimulation.13−15 Simultaneous electrophysiological recording with direct optical stimulation is important for the validation and © XXXX American Chemical Society
Received: January 8, 2018 Revised: March 27, 2018 Published: April 2, 2018 A
DOI: 10.1021/acs.nanolett.8b00087 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 1. Stretchable transparent CNT electrode array. (A) Schematics of stretchable transparent CNT electrode array for simultaneous optical and electrical interrogation of neural circuits. (B) A scanning electron microscopy image of the CNT web-like thin film used in stretchable transparent electrode array. (C) Optical transmittance of a bare PDMS and CNT/PDMS complex, both having PDMS thicknesses of 100 μm. (D) Optical transmittance of a CNT/PDMS complex before stretching and under stretching to a strain of 20%. Thicker CNT film than that in panel C was used here. (E) Schematic illustration of the evolution of the CNT thin film under stretching. (F) A CNT electrode array positioned over a Peking University logo to show its optical transparency. The electrode array was bonded to a heat seal connector (HSC) for amplifier connection. Inset shows the layout of the CNT electrodes in the area marked by the red dashed box. The recording sites are highlighted in red. Logo copyright 2018 Peking University.
sustain multiple cycles of mechanical stretch. We verified the effectiveness of the CNT electrode array in electro-optic neural interfacing by performing simultaneous optical stimulation and imaging with the electrical recording of cortical activities in rodent models. The ability of the CNT electrode array to operate under mechanically active environments was demonstrated by real-time, continuous monitoring of cortical activity prior to, during, and after TBI from rats. Our results highlight the potential application of the stretchable transparent CNT electrode array for combining the optical and electrical modalities in studies of neural circuits of mechanically active neural systems, such as the spinal cord, the peripheral nervous system (PNS), and the brain under injury, etc. The stretchable transparent electrode array we developed integrates an elastic, transparent silicone substrate (polydimethylsiloxane, PDMS, 100 μm in thickness), CNT thin film interconnects and recording sites, and a top-patterned SU-8 layer for interconnects insulation, as shown in Figure 1A. The CNT thin film was prepared through floating catalyst chemical vapor deposition (CVD) and a solvent-induced condensation process (see the Experimental Section in Supporting Information and ref 27 for details). This gives a web-like CNT film consisting of strongly interconnected thin bundles (10−20 nm in diameter) of single-walled carbon nanotubes (SWNTs, 1−2 nm diameter) with high homogeneity and purity over a large area (Figure 1B).27 The void area in CNT film gives high optical transparency, while the low tube-to-tube contact resistance, resulting from the strong interconnection between nanotubes, ensures high electrical conductivity. Plots of the light transmittance versus wavelength for bare PDMS and CNT film on PDMS substrate are shown in Figure 1C. The CNT/PDMS complex maintained a transmittance over 85%
electrode arrays is especially attractive because it enables conformal and compliant interfacing with the soft, curvilinear surfaces of the brain tissue.17,19,20 However, these electrode arrays are not compliant enough to tolerate large mechanical deformations that occur physiologically in many neural tissues. Developing electrode array sufficiently stretchable to adapt to various motions while maintaining reliable operation and stable contact with tissues is important to study the mechanically active neural systems. In humans, both the spinal cord and its meningeal protective membranes can experience as much as 10−20% tensile strain and displacement (relative to the spinal canal) during normal postural movements.21,22 With each beat of the heart or due to movement of the head, the brain experiences up to 5% bulk strain,23,24 and peripheral nerves may experience up to 15% strain.25 Except for these naturally occurring deformations, brain tissue under traumatic brain injury (TBI) also experiences rapid deformation, including