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Simultaneous monitoring of action potentials and neurotransmitter release from neuron-like PC12 cells
Analytica Chimica Acta xxx (xxxx) xxx
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Simultaneous monitoring of action potentials and neurotransmitter release from neuron-like PC12 cells Mei Rong Cui 1, Wei Zhao 1, Xiang Ling Li, Cong Hui Xu**, Jing Juan Xu*, Hong Yuan Chen State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A platform based on ultra-thin microelectrode array and total internal reflection fluorescence microscopy is proposed. The combination of optical and electrical techniques enables completely mapping of neuron connectivity for the first time. Real-time recording of APs and FFN102 release reveals the relevance of electrical and chemical activities of nerve cells.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 October 2019 Received in revised form 12 November 2019 Accepted 21 November 2019 Available online xxx
Simultaneous recording of action potentials (APs) and neurotransmitter release is highly desirable in living neurons since it provides a complete framework of the physiological and pathological statuses of nerve cells. In this work, we proposed an approach coupling ultra-thin microelectrode array (MEA) with total internal reflection fluorescence microscopy (TIRFM), which served as a powerful platform to visualize both APs and vesicular exocytosis in a neuronal circuit model formed by neuron-like PC12 cells. Taking advantages of fluorescent false neurotransmitter (FFN), the transient neurotransmitter transport down an axon could be visualized with high spatial and temporal resolution. The real-time recording of APs burst and neurotransmitter release induced by hypoxia with MEA/TIRFM platform reveals the relevance of electrical and chemical activities in the neuronal model. The combination of the optical and electrical techniques enables mapping of neuron connectivity in an entire neuronal circuit, which may ultimately lead to deeper understanding of nervous system. © 2020 Elsevier B.V. All rights reserved.
Keywords: Action potentials Neurotransmitter MEA/TIRFM
1. Introduction The function of nervous system is intrinsically linked to electrical and chemical impulses, the essential elements for neuronal
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (C.H. Xu), [email protected] (J.J. Xu). 1 These authors contributed equally.
communication [1,2]. Understand the connectivity-map of neuronal circuits and their physiological and pathological functions could help develop better strategies to combat neural disorders [3]. Therefore, it is desirable to achieve visualization of neuronal circuit connectivity, especially precise monitoring of synaptic activity [4,5]. Up to date, the majority of neuronal circuit investigations focus on electrophysiology. Methodologies for recording electrical activity under in vitro or in vivo conditions include sharp micro/ nano-electrodes or patch clamp [6,7], optical imaging using
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Scheme 1. Schematic illustration of TIRFM/MEA coupling for simultaneous measurement of APs and neurotransmitter release from neuron-like PC12 cells.
fluorescent indicators [8], as well as the prime technique, substrateintegrated microelectrode arrays (MEAs) [9e11]. Microelectrode arrays provide real-time, nondestructive measurements of the resting and action potential (AP) signals, which enable simultaneous and long-term recording of extracellular potential from a population of neurons at millisecond time scale, thereby offer robust electrophysiological imaging at the network level [12]. In recent years, number of pioneering researches began to merge the advantages of extracellular microelectrode arrays and intracellular microelectrodes towards the imaging of connectivity-map of neuronal circuits [13,14]. On the other hand, to monitor neurosecretion, amperometric measurements with ultra-microelectrodes [15,16], and optical methods [17e19] to observe synaptic vesicle membrane serve as the major technologies. To date, monitoring of neurotransmitter at individual synapses has been achieved using both technologies [20]. Amperometry provides quantitative detection on the amount of electroactive neurotransmitter with high temporal resolution [21]. However, it lacks spatial resolution to monitor the releases of neurotransmitter from different sites in a living cell or neuron. In contrast, fluorescence method can reveal the distribution of secretory vesicles, and can track their motility during the synaptic release in real-time [22e24]. Nevertheless, the optical method lacks temporal resolution. Recently, fluorescent false neurotransmitters (FFNs), as substrates for the synaptic vesicle monoamine transporter, were firstly reported by Sames and Sulzer’s group [25]. Using FFNs, it is possible to measure key presynaptic processes including the accumulation and release of vesicle transporter substrate. Compared to electrical recordings (patch-clamp membrane capacitance and electrochemical amperometry) of neurotransmitter, optical methods using FFNs provided higher spatial resolution [26]. Combined amperometry with total internal
reflection fluorescence microscopy (TIRFM), Guille-Collignon and co-workers have applied FFNs as a fluorescent/electroactive dual probe for coupled measurements of single vesicular exocytosis events [27]. With the growing interest in mapping the physiological or pathological state of neurons, as well as creating experimental therapy and novel neuroprosthetic devices, it is highly desirable to develop methods for simultaneous recording of both AP waveforms and release of neurotransmitters in an entire living neuron or neural model, as they provide a complete framework of the neuron activities. To achieve such goal, we developed a system for simultaneous monitoring of APs and release of neurotransmitters from living neuron-like rat pheochromocytoma (PC12) cells based on ultrathin transparent MEA (60 TiN microelectrodes) coupled with TIRFM. MEA with ultra-thin thickness of 180 mm facilitated TIRFM observation. As shown in Scheme 1, PC12 cells were in-situ cultured and differentiated with nerve growth factor (NGF) to resemble a dopaminergic neuronal phenotype. Then FFN102, a synthesized analogue of biogenic neurotransmitters, was applied to stain neuron-like PC12 cells and served as an indicator of the neurotransmitter, dopamine. Under external hypoxia stimulation, the APs firing and exocytosis were simultaneously recorded by MEA and TIRFM, respectively, with high temporal and spatial resolutions. And the recorded signals could be coupled in time and space. The proposed MEA/TIRFM coupling platform provides promising opportunity of stimulation and recording of brain circuit activity to understand the molecular and cellular basis of health problems.
