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Papers - Hotta, Kohji

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Cellular thermogenesis compensates environmental temperature fluctuations for maintaining intracellular temperature

Yamanaka R., Shindo Y., Hotta K., Hiroi N., Oka K.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications) 533 ( 1 ) 70 - 76 2020.11

ISSN 0006291X

Temperature governs states and dynamics of all biological molecules, and several cellular processes are often heat sources and/or sinks. Technical achievement of intracellular thermometry enables us to measure intracellular temperature, and it can offer novel perspectives in biology and medicine. However, little is known that changes of intracellular temperature throughout the cell-cycle and the manner of which cells regulates their thermogenesis in response to fluctuation of the environmental temperature. Here, cell-cycle-dependent changes of intracellular temperature were reconstructed from the snapshots of cell population at single-cell resolution using ergodic analysis for asynchronously cultured HeLa cells expressing a genetically encoded thermometry. Intracellular temperature is highest at G1 phase, and it gradually decreases along cell-cycle progression and increases abruptly during mitosis. Cells easily heated up are harder to cool down and vice versa, especially at G1/S phases. Together, intracellular thermogenesis depends on cell-cycle phases and it maintains intracellular temperature through compensating environmental temperature fluctuations.

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Different strategies for tissue scaling in dwarf tailbud embryos revealed by single-cell analysis

Matsumura K.D., Nakamura M.J., Koizumi W.C., Hotta K., Oka K.

Developmental Biology (Developmental Biology) 460 ( 2 ) 215 - 223 2020.04

ISSN 00121606

The tailbud stage is part of the organogenesis period—an evolutionarily conserved developmental period among chordates that is essential for determining the characteristics of the chordate body plan. When the volume of the egg is artificially decreased by cutting, ascidians produce a normal-looking but miniature (dwarf) tailbud embryo. Although cell lineages during ascidian embryogenesis are invariant, the number of cell divisions in the dwarf embryo is altered by a different mechanism in each tissue (Yamada and Nishida, 1999). Here, to elucidate the size-regulation strategies of the Ciona robusta dwarf tailbud embryo, we compared anatomical structure, developmental speed, and cell number/volume in each tissue between dwarf and wild type (WT) embryos. To do this, we constructed a 3D virtual mid-tailbud embryo (Nakamura et al., 2012). We could make a Ciona dwarf tailbud embryo from eggs with a diameter over 108 μm (correspond to > 40% of the wild type egg volume). The timings of cleavage (~St. 12) and subsequent morphogenesis were nearly the same but blastomeres of animal hemisphere slightly delayed the timing of mitosis in the early cleavage period. Intriguingly, the tissue-to-tissue volume ratios of dwarf tailbud embryos were similar to those of wild type embryos suggesting that the ratio of tissue volumes is essential for maintaining the proper shape of the tailbud embryo. The number of cells in the epidermis, nervous system, and mesenchyme was significantly reduced in the dwarf embryos whereas the cell volume distribution of these tissues was similar in the dwarf and wild type. In contrast, the number of cells in the notochord, muscle, heart, and endoderm were maintained in the dwarf embryos; cell volumes were significantly reduced. Neither parameter changed in germline precursors. These results indicate that each tissue uses different scaling strategies to coordinate cell number and cell volume in accordance with the embryo size.

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Neural properties of fundamental function encoding of sound selectivity in the female avian auditory cortex

Inda M., Hotta K., Oka K.

European Journal of Neuroscience (European Journal of Neuroscience) 51 ( 8 ) 1770 - 1783 2020.04

ISSN 0953816X

Zebra finches (Taeniopygia guttata) use their voices for communication. Song structures in the songs of individual males are important for sound recognition in females. The caudomedial mesopallium (CMM) and nidopallium (NCM) are known to be essential higher auditory regions for sound recognition. These two regions have also been discussed with respect to their fundamental functions and song selectivity. To clarify their functions and selectivity, we investigated latencies and spiking patterns and also developed a novel correlation analysis to evaluate the relationship between neural activity and the characteristics of acoustic factors. We found that the latencies and spiking patterns in response to song stimuli differed between the CMM and NCM. In addition, our correlation analysis revealed that amplitude and frequency structures were important temporal acoustic factors for both regions. Although the CMM and NCM have different fundamental functions, they share similar encoding systems for acoustic factors.