stretching, compression, shear strain, etc.26 A stretchable electrode array can move and deform with the tissue while maintaining functionality and recording capabilities. This enables real-time, continuous electrophysiological recording from the same location on the nervous tissues when they are subjected to natural motion during daily activities or under injury, which is not achievable by conventional unstretchable electrode arrays. In this work, we have created a stretchable transparent electrode array from web-like carbon nanotube (CNT) thin film for use in simultaneous electrical and optical neural interfacing under mechanically active environments (Figure 1A). The stretchable transparent CNT electrode retained high optical transparency over a broad wavelength spectrum and excellent electrochemical performance under stretching and can B
DOI: 10.1021/acs.nanolett.8b00087 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 2. Characterization of the stretchable transparent CNT electrode array. (A) Electrochemical impedance magnitude of a CNT and graphene electrode recorded in saline solution (pH 7.4). (B) Cyclic voltammograms of a CNT and graphene electrode recorded in saline solution (pH 7.4). (C) Relative change of impedance at 1 kHz of a CNT and graphene electrode under stretching strain. Insets show a close look of the data. Z0 denotes the impedance values before stretching. The strain is defined as (Ls − L0)/L0 × 100 (Ls, length after stretching; L0, length before stretching). (D) Impedance magnitude of a CNT (top) and graphene (bottom) electrode recorded in saline solution (pH 7.4) before stretching (0%), at elongation of 50% and 0.9%, respectively, and after being released back to the unstretched state [0% (REC)]. (E) Cyclic voltammograms of a CNT electrode before stretching and at 20% and 50% tensile strain. (F) Relative change of impedance at 1 kHz of a CNT electrode at rest and after multiple stretching cycles to 20% strain. Z0 denotes the impedance value before stretching. The recording-site size is 100 μm × 100 μm for all CNT and graphene electrodes.
across a wavelength range of 400 nm to 2.5 μm. This permits efficient light delivery through the CNT electrodes. Importantly, the CNT/PDMS complex retained high optical transparency when being stretched to a strain of 20% (the biologically relevant strain happened in neural systems) (Figure 1D), ensuring its application under mechanically active environments. By the changing of the film thickness using different deposition parameters, the optical transmittance and electrical conductivity of the CNT film can be tuned. Thicker film possesses higher electrical conductivity but lower optical transparency.27 In this work, we used CNT film with transmittance at 550 nm in the range of 70% to 90%, which corresponds to film sheet resistance in the range of 150−500 Ω/sq. Except for the high optical transparency and electrical conductivity, the CNT thin film prepared this way showed superior mechanical strength and robustness, which can be handled in freestanding form and easily transferred to a variety of substrates.27 Furthermore, the entangled web-like structures could maintain their conductive pathways under mechanical deformation by reorientation and repositioning of CNTs within the percolation networks, as schematically shown in Figure 1E.28,29 All of these make the as-prepared CNT web-like thin film an excellent candidate for conducting materials in stretchable transparent neural electrode array. The fabrication process of the CNT stretchable transparent electrode array involved the patterning of the CNT film and SU-8 insulation layer on a sacrificial substrate and the subsequent coating of PDMS film on the CNT/SU-8 complex followed by removing the sacrificial substrate (Figure S1). No opaque metal is needed for the final electrode array. A flexible cable connected to the CNT film on the electrode array was used to send the recorded signals to the outside preamplifier (see the Experimental Section in Supporting Information for details). As shown in Figure 1F, the final electrode array