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2. Experimental section 2.1. Chemicals and materials FFN102 was purchased from Aabcam (Cambridge, UK). Laminin from Engelbreth-Holm-Swam and NGF-b from rat were purchased from Sigma-Aldrich. Poly-L-lysine (PLL) was purchased from Merck KGaA (Germany). Fe12K nutrient mixture, horse serum, fetal bovine serum (FBS) and Celltraker™ green CMFDA are from Gibco Life Technologies. Undifferentiated rat pheochromocytoma (PC12) were purchased from Cellbank (China). All experiments using living cells were performed in phosphate buffer solution (pH 7.4) prepared using double distilled water from a Millipore purification system (18.2 MU cm at 25 C). 2.2. MEA type and layout To fulfill the requirement of high-resolution total internal reflection fluorescence microscopy (TIRFM) imaging, microelectrode array (MEA) with 60 microelectrodes arranged in an 8 8 layout grid embedded in a transparent glass substrate with thickness of 180 mm was purchased from Harvard Bioscience Inc. The MEA type is briefly described as follows: - 180 mm thin recording area - TiN electrodes - SiN isolator
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ITO conductive strips With 1 internal reference electrode Electrode grid 8 8 59 recording electrodes Electrode spacing 100 mm Electrode diameter 10 mm Glass ring 6 mm high (other heights on request).
2.3. Apparatus for characterization Action potential waveforms were performed using MEA 2100System (Harvard Bioscience Inc.) consisted of MEA, interface board, PC with software and temperature controller. The real-time data was collected with the interface board. A low-pass filter with a maximum frequency of 5000 Hz was applied after data acquisition using MATLAB R2016a software, which enables the noise analysis (SNR) and statistical event frequency analysis. Time-lapse imaging was acquired by TIRFM (Olympus, Japan) equipped with a Plan Apochromat 63 /1.4 NA oil immersion objective. In TIRFM, a 405 nm digital laser system employed as the light source to initiate the fluorescence whereas the emission light was filtered before being detected by Prime 95B SCMOS camera (Olympus, Japan). Confocal fluorescence images of cells were acquired with a TCS SP5 confocal microscopy (Leica, Germany).
Fig. 1. (A) Photograph of 60 ThinMEA. (B) Bright field image of 8 8 layout electrodes (scar bar: 100 mm). (C) Confocal image of neuron-like PC12 cells cultured on MEA stained by CelltrakerTM green CMFDA (scar bar: 100 mm). (D) Voltage traces of spontaneous APs at 10 electrodes marked with dashed rectangle in fluorescent image (scar bars: 10 ms horizontal and 50 mV vertical). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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TIRFM. The electrophysiology of the neural network and the release of neurotransmitters from axon varicosities or axon terminals were triggered by adding hypoxic buffer, which was prepared by continuously bubbling 0.1 PBS buffer, sealed in a clean glass flask with pure N2 gas (purity: 5 N) for 30 min. SCMOS and MEA2100System are synchronized by the Digital delay/pulse generator DG535 (Stanford Research Systems, Inc.) with two simultaneous TTL signals to trigger the recording. The rise time of the TTL signal is less than 3 ns and the time delay of the two TTL signals is ca. 5 ps. 2.7. Optical characterization
Fig. 2. Long-term recording of APs from the neuron-like PC12 cells cells on one of the microelectrodes after adding hypoxic buffer (A) and normal PBS solution without anoxic treatment (B).
2.4. MEA pretreatment Before cells culture on the MEA, it was pretreated according to the following process. First, it was bathed in absolute ethanol for 20 min, then rinsed with distilled water and dried in the air. Second, 75% alcohol was applied for disinfection. Third, after naturally dried on ultra-clean table, we applied UV to irradiate the MEA for 6 h (3 h on each side). Forth, 500 m L of 0.1 mg/mL PLL solution was added in the culture chamber, and the MEA was placed in an incubator MCO15AC (Sanyo, Tokyo, Japan) at 37 C, 5% CO2 and saturated humidity for 1 h. Then, it was taken out of the incubator and thoroughly rinsed with distilled water for three times. Finally, it was incubated with laminin at 4 C overnight, and then could be used for cell seeding. 2.5. Cell culture and sample preparation Neuron-like PC12 cells were maintained in Fe12K Nutrient Mixture supplemented with 5% FBS, 10% Horse Serum (Heat-Inactivated) 100 U/mL penicillin and 100 mg/mL streptomycin at 37 C in a 5% CO2-95% air incubator MCO-15AC (Sanyo, Tokyo, Japan). In order to acquire neuron-like PC12 cells model on MEA or the glassbottomed Petri dishes, confluent undifferentiated PC12 cells(105 cells/mL) were counted and cultured in fresh Fe12K Nutrient Mixture supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 C in a 5% CO2 humidified atmosphere. For differentiation, cells were added with nerve growth factor (NGF) (with the final concentration of 50 ng/ml) and cultured for 6 days. 2.6. Simultaneous visualization of AP waveforms and release of neurotransmitters by MEA/TIRFM After neural network cultured on the ultra-thin transparent MEA, we implemented simultaneous visualization of action potential waveforms and releasing of neurotransmitters by MEA/