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Optical Dissection of Synaptic Plasticity for Early Adaptation in Caenorhabditis elegans

Ashida K., Shidara H., Hotta K., Oka K.

Neuroscience (Neuroscience) 428 112 - 121 2020.01

ISSN 03064522

To understand neuronal information processing, it is essential to investigate the input–output relationship and its modulation via detailed dissections of synaptic transmission between pre- and postsynaptic neurons. In Caenorhabditis elegans, pre-exposure to an odorant for five minutes reduces chemotaxis (early adaptation). AWC sensory neurons and AIY interneurons are crucial for this adaptation; AWC neurons sense volatile odors, and AIY interneurons receive glutamatergic inputs from AWC neurons. However, modulations via early adaptation of the input–output relationship between AWC and AIY are not well characterized. Here we use a variety of fluorescent imaging techniques to show that reduced synaptic-vesicle release without Ca2+ modulation in AWC neurons suppresses the Ca2+ response in AIY neurons via early adaptation. First, early adaptation modulates the Ca2+ response in AIY but not AWC neurons. Adaptation in the Ca2+ signal measured in AIY neurons is caused by adaptation in glutamate release from AWC neurons. Further, we found that a G protein γ-subunit, GPC-1, is related to modulation of glutamate input to AIY. Our results dissect the modulation of the pre- and postsynaptic relationship in vivo based on optical methods, and demonstrate the importance of neurotransmitter-release modulation in presynaptic neurons without Ca2+ modulation.

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Inhibition of mg2+ extrusion attenuates glutamate excitotoxicity in cultured rat hippocampal neurons

Shindo Y., Yamanaka R., Hotta K., Oka K.

Nutrients (Nutrients) 12 ( 9 ) 1 - 14 2020

Magnesium plays important roles in the nervous system. An increase in the Mg2+ concentration in cerebrospinal fluid enhances neural functions, while Mg2+ deficiency is implicated in neuronal diseases in the central nervous system. We have previously demonstrated that high concentrations of glutamate induce excitotoxicity and elicit a transient increase in the intracellular concentration of Mg2+ due to the release of Mg2+ from mitochondria, followed by a decrease to below steady-state levels. Since Mg2+ deficiency is involved in neuronal diseases, this decrease presumably affects neuronal survival under excitotoxic conditions. However, the mechanism of the Mg2+ decrease and its effect on the excitotoxicity process have not been elucidated. In this study, we demonstrated that inhibitors of Mg2+ extrusion, quinidine and amiloride, attenuated glutamate excitotoxicity in cultured rat hippocampal neurons. A toxic concentration of glutamate induced both Mg2+ release from mitochondria and Mg2+ extrusion from cytosol, and both quinidine and amiloride suppressed only the extrusion. This resulted in the maintenance of a higher Mg2+ concentration in the cytosol than under steady-state conditions during the ten-minute exposure to glutamate. These inhibitors also attenuated the glutamate-induced depression of cellular energy metabolism. Our data indicate the importance of Mg2+ regulation in neuronal survival under excitotoxicity.

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Serotonin modulates behavior-related neural activity of RID interneuron in Caenorhabditis elegans

Mori H., Ashida K., Shidara H., Nikai T., Hotta K., Oka K.

PLoS ONE (PLoS ONE) 14 ( 12 ) 2019.12

Animals change their behaviors in response to external stimuli, and numerous neurotransmitters are involved in these behavioral changes. In Caenorhabditis elegans, serotonin (5- HT) affects various behaviors such as inhibition of locomotion, stimulation of egg laying, and pharyngeal pumping. Previous research has shown that the neural activity of the RID interneuron increases when the worm moves forward, and the RID is necessary for sustaining forward locomotion. However, the relationship between 5-HT and neural activity of RID, and how it modulates the behavior of the worm has not been investigated. In this article, we reveal the relationship among 5-HT, RID activity, and the behavior of worms using a custom- made tracking and imaging system. We simultaneously measured the neural activity of the RID and behavior in worms with three conditions: mock animals, animals pre-exposed to 5-HT, and 5-HT receptor mod-1 mutants. As shown in previous research, the neural activity of the RID increased during the transition from backward to forward, whereas it decreased during the transition from forward to backward in mock animals. These changes in neural activity were not observed in animals pre-exposed to 5-HT and mod-1 mutants. Moreover, RID activity was correlated with the velocity of the worm in mock animals. However, this correlation was not observed in animals pre-exposed to 5-HT and mod-1 mutants. Our results demonstrate that 5-HT modulates the activity of the RID interneuron, and we infer that the RID plays a role in modulating forward locomotion by changing its activity through 5-HT.

