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. 2020 Feb 11:9:e50345.
doi: 10.7554/eLife.50345.

Organic electrochemical transistor arrays for real-time mapping of evoked neurotransmitter release in vivo

Kai Xie #  1 , Naixiang Wang #  2 , Xudong Lin  1 , Zixun Wang  1 , Xi Zhao  1 , Peilin Fang  1 , Haibing Yue  1 , Junhwi Kim  1 , Jing Luo  3 , Shaoyang Cui  3 , Feng Yan  2 , Peng Shi  1   4   5
Affiliations

Organic electrochemical transistor arrays for real-time mapping of evoked neurotransmitter release in vivo

Kai Xie et al. Elife. .

Abstract

Though neurotransmitters are essential elements in neuronal signal transduction, techniques for in vivo analysis are still limited. Here, we describe an organic electrochemical transistor array (OECT-array) technique for monitoring catecholamine neurotransmitters (CA-NTs) in rat brains. The OECT-array is an active sensor with intrinsic amplification capability, allowing real-time and direct readout of transient CA-NT release with a sensitivity of nanomolar range and a temporal resolution of several milliseconds. The device has a working voltage lower than half of that typically used in a prevalent cyclic voltammetry measurement, and operates continuously in vivo for hours without significant signal drift, which is inaccessible for existing methods. With the OECT-array, we demonstrate simultaneous mapping of evoked dopamine release at multiple striatal brain regions in different physiological scenarios, and reveal a complex cross-talk between the mesolimbic and the nigrostriatal pathways, which is heterogeneously affected by the reciprocal innervation between ventral tegmental area and substantia nigra pars compacta.

Keywords: biochemistry; bioelectronics; brain circuits; chemical biology; dopaminergic signaling; neuroscience; neurotransmitter release; organic electrochemical transistor; organic electronics; rat.

Plain language summary

Cells in the nervous system pass messages using a combination of electrical and chemical signals. When an electrical impulse reaches the end of one cell, it triggers the release of chemicals called neurotransmitters, which pass the message along. Neurotransmitters can be either activating or inhibitory, determining whether the next cell fires its own electrical signal or remains silent. Currently, researchers lack effective methods for measuring neurotransmitters directly. Instead, methods mainly focus on electrical recordings, which can only tell when cells are active. One new approach is to use miniature devices called organic electrochemical transistors. Transistors are common circuit board components that can switch or amplify electrical signals. Organic electrochemical transistors combine these standard components with a semi-conductive material and a flexible membrane. When they interact with certain biological molecules, they release electrons, inducing a voltage. This allows organic electrochemical transistors to detect and measure neurotransmitter release. So far, the technology has been shown to work in tissue isolated from a brain, but no-one has used it to detect neurotransmitters inside a living brain. Xie, Wang et al. now present a new device that can detect the release of the neurotransmitter, dopamine, in real-time in living rats. The device is a miniature microarray of transistors fixed to a blade-shaped film. Xie, Wang et al. implanted this device into the brain of an anaesthetised rat and then stimulated nearby brain cells using an electrode. The device was able to detect the release of the neurotransmitter dopamine, despite there being a range of chemicals released inside the brain. It was sensitive to tiny amounts of the neurotransmitter and could distinguish bursts that were only milliseconds apart. Finally, Xie, Wang et al. also implanted the array across two connected brain areas to show that it was possible to watch different brain regions at the same time. This is the first time that transistor arrays have measured neurotransmitter release in a living brain. The new device works at low voltage, so can track brain cell activity for hours, opening the way for brand new neuroscience experiments. In the future, adaptations could extend the technology even further. More sensors could give higher resolution results, different materials could detect different neurotransmitters, and larger arrays could map larger brain areas.