exhibited high optical transparency in the whole electrode area, including the recording sites and entire connecting lines. We characterized the electrochemical performance of the CNT stretchable transparent electrodes and compared it with electrodes made of single-layer graphene. The graphene electrode array was designed same as the CNT electrode array except that an additional Au layer is applied on top of the graphene layer, covering the pads and partial portions of the connecting lines (Figure S2). A region about 5 mm from the recording sites was left with only graphene and a SU-8 insulation layer to ensure optical transparency at and around the recording sites. This Au layer is necessary to ensure a high yield of functional graphene electrodes in the array and also is important for a reliable connection to the flexible cable used for reading the brain signals into the preamplifier, as will be discussed later. The CNT electrodes displayed lower impedance than the graphene electrodes with same recording site size of 100 μm × 100 μm (0.20 ± 0.03 versus 1.68 ± 0.68 MΩ at 1 kHz; Figure 2A) and larger cathodal charge storage capacity, as calculated from the cyclic voltammogram (8.71 ± 3.3 versus 4.26 ± 1.6 mC/cm2; Figure 2B), indicating the improved electrochemical interfacial properties of the CNT thin film compared to graphene. To test the stretchability needed for use under mechanically active environments, we monitored the electrode impedance under increasing stretch load along the direction parallel to the electrode traces. The impedance of the CNT electrodes remained unchanged when the uniaxial tensile strain was below 5% and then exhibited a ∼26% and ∼55% increase upon a tensile strain of 20% and 50%. Differently, the impedance of graphene electrodes increased steeply under stretching, with an increase of over 10× upon a strain of ∼3% (Figure 2C). This high impedance under ∼3% strain suggests electrochemical failure of the graphene electrodes as neural electrodes. Besides, the impedance spectra of the CNT C
DOI: 10.1021/acs.nanolett.8b00087 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 3. Simultaneous optogenetic stimulation and ECoG recording with a stretchable, transparent CNT electrode array. (A) Schematics showing the experimental setup and position of the CNT electrode array on the cerebral cortex of a Thy1-ChR2-YFP mouse. The exposed cortical surface (marked by circle in the right schematic) was illuminated by blue light stimuli (488 nm). (B) Top, the train of applied rectangular photostimulus pulses (488 nm, 15 ms, 10 Hz, 2.4 mW/mm2, 30 mW applied onto cortical surface). Bottom panel: ECoG signal high-passed at 3 Hz recorded by a CNT electrode shows the light-evoked large negative potential peaks associated with every photostimulus. (C) Left panel: the close-up view of a light pulse (top) and an unfiltered light-evoked negative potential (bottom) shown in panel B. Right panel: ECoG signal recorded from a WT mouse under a light pulse with same stimulation condition as panel B. A total of 300 trials were averaged for each trace. WT mice did not show any response to the photostimulus. (D) Spectrogram of the ECoG signal shown in panel B. (E, F) Light-evoked potentials under photostimulus (indicated by a dot) are mapped according to channel location on the array under different stimulus (E) intensity and (F) duration (panel E, 15 ms, 10 Hz, 15 mW and 30 mW applied, which corresponds to 1.2 mW/mm2 and 2.4 mW/mm2; panel F, 10 Hz, 15 mW applied, 1.2 mW/mm2). Stronger and longer stimuli evoked larger negative potentials. These potentials were spatially uniform with the entire window illuminated. A total of 300 trials were averaged for each trace. The electrode with a row number of 3 and a column number of 3 was nonfunctional, and the data are invalid. (G) The light-induced artifacts in CNT and Au electrode recording with a recording-site size of 100 μm × 100 μm tested in saline under a stimulus pulse (488 nm, 1 Hz, 30 mW, 2.4 mW/mm2, 15 ms). Gray rectangles indicate the start and duration of stimulation pulses. Inset shows the close-up view of the artifact from the CNT recording. The x-scale bars represent 20 ms, and the y-scale bars represent 2 μV. The data shown in this figure were averaged from 400 stimulation trials. The light-induced artifact in CNT electrode recording is negligible compared to the large artifact from the Au electrode recording.