To map the growth-form of neuron-like PC12 cells on transparent MEA, the cells were stained by celltraker™ green CMFDA for 10 min and subsequently imaged by the TCS SP5 confocal microscopy. UVeVis absorption spectra of FFN102 was measured at room temperature on an UVeVis spectrophotometer (UV-3600; Shimadzu Co., Japan). The absorption spectra were recorded from 270 nm to 440 nm with an interval of 1 nm at a scan rate of 1200 nm/min. Fluorescence measurements were acquired on the F7000 (Hitachi) fluorescence spectrometer with a Xeon lamp. For emission scans, the excitation wavelength was set to the maximum absorption wavelength on the basis of UVeVis spectra and the emission wavelengths were scanned from 400 nm to 600 nm with an interval of 1 nm at a scan rate of 1200 nm/min. The emission wavelength was set at 456 nm to record the excitation spectra of FFN102. 2.8. Confocal laser scanning microscopy imaging PC12 cells were digested and incubated in confocal cell dishes overnight. Then, the cells were incubated with FFN102 for 30 min. After that, the cells were washed for three times and confocal laser scanning microscopy (CLSM) images of PC12 cells were acquired. 3. Result and discussion 3.1. Characterization of neuronal circuit model on MEA The implementation of MEA/TIRFM coupling measurements relies mainly on the specific property of the innovative ultra-thin transparent MEA (8 8 layout grid, 10 mm electrode diameter, 100 mm electrode spacing, including one reference electrode) (Fig. 1A and B). The thin MEA is fabricated with ultra-thin optical glass with 180 mm thickness and transparent indium-tin oxide wires embedded as conductive strips, which is ideally suited for high-resolution TIRFM imaging. NGF-differentiated neuron-like PC12 cells were cultured on MEA for 6 days. As well known, differentiated PC12 cells could serve as model for neurosecretion since they closely resemble neurons with smaller vesicle [28e30]. Fig. 1C provides fluorescent image of the neuron-like PC12 cells on MEA. Induced by NGF, PC12 cells show obvious cell adhesion, spreading and neurite outgrowth on the substrate (Fig. S1). The AP waveforms shown in Fig. 1D are the realtime recording data without filtering. The spontaneous APs show typical waveform with 500e2000 ms pulse duration and a 50e200 mV amplitude (Fig. S2), which are consistent with previous research results. 3.2. Hypoxia evokes membrane potential depolarization Adequate oxygen is essential for the survival of mammalian cells. Hypoxic stimulation could inhibit O2-sensitive Kþ channels and consequently trigger the membrane potential depolarization [31]. As PC12 cells contain O2-sensitive channels, they could serve
Please cite this article as: M.R. Cui et al., Simultaneous monitoring of action potentials and neurotransmitter release from neuron-like PC12 cells, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.074
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Fig. 3. (A) TIRFM image of neuron-like PC12 cells stained by FFN102. (B) Sequential pseudocolor images of the area marked with the red rectangle in (A), unit: second. (C) and (D) An individual exocytotic release (fluorescence trace and TIRMF images) from a single fluorescent vesicle indicated by the red circle in (A). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. Time-lapse recording of pseudo color TIRFM images of neuron-like PC 12 cells after adding hypoxic buffer (A) and PBS without anoxic treatment (B), and corresponding fluorescent trace curves (C). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 5. (A) Bright image of NGF-differentiated PC 12 cells cultured on MEA. (B) TIRFM image of the red rectangle in (A). (C) AP waveforms on electrodes shown in the area of (A) at 0.00 s, 20.04 s, 45.40 s. (D) Statistical analysis of the number of APs on 24 microelectrodes in 60 s after hypoxic stimulation. (E) Fluorescent trace (blue curve) and APs waveform (red curve) obtained from FFN102 stained PC 12 neural model indicated by rectangle in (B), and electrode G2, immediately after hypoxic stimulation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
as a chemosensory model for hypoxia [32]. Herein, hypoxic stimulation was applied to investigate the cellular effect by simultaneous monitoring of action potentials and neurotransmitter release from neuron-like PC12 cells. O2 reduced hypoxic buffer was added in the culture chamber to achieve 70% hypoxia ratio, which was sufficient to trigger the membrane potential depolarization [33]. The AP waveforms were recorded in real-time. As shown in the voltage trace in Fig. 2A, after addition of hypoxic buffer, the APs firing frequency recorded by a single electrode shows considerable increase at approximately 5 s, and gradually decreases after 30 s, which consistent with previous works [34]. On the other hand, in the control experiment, no obvious change of spontaneous APs frequency was observed in 60 s after the addition of phosphate buffer without anoxic treatment (Fig. 2B). 3.3. Hypoxia evokes vesicular neurotransmitter release To track the vesicular exocytosis in real-time, the pH-responsive fluorescence substrate, FFN102, which selectively labels secretory vesicles was applied to stain the neuron-like PC12 cells on MEA and the pH-dependent optical properties of FFN102 were examined first (Fig. S3). Considering the pH gradient between the vesicular lumen (pH 5e6) and the extracellular medium (pH 7.4), FFN102 can act as an optical tracer to visualize neurotransmitter release from individual presynaptic terminals [35]. After 30 min incubation in
10 mM FFN102, fluorescent vesicles (300e400 nm in diameter) could be observed in the cell body, axon varicosities and axon terminals (Fig. S4). As Fig. S5 shown, the vesicles adjacent to the cell membrane were presented as stable blue spot under 405 nm laser, so 405 nm laser was selected as the light source to monitor the neurotransmitter release. As reported, hypoxia evokes dopamine secretion from undifferentiated PC12 cells via a mechanism involving Ca2þ influx [32]. Initiated by reducing the concentration of O2 to more than 70%, dopamine release events from neuron-like PC12 cells could be visualized (Fig. S6A, Movie S1), which were easily observed as sparkling fluorescent spots at both cell bodies and neurites when FFN102 diffused from acidic vesicle to the neutral extracellular medium. During transient (30 s) hypoxic stimulation, large amplitude fluorescent spikes were obtained in selected areas of the neuronal network. In the control measurement, no obvious change of fluorescent intensity was observed within 60 s after adding same volume of phosphate buffer without anoxic treatment (Movie S2, Fig. S6B). There are two possible reasons. First, without hypoxic or chemical stimulations, the spontaneous neurotransmitter release with extremely low frequency [36,37] didn’t occur in such short time. Second, the amount of release is low, which hasn’t been captured. Supplementary data related to this article can be found at https://doi.org/10.1016/j.aca.2019.11.074.