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The Input-Output Relationship of AIY Interneurons in Caenorhabditis elegans in Noisy Environment

Ashida K., Hotta K., Oka K.

iScience (iScience) 19 191 - 203 2019.09

Determining how neurotransmitter input causes various neuronal activities is crucial to understanding neuronal information processing. In Caenorhabditis elegans, AIY interneurons receive several sources of sensory information as glutamate inputs and regulate behavior by integrating these inputs. However, the relationship between glutamate input and the Ca2+ response in AIY under environmental noise, in other words, without explicit stimulation, remains unknown. Here, we show that glutamate-input fluctuations evoke a sporadic Ca2+ response in AIY without stimulation. To ensure that Ca2+ response can be considered AIY output, we show that the membrane-potential depolarization precedes Ca2+ responses in AIY. We used an odor as model stimulation to modulate the sensory inputs. Simultaneous imaging of glutamate input and Ca2+ response, together with glutamate transmission mutants, showed that glutamate-input fluctuations evoke sporadic Ca2+ responses. We identified the input-output relationships under environmental noise in vivo, and our results address the relationship between sensory-input fluctuations and behavioral variability. Neuroscience; Systems Neuroscience; Sensory Neuroscience

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Multiple tracking and machine learning reveal dopamine modulation for area-restricted foraging behaviors via velocity change in Caenorhabditis elegans

Ashida K., Kato T., Hotta K., Oka K.

Neuroscience Letters (Neuroscience Letters) 706 68 - 74 2019.07

ISSN 03043940

Food exploration is an essential survival behavior in organisms. To find food efficiently, many organisms use a foraging strategy called area-restricted search (ARS) wherein individuals first turn more frequently, restricting their search to one area, then turn less frequently, moving along a straight path to widen the search area. Previous research suggests that the nematode Caenorhabditis elegans shows ARS behavior by changing turn frequency, and that dopamine is a crucial determinant. However, the effects of dopamine on multiple behavioral parameters have remained unknown. Here, we evaluated turn (pirouette) frequency, moving velocity, and specific area occupancy (cell occupancy) over time by using a multiple-worms tracking system. In the control (mock) experiments, all parameters changed over time, but no changes were observed in experiments with dopamine pre-exposed and dopamine-deficient animals. In inverse reinforcement learning analysis, the value function for specific velocity was found to modulate over time in mock animals only. These results demonstrate that dopamine regulates ARS via changes not only to pirouette frequency change but also to velocity.

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GABA-Induced Intracellular Mg 2+ Mobilization Integrates and Coordinates Cellular Information Processing for the Maturation of Neural Networks

Yamanaka R., Shindo Y., Hotta K., Suzuki K., Oka K.

Current Biology (Current Biology) 28 ( 24 ) 3984 - 3991.e5 2018.12

ISSN 09609822

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14-3-3ϵa directs the pulsatile transport of basal factors toward the apical domain for lumen growth in tubulogenesis

Mizotani Y., Suzuki M., Hotta K., Watanabe H., Shiba K., Inaba K., Tashiro E., Oka K., Imoto M.

Proceedings of the National Academy of Sciences of the United States of America (Proceedings of the National Academy of Sciences of the United States of America) 115 ( 38 ) E8873 - E8881 2018.09

ISSN 00278424

The Ciona notochord has emerged as a simple and tractable in vivo model for tubulogenesis. Here, using a chemical genetics approach, we identified UTKO1 as a selective small molecule inhibitor of notochord tubulogenesis. We identified 14-3-3ϵa protein as a direct binding partner of UTKO1 and showed that 14-3-3ϵa knockdown leads to failure of notochord tubulogenesis. We found that UTKO1 prevents 14-3-3ϵa from interacting with ezrin/radixin/moesin (ERM), which is required for notochord tubulogenesis, suggesting that interactions between 14-3-3ϵa and ERM play a key role in regulating the early steps of tubulogenesis. Using live imaging, we found that, as lumens begin to open between neighboring cells, 14-3-3ϵa and ERM are highly colocalized at the basal cortex where they undergo cycles of accumulation and disappearance. Interestingly, the disappearance of 14-3-3ϵa and ERM during each cycle is tightly correlated with a transient flow of 14-3-3ϵa, ERM, myosin II, and other cytoplasmic elements from the basal surface toward the lumen-facing apical domain, which is often accompanied by visible changes in lumen architecture. Both pulsatile flow and lumen formation are abolished in larvae treated with UTKO1, in larvae depleted of either 14-3-3ϵa or ERM, or in larvae expressing a truncated form of 14-3-3ϵa that lacks the ability to interact with ERM. These results suggest that 14-3-3ϵa and ERM interact at the basal cortex to direct pulsatile basal accumulation and basal-apical transport of factors that are essential for lumen formation. We propose that similar mechanisms may underlie or may contribute to lumen formation in tubulogenesis in other systems.