Due to its unique properties, graphene has recently been exploited as a conducting material in neural electrodes.30 With broad-spectrum transparency, graphene electrodes have successfully recorded electrocorticography (ECoG) responses of the brain tissue to optogenetic stimulation.17,20 Co-localized and simultaneous electrophysiological recording and calcium imaging from hippocampal slices were also demonstrated using graphene electrodes.19 However, despite their superb optical and electrical properties, the graphene electrodes have limited strain sustainability. It was reported that a stretching-induced tensile strain over 6% causes mechanical failure of graphene due to the fractures within the unique honeycomb structure.31 The lack of stretchability restricts the use of transparent graphenebased neural electrode arrays in mechanically active neural systems. Besides, the low mechanical and electrical reliability resulting from the limited strain sustainability makes the graphene vulnerable to damage during the fabrication and implantation processes. Previous studies used multilayer graphene,17 in combination with the use of metal connection traces,17,19,20 to improve the success rate of the electrode
electrodes showed great reversibility at stretching load of 50%, while for the graphene electrodes, for the impedance spectra to be reversible, the stretching loads cannot go beyond 0.9% (Figure 2D). The change of cyclic voltammogram of the CNT electrodes was also modest under uniaxial strains of 20% and 50% (Figure 2E). To demonstrate the robustness of the CNT electrodes against cyclic deformation experienced by many mechanically active neural tissues, we stretched the CNT electrodes to 20% strain over 10 000 cycles. The CNT electrodes withstood the cyclic deformation, displaying minimal variation in impedance over time (Figure 2F). A minimum in impedance at ∼2000 cycles of stretching was shown and also observed in three other tests, which we attribute to a period in which the nanotube bundles adopted their optimum morphology.29 These results demonstrate excellent interfacial property, mechanical robustness, and stretchability of the CNT electrodes, fulfilling the challenging requirements for high-resolution electrophysiological recordings under mechanically active environments. D
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optical stimulation produced readily defined, large negative potential peaks in ECoG recording. These negative potentials persisted for tens of milliseconds after the light pulse turned off and were highly reliable, with similar amplitude being produced under each stimulus (Figure 3B,C). No response was evoked in wild type (WT) mice under the same stimulation (Figure 3C). This suggests that the negative potential originates from the ChR2 activation. The activation of ChR2 causes a net flux of cations into cells, which results in the depolarization of neurons, leaving behind a net negative charge.37 The normalized time-frequency spectral analysis shows a clear increase in power at the stimulation frequency during the stimulation epochs. There were also concomitant increases of power at harmonics of the stimulation frequency (Figure 3D). We suspect that these increases of power at harmonics were a result of the Fourier decomposition of the response waveform, rather than originating from a separate neuronal process or response.38 The evoked potentials were spatially uniform across different channels under photostimulation of the entire cortex window, and the amplitude of the signal depends on both the duration and the intensity of the stimulus, with larger amplitude potentials produced under longer and stronger stimuli (Figure 3E,F). Close-up views of the evoked responses from one representative channel under different stimulus duration and intensity is shown in Figure S3. For simultaneous optogenetic modulation and electrophysiological recording, light-induced artifact is a potential issue because it contaminates the neuronal response signals.16,39 Because the photon energy of the stimulation light is normally below the work function of the electrode materials, the photoelectric effect was not a major concern. The main mechanism of the stimulus artifact is thought to be the photovoltaic effect (i.e., the Becquerel effect) resulting from a differential in photoexcitation between the different conductive layers at a junction.32,33 We characterized the optical stimulus artifact produced from the CNT electrodes in saline. Au electrodes with same size of recording site were tested in parallel for comparison (Figure S4). The artifact amplitude is dependent on the light stimulus power and duration (Figure S5). For a 15 ms pulse with light intensity of 2.4 mW/mm2, the artifact produced in CNT electrode recording was undetectable (Figure 3G) compared with the ∼175 μV neuronal response signals observed in ChR2 mice under the same illumination conditions (Figure 3B,C). Differently, the Au electrodes showed a pronounced photovoltaic effect with artifact amplitude of ∼60 μV under this illumination condition (Figure 3G). Under increased light intensity, light-induced artifacts can be observed from CNT electrode recording, but even at a 10× intensity, the artifact from CNT electrode recording is still much smaller (−12 μV; Figure S5). The small light-induced artifact compared with traditional metal-based electrodes is advantageous for the application of the CNT electrodes in simultaneous optogenetic modulation and electrophysiological recording. The unique optical transmission behavior of SWNTs is critical for the small light-induced artifact. The Becquerel effect requires the photoexcitation of charge carriers into higher energy states.32,33 SWNTs show Van Hove singularities in their band structure in contrast to continuous density of states (DOS) possessed by conventional three-dimensional materials such as metal.40,41 Dipole-allowed optical transitions only occur between mirror image peaks in the DOS, and cross-over transitions are forbidden.42 This makes the photoabsorption
