An In Vitro Assessment Method for Chemotherapy-Induced Peripheral Neurotoxicity Caused by Anti-Cancer Drugs Based on Electrical Measurement of Impedance Value and Spontaneous Activity.

Chemotherapy-induced peripheral neurotoxicity (CIPN) is a major adverse event of anti-cancer drugs, which still lack standardized measurement and treatment methods. In the present study, we attempted to evaluate neuronal dysfunctions in cultured rodent primary peripheral neurons using a microelectrode array system. After exposure to typical anti-cancer drugs (i.e., paclitaxel, vincristine, oxaliplatin, and bortezomib), we successfully detected neurotoxicity in dorsal root ganglia neurons by measuring electrical activities, including impedance value and spontaneous activity. The impedance value decreased significantly for all compounds, even at low concentrations, which indicated cell loss and/or neurite degeneration. The spontaneous activity was also suppressed after exposure, which suggested neurotoxicity again. However, an acute response was observed for paclitaxel and bortezomib before toxicity, which showed different mechanisms based on compounds. Therefore, MEA measurement of impedance value could provide a simple assessment method for CIPN, combined with neuronal morphological changes.

Neuroprotective Potential of L-Glutamate Transporters in Human Induced Pluripotent Stem Cell-Derived Neural Cells against Excitotoxicity.

Human induced pluripotent stem cell (hiPSC)-derived neural cells have started to be used in safety/toxicity tests at the preclinical stage of drug development. As previously reported, hiPSC-derived neurons exhibit greater tolerance to excitotoxicity than those of primary cultures of rodent neurons; however, the underlying mechanisms remain unknown. We here investigated the functions of L-glutamate (L-Glu) transporters, the most important machinery to maintain low extracellular L-Glu concentrations, in hiPSC-derived neural cells. We also clarified the contribution of respective L-Glu transporter subtypes. At 63 days in vitro (DIV), we detected neuronal circuit functions in hiPSC-derived neural cells by a microelectrode array system (MEA). At 63 DIV, exposure to 100 µM L-Glu for 24 h did not affect the viability of neural cells. 100 µM L-Glu in the medium decreased to almost 0 µM in 60 min. Pharmacological inhibition of excitatory amino acid transporter 1 (EAAT1) and EAAT2 suppressed almost 100% of L-Glu decrease. In the presence of this inhibitor, 100 µM L-Glu dramatically decreased cell viability. These results suggest that in hiPSC-derived neural cells, EAAT1 and EAAT2 are the predominant L-Glu transporters, and their uptake potentials are the reasons for the tolerance of hiPSC-derived neurons to excitotoxicity.

Evaluation of neurotoxicity for pesticide-related compounds in human iPS cell-derived neurons using microelectrode array.

In vivo evaluations of chemicals in neurotoxicity have certain limitations due to the considerable time and cost required, necessity of extrapolation from rodents to humans, and limited information on toxicity mechanisms. To address this issue, the development of in vitro test methods using new approach methodologies (NAMs) is important to evaluate the chemicals in neurotoxicity. Microelectrode array (MEA) allows the assessment of changes in neural network activity caused by compound administration. However, studies on compound evaluation criteria are scarce. In this study, we evaluated the impact of pesticides on neural activity using MEA measurements of human iPSC-derived neurons. A principal component analysis was performed on the electrical physiological parameters obtained by MEA measurements, and the influence of excessive neural activity due to compound addition was defined using the standard deviation of neural activity with solvent addition as the reference. By using known seizurogenic compounds as positive controls for neurotoxicity in MEA and evaluating pesticides with insufficient verification of their neurotoxicity in humans, we demonstrated that these pesticides exhibit neurotoxicity in humans. In conclusion, our data suggest that the neurotoxicity evaluation method in human iPSC neurons using MEA measurements could be one of the in vitro neurotoxicity test methods that could replace animal experiments.

Large-Area Field Potential Imaging Having Single Neuron Resolution Using 236 880 Electrodes CMOS-MEA Technology.