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A section of the neuronal model including a long axon is chosen as an example to demonstrate the TIRFM imaging of vesicular exocytosis events with distinct spatial and temporal resolutions (Fig. 3A). As shown in Fig. 3B, FFN102 released with fluorescent signal transiently brightened and disappeared on different sites along the axon (15 mm), in sequence. The direction of nerve impulse transmission could be recognized based on the real-time recording. Furthermore, TIRFM provides high resolution to follow single vesicle fusion. As shown in Fig. 3C and D, single exocytotic event could be captured and visualized within 200e300 ms. Under hypoxic stimulation, the total fluorescent intensity of the stained PC12 neuronal network gradually decreased with time elapse (Fig. 4A), but it remained stable in the control experiment (Fig. 4B), which are consisted with the time-lapse fluorescent curves (Fig. 4C). On the basis of real-time imaging by TIRFM, many important cellular activities including vesicle motility, location of releasing sites and the transmission directions could be visualized with high spatial resolution. Combining TIRFM with transparent MEA enables more a comprehensive information acquisition for the neuron communication.
electrodes, MEA/TIRFM offers a more direct and visualized approach to discover the mechanisms in neuronal communication.
3.4. Simultaneous monitoring of AP waveforms and neurotransmitter release
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.
Two types of spontaneous neurotransmitter release are present in the nervous system, namely AP-dependent release and APindependent release [38]. Simultaneous monitoring of APs and neurotransmitter release may help neuroscientists discovering the underlying mechanisms of neuron conductivity under different types of stimulation, including hypoxia or ischemia. Based on the MEA/TIRFM equipment, simultaneous measurement of APs and dopamine release was performed. As shown in the bright image of MEA (Fig. 5A), neuron-like PC12 cells are growing on the substrate, and partially located on the electrodes. Part of the network containing axon varicosities and terminals was selected for real-time TIRFM imaging (Fig. 5B). The electric activities and FFN102 release events were simultaneously recorded after acute hypoxic stimulation. Fig. 5C shows the voltage traces on part of the microelectrodes of MEA at 0.00 s, 20.04 s and 45.40 s. Compared with the voltage traces at initial time, the frequency change of APs spikes is significant at ca. 20 s, but sharply reduced at 45 s. The frequency of APs on 24 microelectrodes during hypoxic stimulation are statistically analyzed. As the results shown in Fig. 5D, a sudden increase is observed from 5 to 10 s, after 30 s, the number drops significantly. To compare the APs firing and neurotransmitter release, we choose the APs recording from electrode G2 (indicated by red circle in Fig. 5A), which located under a cell body and fluorescence signal derived from the axon varicosity extended from that cell body. As shown in Fig. 5E, fluorescent events occurred immediately after hypoxic stimulation, and decreased after 20e30 s (Movie S3). However, similar to the APs firing statistically analyzed from 24 electrodes, intensive AP firing showed at ca. 5 s at electrode G2. The synaptic release occurs earlier than APs firing. Supplementary data related to this article can be found at https://doi.org/10.1016/j.aca.2019.11.074. It is well agreed that the release of neurotransmitters including dopamine and glutamate under acute hypoxia is triggered by the rapid increases in intracellular Ca2þ levels [39,40]. Previous studies have identified and characterized both AP-dependent and APindependent releases during hypoxia and ischemia, which were mainly mediated by Ca2þ influx from the extracellular environment and Ca2þ efflux from intracellular stores, respectively [41e43]. Therefore, during transient (0e5s) hypoxic stimulation, the vesicular exocytosis might be ascribed to the AP-independent mechanism. Compared with amperometric or cell-attached patch
4. Conclusion In conclusion, a novel coupled technique was constructed by assembling an ultra-thin transparent MEA system with TIRFM for simultaneous visualization of APs firing and neurotransmitter release from neuron-like PC12 cells under hypoxic stimulation. Assisted by FFN102, the sequential images in TIRFM provides high spatial and temporal resolution to follow single vesicle fusion, as well as indicating the direction of nerve impulse transmission. The simultaneous recording of both electric activities and neurotransmitter release induced by acute hypoxia with MEA/TIRFM reveals the possible mechanisms that mediate the neurotransmitter release in this model. The proposed platform enables the mapping of neuron connectivity in an entire circuit, which shows promising potential in the study of the nervous system. Declaration of competing interest
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Simultaneous monitoring of action potentials and neurotransmitter release from neuron-like PC12 cells Mei Rong Cui 1, Wei Zhao 1, Xiang Ling Li, Cong Hui Xu**, Jing Juan Xu*, Hong Yuan Chen State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A platform based on ultra-thin microelectrode array and total internal reflection fluorescence microscopy is proposed. The combination of optical and electrical techniques enables completely mapping of neuron connectivity for the first time. Real-time recording of APs and FFN102 release reveals the relevance of electrical and chemical activities of nerve cells.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 October 2019 Received in revised form 12 November 2019 Accepted 21 November 2019 Available online xxx
Simultaneous recording of action potentials (APs) and neurotransmitter release is highly desirable in living neurons since it provides a complete framework of the physiological and pathological statuses of nerve cells. In this work, we proposed an approach coupling ultra-thin microelectrode array (MEA) with total internal reflection fluorescence microscopy (TIRFM), which served as a powerful platform to visualize both APs and vesicular exocytosis in a neuronal circuit model formed by neuron-like PC12 cells. Taking advantages of fluorescent false neurotransmitter (FFN), the transient neurotransmitter transport down an axon could be visualized with high spatial and temporal resolution. The real-time recording of APs burst and neurotransmitter release induced by hypoxia with MEA/TIRFM platform reveals the relevance of electrical and chemical activities in the neuronal model. The combination of the optical and electrical techniques enables mapping of neuron connectivity in an entire neuronal circuit, which may ultimately lead to deeper understanding of nervous system. © 2020 Elsevier B.V. All rights reserved.