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Developmental Roles and Evolutionary Significance of AMPA-Type Glutamate Receptors

Hirai S., Hotta K., Okado H.

BioEssays (BioEssays) 40 ( 9 ) 2018.09

ISSN 02659247

Organogenesis and metamorphosis require the intricate orchestration of multiple types of cellular interactions and signaling pathways. Glutamate (Glu) is an excitatory extracellular signaling molecule in the nervous system, while Ca 2+ is a major intracellular signaling molecule. The first Glu receptors to be cloned are Ca 2+ -permeable receptors in mammalian brains. Although recent studies have focused on Glu signaling in synaptic mechanisms of the mammalian central nervous system, it is unclear how this signaling functions in development. Our recent article demonstrated that Ca 2+ -permeable AMPA-type Glu receptors (GluAs) are essential for formation of a photosensitive organ, development of some neurons, and metamorphosis, including tail absorption and body axis rotation, in ascidian embryos. Based on findings in these embryos and mammalian brains, we formed several hypotheses regarding the evolution of GluAs, the non-synaptic function of Glu, the origin of GluA-positive neurons, and the neuronal network that controls metamorphosis in ascidians.

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Effect of interactions among individuals on the chemotaxis behaviours of Caenorhabditis elegans

Yoshimizu, Toshiki Shidara, Hisashi Ashida, Keita Hotta, Kohji Oka, Kotaro

The Journal of Experimental Biology (The Journal of experimental biology) 221 ( 11 ) jeb182790 2018.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-0949

In many species, individual social animals interact with others in their group and change their collective behaviours. For the solitary nematode Caenorhabditis elegans strain N2, previous research suggests that individuals can change the behaviour of other worms via pheromones and mechanosensory interactions. In particular, pheromones affect foraging behaviour, so that the chemotactic behaviours of individuals in a group (population) can be modulated by interactions with other individuals in the population. To investigate this, we directly compared the chemotactic behaviours of isolated (single) worms with those of individual animals within a population. We found that worms approached an odour source in a distinct manner depending on whether they were alone or in a population. Analysis of behaviours of the N2 worm and a pheromone production-defective mutant revealed that the 'pirouette' strategy was modulated by interaction of the worms via pheromones. Thus, pheromones play an important role in the characteristic collective behaviours seen in the population condition.

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Characterization of calcium transients during early embryogenesis in ascidians Ciona robusta (Ciona intestinalis type A) and Ciona savignyi

Akahoshi, Taichi Hotta, Kohji Oka, Kotaro

Developmental Biology 431 ( 2 ) 205 - 214 2017.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 1095564X

高感度カルシウムイオンインジケーターGCaMP6を用いてホヤ発生過程における一過的カルシウムイオン濃度上昇を長時間・高い時間解像度で網羅的に明らかにした。その結果、これまで中脳・後脳境界に存在すると考えられていた運動を出力するためのリズム生成子がより後方の運動神経節に存在すること、細胞外カルシウムイオン依存的に尾部表皮細胞のカルシウムイオン濃度上昇がみられることなど多くの新たな発生におけるカルシウムイオン濃度上昇の振る舞いを発見した。

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AMPA glutamate receptors are required for sensory-organ formation and morphogenesis in the basal chordate.