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Conflict of interest statement

KX, NW, XL, ZW, XZ, PF, HY, JK, JL, SC, FY, PS No competing interests declared

Figures

Figure 1.
Figure 1.. System schematic diagram and OECT-array working principle for CA-NT detection.
(a) Systematic diagram of using the OECT for monitoring neurotransmitter release. (b) Illustration of CA-NT’s electro-oxidation reaction on the surface of the Pt-GATE electrode. (c) Diagram showing the working principle of an OECT device. Cvolumetric and CG-E denotes the volumetric capacitance across the PEDOT:PSS active layer and the capacitance between the GATE electrode and the electrolyte respectively. Vvolumetric and VG-E denotes voltage across the active PEDOT:PSS layer and between the GATE electrode and the electrolyte respectively.
Figure 2.
Figure 2.. Ex vivo characterization of the OECT-array.
(a) Photographs showing the overall (left), enlarged (middle) and bent view of the flexible OECT-array. Scale bar indicates 5 mm in left panel and 200 μm in the middle panel. (b) Wiring diagram showing the working setup of an OECT-array. (c) Ex vivo recording of IDS changes in response to artificially added dopamine to ACSF containing a high level of ascorbate acid (1.28 mM; VDS = 0.06 V; VGS = 0.6 V). (d) The calibration curve showing the relationship between the measured ΔVg-eff and changes of dopamine concentrations, n = 3, error bars indicate standard error. The dash line shows linear fitting of the data.
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. The dimension of the OECT-array.
Hchannel = 10 μm, denoting the height of the exposed PEDOT:PSS channel (distance between the SOURCE and DRAIN electrode connected with the channel). Wchannel = 40 μm, denoting the width of the exposed PEDOT:PSS channel (width of the SOURCE and DRAIN electrode connected with the channel). Hgate = 800 μm, denoting the height of the GATE electrode exposed in the patterned SU-8 insulation layer. Wgate = 600 μm, denoting the width of the GATE electrode exposed in the patterned insulation layer. Dunit = 1,200 μm, denoting the distance between the adjacent OECT-units.
Figure 2—figure supplement 2.
Figure 2—figure supplement 2.. Ex vivo characterization of the OECT-array.
(a) Representative IDS curve (black) and corresponding transconductance (red) curve recorded from an OECT-device in PBS. (b–f) Response of an OECT-device to concentration change of different neurotransmitters, including dopamine (b), adrenaline (c), noradrenaline (d), γ-aminobutyric acid (GABA; e) and glutamate (f) as characterized in PBS. n = 3, error bars indicate standard error.
Figure 3.
Figure 3.. OECT-array for monitoring in VTA.
(a) Schematic diagram (upper) and surgical photogram (lower) of the experimental setup. The 1st unit (orange) of an OECT-array was implanted to VTA to monitor somatodendritic dopamine release evoked by electrical stimulation in MFB. (b) Front (upper) and side (lower) view of the device bundle containing an OECT-array and a recording tungsten electrode. Scale bar, 1 mm. (c) Representative transfer (black) and transconductance (red) curve of an OECT device placed ex vivo in the ACSF (upper; VDS = 0.06V; VGS = 0.6V) or in vivo in a rat brain (lower; VDS = 0.06V; VGS = 0.6V). (d) The IDS recording (left) from an OECT-unit in the VTA in response to neural stimulation in MFB using different number of electrical pulses. The corresponding measurements of dopamine release from multiple trials were shown in the right boxplot. The whisker range is 1 ~ 99%, and each box show 25, 50% and 75% percentile of the data collected from three animals. For the control experiments, the stimulation was made off the MFB. (e) The correlation between the intensity of dopamine release in VTA and the number of electrical pulses in MFB, n = 3, error bars indicate standard error. The dash line shows linear fitting of the data.
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Characterization of OECT-array.
(a) Raw transfer curve (VDS = 0.06 V) in brain (black) and ACSF (red). (b) Output characterization upon different VGS (color-coded) in PBS.
Figure 4.
Figure 4.. OECT-array for mapping dopamine release along the mesolimbic pathway.
