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Optogenetic Mapping of Functional Connectivity in Freely Moving Mice via Insertable Wrapping Electrode Array Beneath the Skull Ah Hyung Park, Seung Hyun Lee, Changju Lee, Jeongjin Kim, Han Eol Lee, Se-Bum Paik, Keon Jae Lee, and Daesoo Kim ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b07889 • Publication Date (Web): 06 Jan 2016 Downloaded from http://pubs.acs.org on January 14, 2016
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Optogenetic Mapping of Functional Connectivity in Freely Moving Mice via Insertable Wrapping Electrode Array Beneath the Skull Ah Hyung Park,†,⊥, Seung Hyun Lee,‡,⊥ Changju Lee,§ Jeongjin Kim,† Han Eol Lee,‡ Se-Bum Paik,§ Keon Jae Lee,*,‡ and Daesoo Kim*,† †
Department of Biological Sciences, ‡Department of Materials Science and Engineering, and
§
Department of Bio and Brain Engineering, Korea Advanced Institute of Science and
Technology (KAIST), 291, Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea. ⊥
These authors contributed equally to this work.
* To whom correspondence should be addressed: Email:
[email protected];
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ABSTRACT Spatiotemporal mapping of neural interactions through electrocorticography (ECoG) is the key to understanding brain functions and disorders. For the entire brain cortical areas, this approach has been challenging, especially in freely moving states, owing to the need for extensive craniotomy. Here, we introduce a flexible microelectrode array system, termed iWEBS, which can be inserted through a small cranial slit and stably wrap onto the curved cortical surface. Using iWEBS, we measured dynamic changes of signals across major cortical domains, namely somatosensory, motor, visual and retrosplenial areas, in freely moving mice. iWEBS robustly displayed somatosensory evoked potentials (SEPs) in corresponding cortical areas to specific somatosensory stimuli. We also used iWEBS for mapping functional interactions between cortical areas in the propagation of spike-and-wave discharges (SWDs), the neurological marker of absence seizures, triggered by optogenetic inhibition of a specific thalamic nucleus. This demonstrates that iWEBS represents a significant improvement over conventional ECoG recording methodologies, and therefore is a competitive recording system for mapping wide-range brain connectivity under various behavioral conditions.
KEYWORDS: multi-channel; ECoG; freely moving; optogenetics; flexible electrodes
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Advances in techniques for recording neural activity have facilitated our understanding of brain functions and disorders. In the early 1950s, neurosurgeon Wilder Penfield first developed a method for recording and identifying functional subdomains in the cerebral cortex connected with speech, sensory perception, and motor responses, using electrodes placed directly on the exposed cortical surface (electrocorticography, ECoG) in patients with epilepsy.1, 2 ECoG mapping protocol opened a new era in functional and cognitive neuroscience and has been an essential experimental technique hence.
Electroencephalography (EEG)3, 4 and new brain-imaging technologies such as functional MRI (fMRI)5, 6 and magnetoencephalography (MEG)7, 8 have been used and developed to monitor many brain areas and their functional interactions simultaneously. Yet, these methods have a lower temporal or spatial resolution than direct surface recording through ECoG limiting the reading of precise neural activity. EEG and MEG are affected by signal distortion from volume conduction,9, 10 which is a critical factor in localizing neural activities and mapping their interactions in great detail among brain areas. Moreover, fMRI and MEG demand imaging equipment whose absolute size limits their use in freely moving subjects,11 and thus are burdened with the requirement that experimental animals should be anesthetized12 or head-fixed.13 This creates difficulties in studying behavioral functions.
One recently emerging technology that has been investigated is polyimide (PI)- or silicone-based flexible electrode arrays for ECoG. These flexible electrodes can be attached to the curved surface of the brain, having been used to record cortical activity in various animals such as rat, cat and monkey.14-16 To record ECoG from wide areas of the cortex using flexible electrodes, removing a large portion of the skull is inevitable. This may cause irreversible damage to the brain,17, 18 side effects from harmful chemical fixatives, and even behavioral changes,19 jeopardizing the accuracy of data. It also leads to a loss of landmark information 3 ACS Paragon Plus Environment
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located on the cranial plate (i.e., bregma and lambda in rodents), which is needed for the precise positioning of electrodes. Thus, the development of a less invasive recording system that keeps the cranium intact is necessary for measuring functional connectivity across the wide-range cortical areas in freely moving animals.
Here we propose a solution to such issues: iWEBS (insertable wrapping electrode array beneath the skull), an ECoG system optimized for physiological and pathological optogenetic mapping of functional connectivity in freely moving mice. iWEBS is designed to be inserted through a small cranial slit and stably wraps onto the cortical surface in living mice, minimizing brain damage. Using iWEBS, we precisely measured dynamic changes in cortical activity during physiological and drug-induced epileptic states. Furthermore, the iWEBS is fully compatible with optogenetic mapping techniques in studying long-range functional communication in the brain.