The electrophysiological technology having a high spatiotemporal resolution at the single-cell level and noninvasive measurements of large areas provide insights on underlying neuronal function. Here, a complementary metal-oxide semiconductor (CMOS)-microelectrode array (MEA) is used that uses 236 880 electrodes each with an electrode size of 11.22 × 11.22 µm and 236 880 covering a wide area of 5.5 × 5.9 mm in presenting a detailed and single-cell-level neural activity analysis platform for brain slices, human iPS cell-derived cortical networks, peripheral neurons, and human brain organoids. Propagation pattern characteristics between brain regions changes the synaptic propagation into compounds based on single-cell time-series patterns, classification based on single DRG neuron firing patterns and compound responses, axonal conduction characteristics and changes to anticancer drugs, and network activities and transition to compounds in brain organoids are extracted. This detailed analysis of neural activity at the single-cell level using the CMOS-MEA provides a new understanding of the basic mechanisms of brain circuits in vitro and ex vivo, on human neurological diseases for drug discovery, and compound toxicity assessment.

A functional neuron maturation device provides convenient application on microelectrode array for neural network measurement.

BACKGROUND: Microelectrode array (MEA) systems are valuable for in vitro assessment of neurotoxicity and drug efficiency. However, several difficulties such as protracted functional maturation and high experimental costs hinder the use of MEA analysis requiring human induced pluripotent stem cells (hiPSCs). Neural network functional parameters are also needed for in vitro to in vivo extrapolation. METHODS: In the present study, we produced a cost effective nanofiber culture platform, the SCAD device, for long-term culture of hiPSC-derived neurons and primary peripheral neurons. The notable advantage of SCAD device is convenient application on multiple MEA systems for neuron functional analysis. RESULTS: We showed that the SCAD device could promote functional maturation of cultured hiPSC-derived neurons, and neurons responded appropriately to convulsant agents. Furthermore, we successfully analyzed parameters for in vitro to in vivo extrapolation, i.e., low-frequency components and synaptic propagation velocity of the signal, potentially reflecting neural network functions from neurons cultured on SCAD device. Finally, we measured the axonal conduction velocity of peripheral neurons. CONCLUSIONS: Neurons cultured on SCAD devices might constitute a reliable in vitro platform to investigate neuron functions, drug efficacy and toxicity, and neuropathological mechanisms by MEA.

Responses to antibiotics in human iPSC-derived neurons based on the clinical antibiotic-associated encephalopathy classification.

Antibiotic-associated encephalopathy (AAE) is a central nervous system disorder caused by antibiotics administration and classified into three types based on clinical symptoms. Type 1 AAE causes seizures and myoclonus, type 2 causes psychiatric symptoms, and type 3 is characterized by cerebellar ataxia. In this study, we investigated whether the electrical activity of in vitro human iPSC-derived neurons to antibiotics could be classified based on the 3 types of AAEs classified by clinical symptoms. Glutamatergic, GABAergic neurons and astrocytes differentiated from human iPS cells were seeded on micro-electrode array (MEA). The cumulative administration of 13 different antimicrobials detected changes in neural activity that differed according to AAE type. Next, we classified the antimicrobials by principal component analysis (PCA) and confirmed the AAE type of each agent. We found that Types 1-3 AAE agents were distributed separately. The classification of antibiotics depending on electrophysiological response characteristics was consistent with the clinical practice classification of AAEs. In conclusion, the combination of electrophysiological responses of human iPS cell-derived neural networks measured by MEA plus multivariate analysis methods will effectively detect and classify antibiotics developmental risks.

In Vitro Pain Assay Using Human iPSC-Derived Sensory Neurons and Microelectrode Array.

Drug-induced peripheral neuropathy occurs as an adverse reaction of chemotherapy. However, a highly accurate method for assessing peripheral neuropathy and pain caused by compounds has not been established. The use of human-induced pluripotent stem cell (hiPSC)-derived sensory neurons does not require animal experiments, and it is considered an effective method that can approach extrapolation to humans. In this study, we evaluated the response to pain-related compounds based on neural activities using in vitro microelectrode array (MEA) measurements in hiPSC-derived sensory neurons. Cultured sensory neurons exhibited gene expression of the Nav1.7, TRPV1, TRPA1, and TRPM8 channels, which are typical pain-related channels. Channel-dependent evoked responses were detected using the TRPV1 agonist capsaicin, a TRPA1 agonist, allyl isothiocyanate (AITC), and TRPM8 agonist menthol. In addition, the firing frequency increased with an increase in temperature from 37°C to 46°C, and temperature sensitivity was observed. In addition, the temperature of the peak firing rate differed among individual neurons. Next, we focused on the increase in cold sensitivity, which is a side effect of the anticancer drug oxaliplatin, and evaluated the response to AITC in the presence and absence of oxaliplatin. The response to AITC increased in the presence of oxaliplatin in a concentration-dependent manner, suggesting that the increased cold sensitivity in humans can be reproduced in cultured hiPSC-derived sensory neurons. The in vitro MEA system using hiPSC-derived sensory neurons is an alternative method to animal experiments, and it is anticipated as a method for evaluating peripheral neuropathy and pain induced by compounds.