fabrication and implantation. The use of multilayer graphene decreases the cost- and time-effectiveness, and the metal lead lines are undesirable because they make up opaque areas especially for high-density electrode array.17 Besides, the metal−graphene junction also provides another source for light-induced artifact under optogenetic stimulation.32,33 The stretchability of graphene electrodes can be achieved by imparting some structural configurations to graphene films, such as wrinkled34 or crumpled35 structures. However, the wrinkled and crumpled graphene are not suitable for application in stretchable transparent neural electrodes because they are not mechanically stable and their optical transparency changes upon strain.34,35 ITO was also used to fabricate ECoG electrodes for simultaneous electrical readout and optical stimulation.18 However, the brittle nature of ITO makes it unsustainable to deformation and precludes its use in stretchable transparent electrode arrays. The CNT electrodes maintained excellent electrochemical performances under a strain of up to 50% and during 10 000 cycles of 20% stretching. The percolation network of CNTs plays a critical role in enduring mechanical deformation because the strain can be accommodated by structural reconfiguration through rotating or sliding of CNTs against each other.36 The impedance of the CNT electrodes did not show substantial change when subjected to stretching, indicating that most of the CNTs and CNT connection junctions remained without fracture or breaking. The strong interlocking between neighboring CNT bundles is important for maintaining their connection junctions. The reversibility of the impedance change indicated re-establishment of the CNT percolation network under strain release. It should be noted that due to the much smaller impedance of the CNT electrodes compared to the input impedance of most commercial preamplifiers, the ∼26% increase of impedance under a tensile strain of 20% (the maximum biologically relevant strain for neural systems) should not cause a noticeable effect on amplitude or shape of recorded signals. This is important for continuous electrophysiological monitoring of neural activity under deformation. The CNT electrodes maintained a high optical transmission in the wavelength range of 400 nm to 2.5 μm under a strain of 20%, ensuring their capability of efficient light delivery through the electrodes under deformation. Besides, the CNT electrodes exhibited lower impedance than the graphene electrodes. This is advantageous for scaling down the electrode size for highspatial-resolution electrophysiological mapping of neural activity. The transparent neural surface electrode arrays allow for the delivery of light stimuli through the array directly to the brain region from which the electrical recordings are obtained; this enables unique research capabilities in combining optogenetics with electrophysiology.17,20 With added stretchability, the transparent electrode array thus can find wide applications in deciphering neural circuits of mechanically active systems, such as the spinal cord of freely moving animals. We conducted concurrent optogenetic stimulation and neural recording from Thy1-ChR2-YFP mice using the CNT stretchable transparent electrode array. In a typical recording, a 4 × 4 CNT electrode array was placed over the retrosplenial cortex of a Thy1-ChR2YFP mouse to record ECoG signals under optogenetic stimulation of the exposed cortical surface with an optical fiber delivering blue light stimuli (488 nm; Figure 3A). The CNT electrode array closely and conformly adhered to the brain surface owing to its high compliance with soft tissues. The E
DOI: 10.1021/acs.nanolett.8b00087 Nano Lett. XXXX, XXX, XXX−XXX
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Nano Letters and photoexcitation of SWNTs very selective of incident light wavelength, and only those with photon energy resonant with the electronic transition energy between mirror image peaks in the DOS can be absorbed.43 SWNTs electronic properties exhibit a strong structural dependence. An ensemble normally contains mixed SWNTs with different diameter and chirality [(n,m) values], and only a small fraction can be resonant with a single light wavelength and be photoexcited.41 This would cause a much smaller light-induced artifact under photostimulation than their metal counterpart. In recent years, metal nanomeshes have been explored for stretchable transparent electrode fabrication.44,45 While it is interesting to test their potential application in electro-optic neural interfacing, we expect that a smaller light-induced artifact makes the CNT electrodes more unique and advantageous. The aforementioned results demonstrated the capability of the CNT electrode array in efficient readout of the optogenetic modulation, with the advantage of much-smaller light-induced artifacts. For future applications by applying complex focal photostimuli varying in both time and space, ECoG signals with high spatial resolution could be recorded with the CNT electrode array across extended cortical areas, allowing the extraction of neuronal activation maps that are specific for each site of stimulation. This is not only important for studying the cortical dynamics but also useful in determining the contribution of specific cell types to the ECoG signal, which will further our understanding of the ECoG signal origin. Calcium imaging has the advantage of detecting neural activity at single-cell resolution from large or disperse