Shinobu Hirai, Kohji Hotta, Yoshihiro Kubo, Atsuo Nishino, Shigeo Okabe, Yasushi Okamura, and Haruo Okado

Proc Natl Acad Sci U S A 114 ( 15 ) 3939 - 3944 2017.04

Research paper (scientific journal), Joint Work, Accepted

脊椎動物ではAMPA受容体が４つあり、すべてを取り除く実験が困難であったた。一方、ホヤはAMPA受容体を１つしかもたないため、ノックダウンによる表現型の解析が比較的容易である。今回、ホヤAMAPA受容体のノックダウンおよび変異AMAPA受容体遺伝子導入により、はじめてAMPA受容体の発生における新たな機能を発見した。ホヤのAMPA受容体は中枢神経系の一部にのみ発現しており、その発現を阻害したところ、哺乳類の松果体にあたる光受容感覚器の形成が阻害されたことに加え、成体への変態に必要な尾の退縮が不完全となり、体軸の回転が阻害さることがわかった。

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Compartmentalized cGMP responses of olfactory sensory neurons in Caenorhabditis elegans.

Hisashi Shidara, Kohji Hotta, and Kotaro Oka

Journal of Neuroscience 37 ( 14 ) 3753 - 3763 2017.04

Accepted

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Transitional correlation between inner-membrane potential and ATP levels of neuronal mitochondria

Suzuki, R. Hotta, K. Oka, K.

Scientific Reports (Scientific Reports) 8 ( 1 ) 2017.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 2045-2322

The importance of highly active mitochondria and their contribution to neuronal function has been of recent interest. In most cases, however, mitochondrial activity is estimated using measurements of mitochondrial inner membrane potential (IMPmito), and little is known about the dynamics of native mitochondrial ATP (ATPmito). This study conducted simultaneous imaging of IMPmito and ATPmito in neurons to explore their behaviour and their correlation during physiological mitochondrial/neuronal activity. We found that mitochondrial size, transport velocity and transport direction are not dependent on ATPmito or IMPmito. However, changes in ATPmito and IMPmito during mitochondrial fission/fusion were found; IMPmito depolarized via mitochondrial fission and hyperpolarized via fusion, and ATPmito levels increased after fusion. Because the density of mitochondria is higher in growth cones (GCs) than in axonal processes, integrated ATPmito signals (density × ATPmito) were higher in GCs. This integrated signal in GCs correlated with axonal elongation. However, while the averaged IMPmito was relatively hyperpolarized in GCs, there was no correlation between IMPmito in GCs and axonal elongation. A detailed time-course analysis performed to clarify the reason for these discrepancies showed that IMPmito and ATPmito levels did not always correlate accurately; rather, there were several correlation patterns that changed over time.

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Altered expression of Mg²⁺ transport proteins during Parkinson's disease-like dopaminergic cell degeneration in PC12 cells

Shindo Yutaka, Yamanaka Ryu, Suzuki Koji, Hotta Kohji, Oka Kotaro

Biochimica et Biophysica Acta - Molecular Cell Research 1863 ( 8 ) 1979 - 1984 2016.08

ISSN 0167-4889

Mg²⁺ is an essential cation to maintain cellular functions, and intracellular Mg²⁺ concentration ([Mg²⁺]_i) is regulated by Mg²⁺ channels and transporters. In our previous study, we demonstrated that MPP⁺ elicits Mg²⁺ influx across the cell membrane and Mg²⁺ mobilization from mitochondria, and the resulting [Mg²⁺]_i is an important determinants of the cell viability in MPP⁺ model of Parkinson's disease (PD). It indicates that cellular Mg²⁺ transport is one of the important factors to determine the progress of PD. However, whether the expression levels of Mg²⁺ transport proteins change in the progress of PD has still been obscure. In this study, we estimated the mRNA expression levels of Mg²⁺ transport proteins upon the exposure to MPP⁺. In thirteen Mg²⁺ transport proteins examined, mRNA expression level of SLC41A2 was increased and that of ACDP2, NIPA1 and MMgT2 were decreased. Knockdown of SLC41A2, ACDP2 or NIPA1 accelerated the MPP⁺-induced cell degeneration, and overexpression attenuated it. The decrease in the mRNA expression levels of NIPA1 and MMgT2 were also elicited by rotenone, H₂O₂ and FCCP, indicating that mitochondrial dysfunction related to this down-regulation. The increase in that of SLC41A2 was induced by an uncoupler, FCCP, as well as MPP⁺, suggesting that it is an intrinsic protection mechanism against depolarized mitochondrial membrane potential and/or cellular ATP depletion. Our results shown here indicate that alteration of Mg²⁺ transport proteins is implicated in the MPP⁺ model of PD, and it affects cell degeneration.