Detection of astrocytic slow oscillatory activity and response to seizurogenic compounds using planar microelectrode array.

Since the development of the planar microelectrode array (MEA), it has become popular to evaluate compounds based on the electrical activity of rodent and human induced pluripotent stem cell (iPSC)-derived neurons. However, there are no reports recording spontaneous human astrocyte activity from astrocyte-only culture sample by MEA. It is becoming clear that astrocytes play an important role in various neurological diseases, and astrocytes are expected to be excellent candidates for targeted therapeutics for the treatment of neurological diseases. Therefore, measuring astrocyte activity is very important for drug development for astrocytes. Recently, astrocyte activity has been found to be reflected in the low-frequency band < 1 Hz, which is much lower than the frequency band for recording neural activity. Here, we separated the signals obtained from human primary astrocytes cultured on MEA into seven frequency bands and successfully recorded the extracellular electrical activity of human astrocytes. The slow waveforms of spontaneous astrocyte activity were observed most clearly in direct current potentials < 1 Hz. We established nine parameters to assess astrocyte activity and evaluated five seizurogenic drug responses in human primary astrocytes and human iPSC-derived astrocytes. Astrocytes demonstrated the most significant dose-dependent changes in pilocarpine. Furthermore, in a principal component analysis using those parameter sets, the drug responses to each seizurogenic compound were separated. In this paper, we report the spontaneous electrical activity measurement of astrocytes alone using MEA for the first time and propose that the MEA measurement focusing on the low-frequency band could be useful as one of the methods to assess drug response in vitro.

Constructing the 'automatic' Greenwich time system: George Biddell Airy and the telegraphic distribution of time, c.1852-1880.

In the context of the telegraphic distribution of Greenwich time, while the early experiments, the roles of successive Astronomers Royal in its expansion, and its impacts on the standardization of time in Victorian Britain have all been evaluated, the attempts of George Biddell Airy and his collaborators in constructing the Royal Observatory's time signals as the authoritative source of standard time have been underexplored within the existing historical literature. This paper focuses on the wide-ranging activities of Airy, his assistant astronomers, telegraph engineers, clockmakers and others, which served to increase the reliability of the Royal Observatory's time service between the 1850s and 1870s. Airy and his collaborators aimed to mechanize and automate their telegraphic time distribution system in order to improve its accuracy and reliability. The accomplishment of such technological innovations was disseminated via public lectures, journal articles and correspondence with experts, secondary distributors of standard time and the general public. These communications were used to build public trust in the Greenwich time service. However, the unexplored archival material used in the present paper provides fresh insight into the unstable nature of the Greenwich time system, including its clear limits in terms of its scale of automation and degree of accuracy.

In pursuit of accurate timekeeping: Liverpool and Victorian electrical horology.

This paper explores how nineteenth-century Liverpool became such an advanced city with regard to public timekeeping, and the wider impact of this on the standardisation of time. From the mid-1840s, local scientists and municipal bodies in the port city were engaged in improving the ways in which accurate time was communicated to ships and the general public. As a result, Liverpool was the first British city to witness the formation of a synchronised clock system, based on an invention by Robert Jones. His method gained a considerable reputation in the scientific and engineering communities, which led to its subsequent replication at a number of astronomical observatories such as Greenwich and Edinburgh. As a further key example of developments in time-signalling techniques, this paper also focuses on the time ball established in Liverpool by the Electric Telegraph Company in collaboration with George Biddell Airy, the Astronomer Royal. This is a particularly significant development because, as the present paper illustrates, one of the most important technologies in measuring the accuracy of the Greenwich time signal took shape in the experimental operation of the time ball. The inventions and knowledge which emerged from the context of Liverpool were vital to the transformation of public timekeeping in Victorian Britain.