neuron populations simultaneously, which can be complementary to electrophysiological recording in many applications.46 Besides, imaging calcium signal in the brain region from which the electrophysiological signals are recorded is also important for investigating the basis of the field potential and evaluating the performance of calcium sensitive proteins, etc. To perform simultaneous transcranial two-photon calcium imaging and ECoG recording, we used transgenic mice that express improved genetically encoded green fluorescent protein based calcium sensor GCaMP3 in subsets of excitatory neurons in the brain under the control of the Thy1 promoter.47 In an acute study, a ∼5 mm × 5 mm cranial window was opened over the right hemisphere of an anaesthetized transgenic mouse between the Lambda and Bregma to expose the cortex. A 4 × 4 CNT electrode array was placed over the primary visual cortex above the dura mater and covered by a glass coverslip around same size of the cranial window (Figure 4A). The electrode array formed an intimate interface with the targeted neural tissues. The high broad-band optical transparency allowed the penetration of the excitation light (920 nm) as well as fluorescence emission light (495−540 nm) through the CNT electrodes, which enabled the calcium imaging of the brain region under the electrodes. Figure 4B shows the transcranial two-photon fluorescent images of the GCaMP3-expressing cortical neurons underneath a CNT recording site at two different depths. The existence of the CNT electrode on the cortex did not cause any obvious adverse effect on the twophoton calcium imaging. Individual GCaMP3-expressing neurons are clearly visible and resolvable in both layers, having a GCaMP3-expression perimembrane and being undetectable in the nucleus. Dynamic calcium imaging can be conducted while simultaneously recording the neural signals with the CNT electrodes. The K+ channel blocker 4-aminopyridine (4-AP),
Figure 4. Simultaneous two-photon calcium imaging and ECoG recording with stretchable, transparent CNT electrode array. (A) Schematic showing the position of the CNT electrode array on the cerebral cortex of a Thy1-GCaMP3 transgenic mouse. The rectangle marks the cranial window and the blue dot indicates the position for 4AP delivery. (B) In vivo two-photon images of GCaMP3-expressing neurons underneath the recording site of a CNT electrode in the visual cortex of a Thy1-GCaMP3 mouse. The depth below the dura mater is shown in each panel. (C) ECoG recording from a CNT electrode showing normal baseline brain activity (left trace) and interictal-like activity (right trace). The data was high-pass filtered at 3 Hz. Bottom images show the two-photon calcium images of the brain region underneath the recording site of the electrode used to record the top traces, taken at the time points marked by the red dashed lines. Individual activated neurons (indicated by the white dashed circles) with calcium transients can be identified coinciding with the interictallike event. (D) ECoG signal recorded by the CNT electrode showing seizure-like ictal discharges. The data was high-pass filtered at 3 Hz. (E) ΔF/F0 traces for the entire brain region under the recording site (labeled as ROI) and several individual cells during the ictal-like events. (F) Two-photon calcium images of the brain region underneath the recording site taken during the ictal-like events. The four images correspond to the time points indicated by the dashed lines in E. The individual activated neurons shown in E are indicated by the white dashed circles.
which is used to induce seizures,48 was delivered into the cortex of a transgenic mouse at the edge of the glass coverslip, ∼2 mm away from the electrode array (Figure 4A). The calcium imaging did not cause any additional noise or artifact in the electrophysiological recordings with the CNT electrode. Upon application of 4-AP, the recording first showed normal basal brain activity that corresponds to the baseline fluorescence intensity of GCaMP3. Interictal spikes were then observed ∼10 min after 4-AP application. Individual activated neurons with calcium transients can be identified in the region underneath the recording site coinciding with the interictal-like event (Figure 4C). The electrical signal recorded by the CNT electrode evolved into seizure-like ictal event, which are characterized by high-frequency, large-amplitude discharges, ∼15 min after application of the 4-AP (Figure 4D). The seizure discharges were generated by synchronously firing of neuronal clusters. We observed this epileptiform discharges from all functional channels in the CNT electrode array (Figure S6). As shown from the simultaneous two-photon calcium imaging, the calcium fluorescence within the recording site area (labeled as F
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Figure 5. ECoG recording with TBI. (A) Schematic showing the CNT electrode array placement and weight drop location (indicated by the blue circle). The rectangle marks the position of the craniotomy. (B) A representative continuous ECoG trace recorded by a CNT electrode before, during, and after the TBI high-pass filtered at 3 Hz. The TBI was induced at the time indicated by the arrow. (C) Power-density spectrum of the recorded ECoG signal before TBI (red curve), after the TBI (blue curve, averaged from the data collected during the first 30 s after TBI), and 2 min after the TBI (black curve), in which the ECoG activity was fully recovered to the preinjury level. (D) Timefrequency spectral analysis of the ECoG signal recorded by a CNT electrode before, during, and after the TBI. The TBI was induced at the time indicated by the white dashed line. The white arrow indicates the period in which the low-frequency ECoG activity was suppressed due to TBI.