(a) Schematic diagram of using an OECT-array to map dopamine release around NAc region in response to neural stimulation of VTA. (b) Immunostaining of brain tissues in NAc (upper) or VTA (lower) after the electrochemical measurements. The NAc brain slices were stained for dopaminergic axon terminals (TH+, red) and cell nuclei (DAPI, cyan); the VTA brain slices were stained for dopaminergic neurons (TH+, red) and neural activation marker (c-Fos+, green). The implantation track of the OECT-array (in NAc) and the stimulation electrode (in VTA) were indicated by the dashed lines. Scale bar, 100 μm. (c) The IDS recorded from the OECT-unit placed in NAc in response to electrical stimulation of VTA (2 ms pulse width, 50 Hz, 50 pulses; VDS = 0.05V; VGS = 0.65V). The gray curves are the raw IDS recording from multiple VTA-stimulation trials, the solid red curve shows the average of all 34 trials, and the red shade indicates the range of standard error. (d) Quantitative measurements of dopamine release in NAc in response to VTA-stimulation, n = 3, * indicates p<0.05 by student t-test. (e) Frequency-dependent dopamine release in the NAc as evoked by VTA stimulation (2 ms pulse width, 1 s duration, 30, 50, or 100 Hz), n = 3. (f) Comparison of dopamine release simultaneously measured by the 1st (inside NAc) and the 2nd (outside NAc) OECT-unit in response to VTA-stimulation (2 ms pulse width, 50 Hz, 1 s duration), n = 8, ** indicates p<0.005 by student t-test. For panel d-f, error bars indicate standard error.
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. Confirmation of correct OECT-unit placement in NAc.
Nissl blue staining of the coronal section of NAc region showing the surgical track of an OECT-array (left) and a tungsten electrode (right) after an experiment. Slice thickness, 50 μm. Scale bar, 1 mm.
Figure 4—figure supplement 2.
Figure 4—figure supplement 2.. Electrophysiological characterization in the NAc in response to VTA-stimulation.
Electrophysiological recording in parallel with dopamine monitoring in NAc upon VTA-stimulation. The raster plot (upper) and post-stimulation time histogram (lower) in VTA (a) and NAc (b) upon electrical stimulation in VTA. Data from 38 trials were shown.
Figure 5.
Figure 5.. Dopamine mapping across the mesolimbic and nigrostriatal pathways.
(a) Immunohistochemical staining for TH in the CPu and the NAc after an electrochemical measurement. The surgical track of the OECT-array was indicated by the dashed box. The location of each OECT-unit was denoted by dashed lines. Scale bar, 1 mm. (b) Immunohistochemical staining for TH in the VTA and SNc. The surgerical tracks of the stimulation electrodes in VTA or SNc were indicated by dashed boxes. Scale bar, 1 mm. (c) Quantitative analysis of dopamine release in NAc and different parts of CPu simultaneously measured by multiple OECT-units upon electrical stimulation of the mesolimbic (in VTA) or the nigrostriatal (in SNc) pathways. n = 10, the error bars indicate error, ** indicates p<0.005 by student t-test, n.s. stands for ‘not significant’.
Figure 6.
Figure 6.. Electrochemical cross-talk between the mesolimbic and nigrostriatal signalling.
(a) Schematic diagram of dopamine mapping in the NAc and CPu in response to surgically isolated VTA- or SNc-stimulation. (b) Immunohistochemical staining for TH in a brain slice showing the surgical tracks of the stimulation electrode in the VTA or SNc, and the mechanical lesion between these two regions. Scale bar, 1 mm. (c–e) Quantitative analysis of the change in dopamine release pattern at different brain regions across mesolimbic and nigrostriatal pathways, including the NAc (c), lower CPu (d) and upper CPu (e), in response to VTA- or SNc-stimulation before and after the surgical lesion to mechanically break the mutual connections between VTA and SNc. n = 6, error bars indicate standard error, *indicates p<0.05 by student t-test; n.s. stands for ‘not significant’. +/- indicates that the measurements were conducted with/without the VTA-SNc surgical lesion. (f) Summary of the identified complex cross-talk between the mesolimbic and nigrostriatal pathways as regulated by the mutual connection between VTA and SNc.
Figure 6—figure supplement 1.
Figure 6—figure supplement 1.. Reciprocal connection between VTA and SNc characterized by electrophysiology.
(a) The raster plot (upper) and post stimulation time histogram (PSTH; lower) in SNc upon electrical stimulation in VTA. (b) The raster plot (upper) and PSTH (lower) in VTA upon electrical stimulation in SNc. (c) Representative waveform of the spontaneous spiking of the dopaminergic neurons (>100 spikes). These spikes typically have a long action potential duration greater than 3 ms. (d) Quantitative analysis of the spike count 100 ms after electrical stimulation. The post-stimulation counts were normalized to before-stimulation counts, n = 6, error bars indicate standard error, * indicates p<0.05 by student t-test. For panel (a) and (b), the stimulation is 2 ms pulse width, 50 Hz, 5 pulses, 200 μA amplitude.
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