Bimodal neural probe for highly co-localized chemical and electrical monitoring of neural activities in vivo

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Highlights

  • The bimodal neural probe enables the observation of co-localized electrical and chemical signals from the cells in vivo in a live animal.

  • The bimodal neural probe has the capabilities to deliver multiple drugs for neuromodulation and to isolate collected samples without interrupting in vivo experiments.

  • This paper provides the experimental and simulation results for the characteristics of the bimodal neural probe to verify the feasibility of in vivo experiments.

  • This paper provides results that simultaneously observe co-localized multimodal signals of cells in vivo in the intact mouse brain.

Abstract

Investigation of the chemical and electrical signals of cells in vivo is critical for studying functional connectivity and brain diseases. Most previous studies have observed either the electrical signals or the chemical signals of cells because recording electrical signals and neurochemicals are done by fundamentally different methods. Herein, we present a bimodal MEMS neural probe that is monolithically integrated with an array of microelectrodes for recording electrical activity, microfluidic channels for sampling extracellular fluid, and a microfluidic interface chip for multiple drug delivery and sample isolation from the localized region at the cellular level. In this work, we successfully demonstrated the functionality of our probe by monitoring and modulating bimodal (electrical and chemical) neural activities through the delivery of chemicals in a co-localized brain region in vivo. We expect our bimodal probe to provide opportunities for a variety of in-depth studies of brain functions as well as for the investigation of neural circuits related to brain diseases.

Introduction

The neural circuits in the brain are composed of connections among the different regions of the brain, and the investigation of the roles of these different regions in neural circuits is essential in order to understand the fundamental mechanisms of the functions of the brain (Holtmaat and Svoboda, 2009; Kolb and Whishaw, 2013). Over the past several decades, many research groups have attempted to decode the communications among the cells that comprise the neural circuits in specific regions of the brain. It is well known that cells use electrical signals and chemicals to communicate (Carter and Shieh, 2015; Schwartz, 2004), and each plays a pivotal role in the function of neural circuits. For example, brain diseases, such as Parkinson's disease and schizophrenia, have been reported to be closely associated with abnormal levels of various neurotransmitters (Baranwal and Chandra, 2018; Donaldson and Young, 2008; Drevets et al., 2008; Luchtman et al., 2009; Meldrum, 2000). Therefore, determining the concentrations of neurochemicals in the brain is essential for understanding and treating brain diseases and for the study of neural circuits. The measurement of the electrophysiological signals with neurochemicals enables both the study of the neural activity that corresponds to specific neurochemicals and the study of the neural activities associated with specific types of cells, such as glutamatergic neurons or GABAergic neurons.

Recording electrical activity is considered less challenging than determining the concentrations of various chemicals in vivo. Electrical signals typically are recorded with metal electrodes or microelectromechanical systems (MEMS)-based, neural probes implanted in target regions of the brain (Altuna et al., 2012; Chen et al., 2009; Fiáth et al., 2018; Lee et al., 2015b; Royer et al., 2010; Son et al., 2015; Wu et al., 2015). However, the conventional method for monitoring chemical signals in vivo is to collect extracellular fluid (ECF) from target regions in the brain and analyze the fluid with precise external analyzers (e.g., mass spectroscopy). The microdialysis method is used to obtain ECF from the brain because it enables the collection of the diffused neurochemicals through a semipermeable membrane due to the concentration gradient that exists between a region in the brain and the fluidic channel inside an inserted probe (Ao and Stenken, 2006; Duo et al., 2006; Kehr, 1993; Lee et al., 2016; Watson et al., 2006). While microdialysis allows the extraction of ECF at relatively high flow rates without net fluid loss from tissues, the large membrane (0.2 mm × 1 mm) on traditional microdialysis probes induces significant tissue damage and also prevents the high-spatial-resolution chemical signal analysis (Kennedy, 2013; Szarowski et al., 2003).

Recently, a few devices have been developed for high spatial-resolution in vivo neurochemistry that overcome the disadvantages of the traditional microdialysis technique. A miniaturized push-pull type sampling probe fabricated using MEMS fabrication processes was introduced recently (Lee et al., 2013; Ngernsutivorakul et al., 2018). The push-pull sampling probes collect ECF from the sampling area through a pull channel while injecting buffer solution (i.e., artificial cerebrospinal fluid (aCSF)), through a push channel to prevent the depletion of the fluid in the brain (Cellar et al., 2005; Myers et al., 1998). The push-pull sampling probes are only 85 μm wide and 70 μm thick, and they reduced the damage to the tissue and increased the spatial resolution of the chemical signal. Unfortunately, these sampling probes cannot record electrical signals, and one previous study proposed integrating the sampling probe with a single collection channel with recording electrodes (Petit-Pierre et al., 2016). This approach provided the capability of recording electrical signals and sampling the ECF from the same small region of the brain. However, the device is not suitable for long-term experiments due to the possibility of depleting extracellular brain fluid during extended neurochemical monitoring (Petit-Pierre et al., 2016).