Keywords: Action potentials Neurotransmitter MEA/TIRFM
1. Introduction The function of nervous system is intrinsically linked to electrical and chemical impulses, the essential elements for neuronal
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (C.H. Xu), [email protected] (J.J. Xu). 1 These authors contributed equally.
communication [1,2]. Understand the connectivity-map of neuronal circuits and their physiological and pathological functions could help develop better strategies to combat neural disorders [3]. Therefore, it is desirable to achieve visualization of neuronal circuit connectivity, especially precise monitoring of synaptic activity [4,5]. Up to date, the majority of neuronal circuit investigations focus on electrophysiology. Methodologies for recording electrical activity under in vitro or in vivo conditions include sharp micro/ nano-electrodes or patch clamp [6,7], optical imaging using
https://doi.org/10.1016/j.aca.2019.11.074 0003-2670/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: M.R. Cui et al., Simultaneous monitoring of action potentials and neurotransmitter release from neuron-like PC12 cells, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.074
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Scheme 1. Schematic illustration of TIRFM/MEA coupling for simultaneous measurement of APs and neurotransmitter release from neuron-like PC12 cells.
fluorescent indicators [8], as well as the prime technique, substrateintegrated microelectrode arrays (MEAs) [9e11]. Microelectrode arrays provide real-time, nondestructive measurements of the resting and action potential (AP) signals, which enable simultaneous and long-term recording of extracellular potential from a population of neurons at millisecond time scale, thereby offer robust electrophysiological imaging at the network level [12]. In recent years, number of pioneering researches began to merge the advantages of extracellular microelectrode arrays and intracellular microelectrodes towards the imaging of connectivity-map of neuronal circuits [13,14]. On the other hand, to monitor neurosecretion, amperometric measurements with ultra-microelectrodes [15,16], and optical methods [17e19] to observe synaptic vesicle membrane serve as the major technologies. To date, monitoring of neurotransmitter at individual synapses has been achieved using both technologies [20]. Amperometry provides quantitative detection on the amount of electroactive neurotransmitter with high temporal resolution [21]. However, it lacks spatial resolution to monitor the releases of neurotransmitter from different sites in a living cell or neuron. In contrast, fluorescence method can reveal the distribution of secretory vesicles, and can track their motility during the synaptic release in real-time [22e24]. Nevertheless, the optical method lacks temporal resolution. Recently, fluorescent false neurotransmitters (FFNs), as substrates for the synaptic vesicle monoamine transporter, were firstly reported by Sames and Sulzer’s group [25]. Using FFNs, it is possible to measure key presynaptic processes including the accumulation and release of vesicle transporter substrate. Compared to electrical recordings (patch-clamp membrane capacitance and electrochemical amperometry) of neurotransmitter, optical methods using FFNs provided higher spatial resolution [26]. Combined amperometry with total internal
reflection fluorescence microscopy (TIRFM), Guille-Collignon and co-workers have applied FFNs as a fluorescent/electroactive dual probe for coupled measurements of single vesicular exocytosis events [27]. With the growing interest in mapping the physiological or pathological state of neurons, as well as creating experimental therapy and novel neuroprosthetic devices, it is highly desirable to develop methods for simultaneous recording of both AP waveforms and release of neurotransmitters in an entire living neuron or neural model, as they provide a complete framework of the neuron activities. To achieve such goal, we developed a system for simultaneous monitoring of APs and release of neurotransmitters from living neuron-like rat pheochromocytoma (PC12) cells based on ultrathin transparent MEA (60 TiN microelectrodes) coupled with TIRFM. MEA with ultra-thin thickness of 180 mm facilitated TIRFM observation. As shown in Scheme 1, PC12 cells were in-situ cultured and differentiated with nerve growth factor (NGF) to resemble a dopaminergic neuronal phenotype. Then FFN102, a synthesized analogue of biogenic neurotransmitters, was applied to stain neuron-like PC12 cells and served as an indicator of the neurotransmitter, dopamine. Under external hypoxia stimulation, the APs firing and exocytosis were simultaneously recorded by MEA and TIRFM, respectively, with high temporal and spatial resolutions. And the recorded signals could be coupled in time and space. The proposed MEA/TIRFM coupling platform provides promising opportunity of stimulation and recording of brain circuit activity to understand the molecular and cellular basis of health problems.
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2. Experimental section 2.1. Chemicals and materials FFN102 was purchased from Aabcam (Cambridge, UK). Laminin from Engelbreth-Holm-Swam and NGF-b from rat were purchased from Sigma-Aldrich. Poly-L-lysine (PLL) was purchased from Merck KGaA (Germany). Fe12K nutrient mixture, horse serum, fetal bovine serum (FBS) and Celltraker™ green CMFDA are from Gibco Life Technologies. Undifferentiated rat pheochromocytoma (PC12) were purchased from Cellbank (China). All experiments using living cells were performed in phosphate buffer solution (pH 7.4) prepared using double distilled water from a Millipore purification system (18.2 MU cm at 25 C). 2.2. MEA type and layout To fulfill the requirement of high-resolution total internal reflection fluorescence microscopy (TIRFM) imaging, microelectrode array (MEA) with 60 microelectrodes arranged in an 8 8 layout grid embedded in a transparent glass substrate with thickness of 180 mm was purchased from Harvard Bioscience Inc. The MEA type is briefly described as follows: - 180 mm thin recording area - TiN electrodes - SiN isolator
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ITO conductive strips With 1 internal reference electrode Electrode grid 8 8 59 recording electrodes Electrode spacing 100 mm Electrode diameter 10 mm Glass ring 6 mm high (other heights on request).