The experiments here highlight the potential application of the CNT stretchable transparent electrode array in combining optical and electrical modalities to study neural circuits, especially those of mechanically active neural systems, including brains under injury, spinal cords, and the PNS. TBI is associated with tissue damage, apoptotic and necrotic cell death, and blood−brain-barrier disruption, etc. in the lesion. Optical imaging can assess morphological and molecular changes at the cellular level in brain tissue under injury. Combined with simultaneous, continuous electrical recording of neural signals from the injury site, important insights regarding the mechanisms, progression, or treatment of TBI could be provided. Optogenetics offers promise for dissecting the complex neural circuits of the spinal cord and PNS and has therapeutic potential for addressing many clinical needs. Although imposed with unique challenges, optogenetic control in the spinal cord and PNS has been successfully demonstrated.51 Soft stretchable electrode array meets the demanding static and dynamic mechanical properties of spinal cord and PNS and has shown stable long-term neural interfacing capability with limited foreign-body reactions.52 With combined optical transparency and stretchability, the CNT electrode array could enable reliable observation of neural response to optogenetic modulation in the spinal cord and PNS. This will G
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increase our knowledge regarding the connectivity and function of peripheral and spinal sensory neurons under both normal and pathological conditions. In conclusion, we have created an electrode array based on CNT web-like thin film that exhibited broad-spectrum optical transparency, excellent electrochemical interfacial properties, mechanical robustness, and stretching durability. With the capability of conducting electrical recording with negligible light-induced artifacts under simultaneous optical stimulation and imaging, and real-time continuous recording under mechanically active environments, we expect that the electrode technology presented here could be a powerful tool in neuroscience with which to study neural mechanotransduction and traumatic injuries of the nervous system.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.8b00087. Additional experimental details. Figures showing a fabrication process schematic, a diagram of array construction, evoked responses, an electrode used for light-induced artifact comparison, light-induced artifacts in CNT electrode recording, representative recording of epileptiform discharges, schematics showing the placement of the CNT electrode array, and ECoG recording with TBI. (PDF)
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Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Anyuan Cao: 0000-0002-2282-716X Xiaojie Duan: 0000-0001-7799-3897 Author Contributions
X.D. and J.Z. conceived the idea and designed the experiments. J.Z., W.X., L.X., and A.C. prepared the CNT film. J.Z. and F.C. fabricated the device. J.Z. did the characterization of the device. J.Z., X.L., W.L., and J.G. performed the optogenetics experiment. J.Z., X.L., M.L., and S.T. conducted the two-photon calcium imaging experiment. J.Z. and F.C. performed the TBI experiment. J.Z. and X.L. analyzed the data. X.D and J.Z. wrote the manuscript with inputs from all authors. All authors approved the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Microelectronics Research Center at Peking University for the microfabrication facility and support, the Animal Resources Center at Peking University for animal housing and care, and the Core Facilities at the School of Life Sciences, Peking University, for assistance with two-photon imaging. This work was supported by grants from the National Natural Science Foundation of China (grant nos. 21422301, 91648207, and 21373013), the National Basic Research Program of China (grant nos. 2016YFA0200103 and 2014CB932500), and China’s 1000 Young Talent Award program. H
DOI: 10.1021/acs.nanolett.8b00087 Nano Lett. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.nanolett.8b00087 Nano Lett. XXXX, XXX, XXX−XXX