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Mitochondrial Mg²⁺ homeostasis decides cellular energy metabolism and vulnerability to stress

Yamanaka Ryu, Tabata Sho, Shindo Yutaka, Hotta Kohji, Suzuki Koji, Soga Tomoyoshi, Oka Kotaro

Scientific Reports 6 2016.07

ISSN 2045-2322

Cellular energy production processes are composed of many Mg²⁺ dependent enzymatic reactions. In fact, dysregulation of Mg²⁺ homeostasis is involved in various cellular malfunctions and diseases. Recently, mitochondria, energy-producing organelles, have been known as major intracellular Mg²⁺ stores. Several biological stimuli alter mitochondrial Mg²⁺ concentration by intracellular redistribution. However, in living cells, whether mitochondrial Mg²⁺ alteration affect cellular energy metabolism remains unclear. Mg²⁺ transporter of mitochondrial inner membrane MRS2 is an essential component of mitochondrial Mg²⁺ uptake system. Here, we comprehensively analyzed intracellular Mg²⁺ levels and energy metabolism in Mrs2 knockdown (KD) cells using fluorescence imaging and metabolome analysis. Dysregulation of mitochondrial Mg²⁺ homeostasis disrupted ATP production via shift of mitochondrial energy metabolism and morphology. Moreover, Mrs2 KD sensitized cellular tolerance against cellular stress. These results indicate regulation of mitochondrial Mg²⁺ via MRS2 critically decides cellular energy status and cell vulnerability via regulation of mitochondrial Mg²⁺ level in response to physiological stimuli.

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An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation

Shindo Yutaka, Komatsu Hirokazu, Hotta Kohji, Ariga Katsuhiko, Oka Kotaro

Scientific Reports 6 2016.07

ISSN 2045-2322

Acetylation, which modulates protein function, is an important process in intracellular signalling. In mitochondria, protein acetylation regulates a number of enzymatic activities and, therefore, modulates mitochondrial functions. Our previous report showed that tributylphosphine (PBu₃), an artificial reaction promoter that promotes acetylransfer reactions in vitro, also promotes the reaction between acetyl-CoA and an exogenously introduced fluorescent probe in mitochondria. In this study, we demonstrate that PBu₃ induces the acetylation of mitochondrial proteins and a decrease in acetyl-CoA concentration in PBu₃-treated HeLa cells. This indicates that PBu₃ can promote the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins in living cells. PBu₃-induced acetylation gradually reduced mitochondrial ATP concentrations in HeLa cells without changing the cytoplasmic ATP concentration, suggesting that PBu₃ mainly affects mitochondrial functions. In addition, pyruvate, which is converted into acetyl-CoA in mitochondria and transiently increases ATP concentrations in the absence of PBu₃, elicited a further decrease in mitochondrial ATP concentrations in the presence of PBu₃. Moreover, the application and removal of PBu₃ reversibly alternated mitochondrial fragmentation and elongation. These results indicate that PBu₃ enhances acetyltransfer reactions in mitochondria and modulates mitochondrial functions in living cells.

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Qualitative and quantitative estimation of comprehensive synaptic connectivity in short- and long-term cultured rat hippocampal neurons with new analytical methods inspired by Scatchard and Hill plots

Tanamoto Ryo, Shindo Yutaka, Niwano Mariko, Matsumoto Yoshinori, Miki Norihisa, Hotta Kohji, Oka Kotaro

Biochemical and Biophysical Research Communications 471 ( 4 ) 486 - 491 2016.03

ISSN 0006-291X

To investigate comprehensive synaptic connectivity, we examined Ca²⁺ responses with quantitative electric current stimulation by indium-tin-oxide (ITO) glass electrode with transparent and high electro-conductivity. The number of neurons with Ca²⁺ responses was low during the application of stepwise increase of electric current in short-term cultured neurons (less than 17 days in-vitro (DIV)). The neurons cultured over 17 DIV showed two-type responses: S-shaped (sigmoid) and monotonous saturated responses, and Scatchard plots well illustrated the difference of these two responses. Furthermore, sigmoid like neural network responses over 17 DIV were altered to the monotonous saturated ones by the application of the mixture of AP5 and CNQX, specific blockers of NMDA and AMPA receptors, respectively. This alternation was also characterized by the change of Hill coefficients. These findings indicate that the neural network with sigmoid-like responses has strong synergetic or cooperative synaptic connectivity via excitatory glutamate synapses.

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