2.3. Apparatus for characterization Action potential waveforms were performed using MEA 2100System (Harvard Bioscience Inc.) consisted of MEA, interface board, PC with software and temperature controller. The real-time data was collected with the interface board. A low-pass filter with a maximum frequency of 5000 Hz was applied after data acquisition using MATLAB R2016a software, which enables the noise analysis (SNR) and statistical event frequency analysis. Time-lapse imaging was acquired by TIRFM (Olympus, Japan) equipped with a Plan Apochromat 63 /1.4 NA oil immersion objective. In TIRFM, a 405 nm digital laser system employed as the light source to initiate the fluorescence whereas the emission light was filtered before being detected by Prime 95B SCMOS camera (Olympus, Japan). Confocal fluorescence images of cells were acquired with a TCS SP5 confocal microscopy (Leica, Germany).
Fig. 1. (A) Photograph of 60 ThinMEA. (B) Bright field image of 8 8 layout electrodes (scar bar: 100 mm). (C) Confocal image of neuron-like PC12 cells cultured on MEA stained by CelltrakerTM green CMFDA (scar bar: 100 mm). (D) Voltage traces of spontaneous APs at 10 electrodes marked with dashed rectangle in fluorescent image (scar bars: 10 ms horizontal and 50 mV vertical). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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TIRFM. The electrophysiology of the neural network and the release of neurotransmitters from axon varicosities or axon terminals were triggered by adding hypoxic buffer, which was prepared by continuously bubbling 0.1 PBS buffer, sealed in a clean glass flask with pure N2 gas (purity: 5 N) for 30 min. SCMOS and MEA2100System are synchronized by the Digital delay/pulse generator DG535 (Stanford Research Systems, Inc.) with two simultaneous TTL signals to trigger the recording. The rise time of the TTL signal is less than 3 ns and the time delay of the two TTL signals is ca. 5 ps. 2.7. Optical characterization
Fig. 2. Long-term recording of APs from the neuron-like PC12 cells cells on one of the microelectrodes after adding hypoxic buffer (A) and normal PBS solution without anoxic treatment (B).
2.4. MEA pretreatment Before cells culture on the MEA, it was pretreated according to the following process. First, it was bathed in absolute ethanol for 20 min, then rinsed with distilled water and dried in the air. Second, 75% alcohol was applied for disinfection. Third, after naturally dried on ultra-clean table, we applied UV to irradiate the MEA for 6 h (3 h on each side). Forth, 500 m L of 0.1 mg/mL PLL solution was added in the culture chamber, and the MEA was placed in an incubator MCO15AC (Sanyo, Tokyo, Japan) at 37 C, 5% CO2 and saturated humidity for 1 h. Then, it was taken out of the incubator and thoroughly rinsed with distilled water for three times. Finally, it was incubated with laminin at 4 C overnight, and then could be used for cell seeding. 2.5. Cell culture and sample preparation Neuron-like PC12 cells were maintained in Fe12K Nutrient Mixture supplemented with 5% FBS, 10% Horse Serum (Heat-Inactivated) 100 U/mL penicillin and 100 mg/mL streptomycin at 37 C in a 5% CO2-95% air incubator MCO-15AC (Sanyo, Tokyo, Japan). In order to acquire neuron-like PC12 cells model on MEA or the glassbottomed Petri dishes, confluent undifferentiated PC12 cells(105 cells/mL) were counted and cultured in fresh Fe12K Nutrient Mixture supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 C in a 5% CO2 humidified atmosphere. For differentiation, cells were added with nerve growth factor (NGF) (with the final concentration of 50 ng/ml) and cultured for 6 days. 2.6. Simultaneous visualization of AP waveforms and release of neurotransmitters by MEA/TIRFM After neural network cultured on the ultra-thin transparent MEA, we implemented simultaneous visualization of action potential waveforms and releasing of neurotransmitters by MEA/
To map the growth-form of neuron-like PC12 cells on transparent MEA, the cells were stained by celltraker™ green CMFDA for 10 min and subsequently imaged by the TCS SP5 confocal microscopy. UVeVis absorption spectra of FFN102 was measured at room temperature on an UVeVis spectrophotometer (UV-3600; Shimadzu Co., Japan). The absorption spectra were recorded from 270 nm to 440 nm with an interval of 1 nm at a scan rate of 1200 nm/min. Fluorescence measurements were acquired on the F7000 (Hitachi) fluorescence spectrometer with a Xeon lamp. For emission scans, the excitation wavelength was set to the maximum absorption wavelength on the basis of UVeVis spectra and the emission wavelengths were scanned from 400 nm to 600 nm with an interval of 1 nm at a scan rate of 1200 nm/min. The emission wavelength was set at 456 nm to record the excitation spectra of FFN102. 2.8. Confocal laser scanning microscopy imaging PC12 cells were digested and incubated in confocal cell dishes overnight. Then, the cells were incubated with FFN102 for 30 min. After that, the cells were washed for three times and confocal laser scanning microscopy (CLSM) images of PC12 cells were acquired. 3. Result and discussion 3.1. Characterization of neuronal circuit model on MEA The implementation of MEA/TIRFM coupling measurements relies mainly on the specific property of the innovative ultra-thin transparent MEA (8 8 layout grid, 10 mm electrode diameter, 100 mm electrode spacing, including one reference electrode) (Fig. 1A and B). The thin MEA is fabricated with ultra-thin optical glass with 180 mm thickness and transparent indium-tin oxide wires embedded as conductive strips, which is ideally suited for high-resolution TIRFM imaging. NGF-differentiated neuron-like PC12 cells were cultured on MEA for 6 days. As well known, differentiated PC12 cells could serve as model for neurosecretion since they closely resemble neurons with smaller vesicle [28e30]. Fig. 1C provides fluorescent image of the neuron-like PC12 cells on MEA. Induced by NGF, PC12 cells show obvious cell adhesion, spreading and neurite outgrowth on the substrate (Fig. S1). The AP waveforms shown in Fig. 1D are the realtime recording data without filtering. The spontaneous APs show typical waveform with 500e2000 ms pulse duration and a 50e200 mV amplitude (Fig. S2), which are consistent with previous research results. 3.2. Hypoxia evokes membrane potential depolarization Adequate oxygen is essential for the survival of mammalian cells. Hypoxic stimulation could inhibit O2-sensitive Kþ channels and consequently trigger the membrane potential depolarization [31]. As PC12 cells contain O2-sensitive channels, they could serve