Results and Discussion
Development of the iWEBS for recording cortical surface activity
The stable attachment of electrodes onto the cortical surface without inducing structural changes in the brain is the key to acquiring high-fidelity neural signals containing specific behavioral information. To this end, we developed a novel PI-based flexible microelectrode array system which can be inserted through very small cranial slits and firmly adhere to the cortical surface for the purpose of mapping cortical connectivity across a wide surface during freely moving states (iWEBS, Figure 1A).
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This microelectrode array consists of gold (Au) metal lines patterned on a flexible substrate passivated with SU-8 photoresist which is easily patternable and chemically stable.20 PI and SU-8 are widely used materials for biomedical application and proved for their biocompatibility21, 22 and stability in long-term implantation.14, 23 The total thickness of this electrode array was adjusted to 14.5 µm using a 12.5-µm PI base and 2-µm SU-8 passivation layer (Figure 1B and Figure S1). The SU-8 layer was set to the minimum thickness that stably coated Au metal lines. This 2-µm passivation also maximizes the robust contact of recording units on the brain surface without using additional electrode tip materials for conduction.24, 25 In addition, we adopted a bifurcated flap shape, which enabled robust penetration and attachment to both hemispheres without damaging the blood vessels located at the midline (superior sagittal sinus) of the brain (Figure 1C, left).
In neural signal recording, reducing electrode impedance is the most important for acquiring high-quality signal since it is a critical factor in increasing signal to noise ratio26, 27 and preventing Faradaic reaction – undesirable chemical reaction of metal electrode.28 The size of exposed recording units (100 × 100 µm2) and width of connected metal lines (100 µm) were chosen to achieve low impedance values without increasing the thickness of Au metal lines (Figure 1C, right). Using a simple fabrication method, we obtained electrodes with a robust frequency-impedance relationship, which certifies iWEBS for the application in physiological signal recording (Figure 1D). The recording units of iWEBS had acceptably low and uniform impedance values (26.6 ± 0.2 kΩ at 1 kHz, Figure 1E), considering the impedance values between 1 - 600 kΩ at 1 kHz have been used to record ECoG signals.14, 29 These results indicate that the iWEBS is of sufficiently high quality for the acquisition of consistent signals across many cortical subdomains.
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For insertional implantation, bending stiffness of iWEBS is a critical parameter: it should be flexible enough to make efficient contact with curved cortical surfaces, yet rigid enough to slide into the subcranial space. To achieve the appropriate balance between flexibility and rigidity, we determined the minimum value for bending stiffness (~1.12 × 10-9 Nm2) using reference films (Figure S2). This could only be achieved using a complicated solution-processed PI (8.5 µm) fabrication procedure. At ~1.85 × 10-9 Nm2, the bending stiffness of iWEBS is ~3-fold lower than previously reported flexible recording systems and approaches a minimum achievable value (Figure 1F).15
Implantation of the iWEBS causes minimal brain damage in mice
The iWEBS is designed to cover all major cortical subdomains, including somatosensory, visual, motor and retrosplenial cortices. The position of recording sites was adjusted along the coordinates set by an anatomical reference point (bregma) to precisely affix electrode tips to target cortical subdomains (Figure 2A). To insert the iWEBS beneath the skull, we made a narrow cranial slit (0.7 × 2.5 mm) in each hemisphere (Figure 2B, left), then slowly slid the two bifurcated flaps of the iWEBS through the meningeal space (Figure 2B, right). To confirm the advantage of the cranial slit method, we compared cross-sectional images from brains 20 minutes after craniotomy. The sections show that the cranial slit method caused little damage, leaving the target cortical surface intact (Figure 2C and D). In striking contrast, removal of the entire skull (mass craniotomy) induced edema and deformation in the brain, which may cause significant dislocations in targeted regions of cortex. The iWEBS system also caused only minute damage to the cortex 3 weeks after implantation (Figure 2E and Figure S4).
Stable recoding of cortical activity using the iWEBS during various brain states in freely moving mice 6 ACS Paragon Plus Environment
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To test signal quality and stability, we recorded cortical activity using the iWEBS, together with an electromyogram (EMG) and a video-monitoring system, in freely moving mice. Both of these latter tools were used to identify changes in behavioral states. Using the iWEBS, we were able to successfully record various cortical activity markers known to represent specific behavioral states (Figure 3A and Movie S1). First, cortical areas showed high-frequency, lowamplitude activities during resting states (Figure 3A), as described previously.30 Although the signal were generally synchronized, there were precise differences within the waveforms among the signals (Figure S5). Second, during exploratory movements, many cortical areas showed synchronous type I theta rhythms (~9 Hz), which were absent during non-exploratory (stereotypic) behaviors such as digging and gnawing (Figure 3B).31 We also found a difference in the high-frequency component (37–57 Hz gamma wave power), which is known to be associated with attention32, 33 and associative learning,34 between physiological states (Figure S6). Gamma wave power in mice was significantly higher during exploration, a state that involves frequent attention to surroundings. Third, the iWEBS signals accurately displayed transitions between brain states—wakefulness, non-rapid eye movement (NREM) and rapid eye movement (REM) sleep (Figure 3B and C)—defined by their EMG tones (Figure S7). In NREM sleep, mice showed low frequency delta (