Herein, we present the first-of-a-kind device that enables the measurement of the electrical and the chemical signals from the same local region of the brain in a live animal. The bimodal MEMS neural probe we developed provides the capabilities of 1) co-localized bimodal (electrical and chemical) monitoring of neural signals by simultaneous recording of electrical signals and the sampling of ECF, 2) real-time delivery of multiple drugs, and 3) isolation of the samples. The monolithic integration of the dedicated delivery (i.e., push) and collection (i.e., pull) microfluidic channels in the vicinity of the recording electrodes allows highly co-localized observation of both the electrical and chemical signals. A microfluidic interface chip that is attached to the body of the probe provides the capabilities of delivering multiple drugs and the isolation of the sample, which enable the high-temporal-resolution study of the pharmaceutical effect in neural circuits with modulation by drugs (Fig. 1). We used a previously-reported glass reflow process (Lee et al. 2015a, 2015b) to embed the microfluidic channels onto the probe and integrate them with the array of electrodes. The electrodes were located in close proximity to the inlet of the collection channel to enable the highly co-localized monitoring of the electrical and chemical activities. Due to the high-density monolithic integration, the bimodal neural probe is only 40-μm thick, and it has a cross-sectional area that is 3–4 times smaller than those of the previously developed sampling probes (Petit-Pierre et al., 2016), which minimizes the damage to tissue during the insertion and facilitates the additional localized collection of the ECF.

We demonstrated the capabilities of the bimodal neural probe by measuring the chemical and electrical signals from the hippocampus and the thalamus of intact mouse brains and by investigating the correlation of the electrical and chemical activities in the region. The electrical and chemical activities were monitored for several hours before and after the delivery of the drug to modulate the activities of the brain. The changes in the concentrations of neurotransmitters, such as glutamate and y-Aminobutyric acid, were measured using ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS). We expect our novel technology to provide opportunities for a variety of studies for the in-depth study of brain functions as well as for the investigation of neural circuits related to brain diseases.

Section snippets

Materials

For the push-pull sampling operation in vivo, aCSF was used as the buffer solution, and it contained 145 mM NaCl, 2.68 mM KCl, 1.01 mM MgSO4, 1.22 mM CaCl2, 1.55 mM Na2HPO4, and 0.45 mM NaH2PO4. The pH of the buffer solution was 7.4. Chloroplatinic acid hydrate, 0.1 M HCl, and lead acetate trihydrate were used for the Pt black electroplating on the recording electrode. L-Glutamic acid monosodium salt monohydrate was used for the characterization of our device. All of the reagents were purchased

Design of the bimodal MEMS neural probe

The bimodal MEMS neural probe consists of a shank and a probe body. The shank contains microfluidic channels for push-pull perfusion and twelve electrodes at its tip for the electrical recording. A polydimethylsiloxane (PDMS) microfluidic interface chip is attached to the body of the probe. The length of the shank was designed to be 6 mm long so that the tip of the probe can reach any deep region in the brain of a mouse. Also, the probe tip has a sharp end to focus the insertion force to the

Conclusions

We present a bimodal MEMS neural probe that enables the concurrent monitoring of the electrical and chemical activities of cells in vivo. The probe contains monolithically-integrated microfluidic channels for the collection of ECF, an array of twelve electrodes for recording electrical neural signals, and a microfluidic interface chip that provides the chemical delivery and sample isolation capabilities. The configuration at the tip of the probe, where the electrodes and the sampling site are

CRediT authorship contribution statement

Uikyu Chae: Conceptualization, Methodology, Software, Validation, Formal analysis, Writing – original draft, Visualization. Hyogeun Shin: Resources. Nakwon Choi: Writing – review & editing. Mi-Jung Ji: Resources. Hyun-Mee Park: Resources. Soo Hyun Lee: Resources. Jiwan Woo: Resources. Yakdol Cho: Resources. Kanghwan Kim: Writing – review & editing. Seulkee Yang: Resources. Min-Ho Nam: Resources. Hyun-Yong Yu: Writing – review & editing. Il-Joo Cho: Conceptualization, Methodology, Writing –

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Brain Research Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (NRF-2017M3C7A1028854). Also, this work was supported by the Brain Convergence Research Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (NRF-2019M3E5D2A01063814), Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (NRF-2017M3A9B3061319), the

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