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Fig. 3. (A) TIRFM image of neuron-like PC12 cells stained by FFN102. (B) Sequential pseudocolor images of the area marked with the red rectangle in (A), unit: second. (C) and (D) An individual exocytotic release (fluorescence trace and TIRMF images) from a single fluorescent vesicle indicated by the red circle in (A). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. Time-lapse recording of pseudo color TIRFM images of neuron-like PC 12 cells after adding hypoxic buffer (A) and PBS without anoxic treatment (B), and corresponding fluorescent trace curves (C). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 5. (A) Bright image of NGF-differentiated PC 12 cells cultured on MEA. (B) TIRFM image of the red rectangle in (A). (C) AP waveforms on electrodes shown in the area of (A) at 0.00 s, 20.04 s, 45.40 s. (D) Statistical analysis of the number of APs on 24 microelectrodes in 60 s after hypoxic stimulation. (E) Fluorescent trace (blue curve) and APs waveform (red curve) obtained from FFN102 stained PC 12 neural model indicated by rectangle in (B), and electrode G2, immediately after hypoxic stimulation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
as a chemosensory model for hypoxia [32]. Herein, hypoxic stimulation was applied to investigate the cellular effect by simultaneous monitoring of action potentials and neurotransmitter release from neuron-like PC12 cells. O2 reduced hypoxic buffer was added in the culture chamber to achieve 70% hypoxia ratio, which was sufficient to trigger the membrane potential depolarization [33]. The AP waveforms were recorded in real-time. As shown in the voltage trace in Fig. 2A, after addition of hypoxic buffer, the APs firing frequency recorded by a single electrode shows considerable increase at approximately 5 s, and gradually decreases after 30 s, which consistent with previous works [34]. On the other hand, in the control experiment, no obvious change of spontaneous APs frequency was observed in 60 s after the addition of phosphate buffer without anoxic treatment (Fig. 2B). 3.3. Hypoxia evokes vesicular neurotransmitter release To track the vesicular exocytosis in real-time, the pH-responsive fluorescence substrate, FFN102, which selectively labels secretory vesicles was applied to stain the neuron-like PC12 cells on MEA and the pH-dependent optical properties of FFN102 were examined first (Fig. S3). Considering the pH gradient between the vesicular lumen (pH 5e6) and the extracellular medium (pH 7.4), FFN102 can act as an optical tracer to visualize neurotransmitter release from individual presynaptic terminals [35]. After 30 min incubation in
10 mM FFN102, fluorescent vesicles (300e400 nm in diameter) could be observed in the cell body, axon varicosities and axon terminals (Fig. S4). As Fig. S5 shown, the vesicles adjacent to the cell membrane were presented as stable blue spot under 405 nm laser, so 405 nm laser was selected as the light source to monitor the neurotransmitter release. As reported, hypoxia evokes dopamine secretion from undifferentiated PC12 cells via a mechanism involving Ca2þ influx [32]. Initiated by reducing the concentration of O2 to more than 70%, dopamine release events from neuron-like PC12 cells could be visualized (Fig. S6A, Movie S1), which were easily observed as sparkling fluorescent spots at both cell bodies and neurites when FFN102 diffused from acidic vesicle to the neutral extracellular medium. During transient (30 s) hypoxic stimulation, large amplitude fluorescent spikes were obtained in selected areas of the neuronal network. In the control measurement, no obvious change of fluorescent intensity was observed within 60 s after adding same volume of phosphate buffer without anoxic treatment (Movie S2, Fig. S6B). There are two possible reasons. First, without hypoxic or chemical stimulations, the spontaneous neurotransmitter release with extremely low frequency [36,37] didn’t occur in such short time. Second, the amount of release is low, which hasn’t been captured. Supplementary data related to this article can be found at https://doi.org/10.1016/j.aca.2019.11.074.
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A section of the neuronal model including a long axon is chosen as an example to demonstrate the TIRFM imaging of vesicular exocytosis events with distinct spatial and temporal resolutions (Fig. 3A). As shown in Fig. 3B, FFN102 released with fluorescent signal transiently brightened and disappeared on different sites along the axon (15 mm), in sequence. The direction of nerve impulse transmission could be recognized based on the real-time recording. Furthermore, TIRFM provides high resolution to follow single vesicle fusion. As shown in Fig. 3C and D, single exocytotic event could be captured and visualized within 200e300 ms. Under hypoxic stimulation, the total fluorescent intensity of the stained PC12 neuronal network gradually decreased with time elapse (Fig. 4A), but it remained stable in the control experiment (Fig. 4B), which are consisted with the time-lapse fluorescent curves (Fig. 4C). On the basis of real-time imaging by TIRFM, many important cellular activities including vesicle motility, location of releasing sites and the transmission directions could be visualized with high spatial resolution. Combining TIRFM with transparent MEA enables more a comprehensive information acquisition for the neuron communication.
electrodes, MEA/TIRFM offers a more direct and visualized approach to discover the mechanisms in neuronal communication.
3.4. Simultaneous monitoring of AP waveforms and neurotransmitter release
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.
Two types of spontaneous neurotransmitter release are present in the nervous system, namely AP-dependent release and APindependent release [38]. Simultaneous monitoring of APs and neurotransmitter release may help neuroscientists discovering the underlying mechanisms of neuron conductivity under different types of stimulation, including hypoxia or ischemia. Based on the MEA/TIRFM equipment, simultaneous measurement of APs and dopamine release was performed. As shown in the bright image of MEA (Fig. 5A), neuron-like PC12 cells are growing on the substrate, and partially located on the electrodes. Part of the network containing axon varicosities and terminals was selected for real-time TIRFM imaging (Fig. 5B). The electric activities and FFN102 release events were simultaneously recorded after acute hypoxic stimulation. Fig. 5C shows the voltage traces on part of the microelectrodes of MEA at 0.00 s, 20.04 s and 45.40 s. Compared with the voltage traces at initial time, the frequency change of APs spikes is significant at ca. 20 s, but sharply reduced at 45 s. The frequency of APs on 24 microelectrodes during hypoxic stimulation are statistically analyzed. As the results shown in Fig. 5D, a sudden increase is observed from 5 to 10 s, after 30 s, the number drops significantly. To compare the APs firing and neurotransmitter release, we choose the APs recording from electrode G2 (indicated by red circle in Fig. 5A), which located under a cell body and fluorescence signal derived from the axon varicosity extended from that cell body. As shown in Fig. 5E, fluorescent events occurred immediately after hypoxic stimulation, and decreased after 20e30 s (Movie S3). However, similar to the APs firing statistically analyzed from 24 electrodes, intensive AP firing showed at ca. 5 s at electrode G2. The synaptic release occurs earlier than APs firing. Supplementary data related to this article can be found at https://doi.org/10.1016/j.aca.2019.11.074. It is well agreed that the release of neurotransmitters including dopamine and glutamate under acute hypoxia is triggered by the rapid increases in intracellular Ca2þ levels [39,40]. Previous studies have identified and characterized both AP-dependent and APindependent releases during hypoxia and ischemia, which were mainly mediated by Ca2þ influx from the extracellular environment and Ca2þ efflux from intracellular stores, respectively [41e43]. Therefore, during transient (0e5s) hypoxic stimulation, the vesicular exocytosis might be ascribed to the AP-independent mechanism. Compared with amperometric or cell-attached patch
4. Conclusion In conclusion, a novel coupled technique was constructed by assembling an ultra-thin transparent MEA system with TIRFM for simultaneous visualization of APs firing and neurotransmitter release from neuron-like PC12 cells under hypoxic stimulation. Assisted by FFN102, the sequential images in TIRFM provides high spatial and temporal resolution to follow single vesicle fusion, as well as indicating the direction of nerve impulse transmission. The simultaneous recording of both electric activities and neurotransmitter release induced by acute hypoxia with MEA/TIRFM reveals the possible mechanisms that mediate the neurotransmitter release in this model. The proposed platform enables the mapping of neuron connectivity in an entire circuit, which shows promising potential in the study of the nervous system. Declaration of competing interest
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21327902, 21535003, and 21605079) of China, the Natural Science Foundation (BK20160637) of Jiangsu Province, and Fundamental Research Funds for the Central Universities. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.11.074. References [1] N. Kriegeskorte, R. Goebel, P. Bandettini, Information-based functional brain mapping, Proc. Natl. Acad. Sci. U.S.A. 103 (2006) 3863e3868. [2] R.G. Andrzejak, K. Lehnertz, F. Mormann, C. Rieke, P. David, C.E. Elger, Indications of nonlinear deterministic and finite-dimensional structures in time series of brain electrical activity: dependence on recording region and brain state, Phys. Rev. 64 (2001), 061907. [3] K. Zhang, Q. Yang, Z. Fan, J. Zhao, H. Li, Platelet-driven formation of interface peptide nano-network biosensor enabling a non-invasive means for early detection of Alzheimer’s disease, Biosens. Bioelectron. 145 (2019), 111701111701. [4] Y.T. Li, S.H. Zhang, X.Y. Wang, X.W. Zhang, A.I. Oleinick, I. Svir, C. Amatore, W.H. Huang, Real-time monitoring of discrete synaptic release events and excitatory potentials within self-reconstructed neuromuscular junctions, Angew. Chem. Int. Ed. 54 (2015) 9313e9318. [5] Y. Xu, L. Peng, S. Wang, A. Wang, R. Ma, Y. Zhou, J. Yang, D.E. Sun, W. Lin, X. Chen, P. Zou, Hybrid indicators for fast and sensitive voltage imaging, Angew. Chem. Int. Ed. 57 (2018) 3949e3953. [6] H.G. Jahnke, D. Krinke, D. Seidel, K. Lilienthal, S. Schmidt, R. Azendorf, M. Fischer, T. Mack, F. Striggow, H. Althaus, A. Schober, A.A. Robitzki, A novel 384-multiwell microelectrode array for the impedimetric monitoring of Tau protein induced neurodegenerative processes, Biosens. Bioelectron. 88 (2017) 78e84. [7] Y. Mourzina, D. Kaliaguine, P. Schulte, A. Offenhaeusser, Patterning chemical stimulation of reconstructed neuronal networks, Anal. Chim. Acta 575 (2006) 281e289. [8] R. Chereau, G.E. Saraceno, J. Angibaud, D. Cattaert, U.V. Nagerl, Superresolution imaging reveals activity-dependent plasticity of axon morphology linked to changes in action potential conduction velocity, Proc. Natl. Acad. Sci. U.S.A. 114 (2017) 1401e1406. [9] C. Kokkinos, A. Economou, I. Raptis, Microfabricated disposable lab-on-a-chip sensors with integrated bismuth microelectrode arrays for voltammetric determination of trace metals, Anal. Chim. Acta 710 (2012) 1e8.
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