The analysis of dynamics and the relationship between spontaneous and evoked activity as cell assemblies in a cultured neuronal network.

In a brain, it is considered that the synchronous activity of neurons expresses a representation of information. Hebb named the synchronous active group of neurons "Cell Assembly". In this study, we hypothesized that a repeatedly expressed pattern is a cell assembly representing a certain kind of information and attempted to extract such "Cell Assembly" by X-means clustering based on spatiotemporal continuity of spontaneous spikes. Moreover, we divided cell assemblies into classes consist of similar types of cell assemblies, using the indiscernibility-based clustering, "rough clustering". As the result, it showed that cell assemblies did not have a large temporal extent, but a spatial extent. Additionally, we analyzed the neuronal network activity as the stochastic process whose state space is the set of cell assembly, finding out that similar patterns appear consecutively. According to these results, information processing in the neuronal network is suggested to be the hierarchical process. Finally, the clustering method was adopted for spontaneous activity and the evoked responses. It is suggested that spontaneous activities and evoked responses are not completely independent, but share resemble activities.

Bisphenol A and rotenone induce S-nitrosylation of protein disulfide isomerase (PDI) and inhibit neurite outgrowth of primary cultured cells of the rat hippocampus and PC12 cells.

Bisphenol A (BPA) interferes the function and development of the central nervous system (CNS), resulting in behavioral abnormalities and memory loss. S-nitrosylation of protein disulfide isomerase (PDI) is increased in brains with sporadic Alzheimer's disease and Parkinson's disease. The aim of the present study was to clarify the role of nitric oxide (NO) in BPA-induced neurotoxicity. Since rotenone induces NO-mediated neurodegeneration through S-nitrosylation of PDI, it was used as a positive control. First, rats were treated with BPA and rotenone, and S-nitrosylation of PDI was detected in rat brain microsomes. BPA and rotenone decreased RNase oxidation activity of PDI concomitant with S-nitrosylation of PDI. Next, to clarify S-nitrosylation of PDI by BPA and rotenone in rat brains, we treated the rat pheochromocytoma cell line PC12 and primary cultured neuron cells from the rat hippocampus with BPA (5 and 10 µM) and rotenone (100 or 200 nM). BPA induced S-nitrosylation of PDI, while NG-monomethyl-L-arginine (L-NMMA), a NOS inhibitor, exerted the opposite effects. Finally, to evaluate the toxicity of BPA in the CNS, we investigated its effects on neurite outgrowth of PC12 and primary cultured neuron cells. BPA inhibited neurite outgrowth of these cells, while L-NMMA reversed this inhibition. The involvement of PDI activity in neurite outgrowth was also examined, and bacitracin, a PDI inhibitor, is shown to decrease neurite outgrowth. Furthermore, the overexpression of PDI, but not a catalytically inactive PDI mutant, enhanced neurite outgrowth. These results suggested that S-nitrosylation of PDI induced by excessive NO caused BPA-induced neurotoxicity.

Real-time fluorescence measurement of spontaneous activity in a high-density hippocampal network cultivated on a plasmonic dish.

High-density cultured neuronal networks have been used to evaluate synchronized features of neuronal populations. Voltage-sensitive dye (VSD) imaging of a dissociated cultured neuronal network is a critical method for studying synchronized neuronal activity in single cells. However, the signals of VSD are generally too faint-that is, the signal-to-noise ratio (S/N) is too low-to detect neuronal activity. In our previous research, a silver (Ag) plasmonic chip enhanced the fluorescence intensity of VSD to detect spontaneous neural spikes on VSD imaging. However, no high-density network was cultivated on the Ag plasmonic chip, perhaps because of the chemical instability of the Ag surface. In this study, to overcome the instability of the chip, we used a chemically stable gold (Au) plasmonic dish, which was a plastic dish with a plasmonic chip pasted to the bottom, to observe neuronal activity in a high-density neuronal network. We expected that the S/N in real-time VSD imaging of the Au plasmonic chip would be improved compared to that of a conventional glass-bottomed dish, and we also expected to detect frequent neural spikes. The increase in the number of spikes when inhibitory neurotransmitter receptors were inhibited suggests that the spikes corresponded to neural activity. Therefore, real-time VSD imaging of an Au plasmonic dish was effective for measuring spontaneous network activity in a high-density neuronal network at the spatial resolution of a single cell.

Elucidation of EEG Characteristics of Fuzzy Reasoning-Based Heuristic BCI and Its Application to Patient With Brain Infarction.

Non-invasive brain-computer interfaces (BCIs) based on common electroencephalography (EEG) are limited to specific instrumentation sites and frequency bands. These BCI induce certain targeted electroencephalographic features of cognitive tasks, identify them, and determine BCI's performance, and use machine-learning to extract these electroencephalographic features, which makes them enormously time-consuming. In addition, there is a problem in which the neurorehabilitation using BCI cannot receive ambulatory and immediate rehabilitation training. Therefore, we proposed an exploratory BCI that did not limit the targeted electroencephalographic features. This system did not determine the electroencephalographic features in advance, determined the frequency bands and measurement sites appropriate for detecting electroencephalographic features based on their target movements, measured the electroencephalogram, created each rule (template) with only large "High" or small "Low" electroencephalograms for arbitrarily determined thresholds (classification of cognitive tasks in the imaginary state of moving the feet by the size of the area constituted by the power spectrum of the EEG in each frequency band), and successfully detected the movement intention by detecting the electroencephalogram consistent with the rules during motor tasks using a fuzzy inference-based template matching method (FTM). However, the electroencephalographic features acquired by this BCI are not known, and their usefulness for patients with actual cerebral infarction is not known. Therefore, this study clarifies the electroencephalographic features captured by the heuristic BCI, as well as clarifies the effectiveness and challenges of this system by its application to patients with cerebral infarction.

14,15-epoxyeicosatrienoic acid produced by cytochrome P450s enhances neurite outgrowth of PC12 and rat hippocampal neuronal cells.

Polyunsaturated fatty acids, such as arachidonic acid, are accumulated in brain and induce neuronal differentiation. Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) by cytochrome P450s. In this study, we found that 14,15-EET and 20-HETE-enhanced NGF-induced rat pheochromocytoma PC12 cell neurite outgrowth even at the concentration of 100 nmol L-1. LC-MS analysis revealed that 14,15-EET was effectively produced from arachidonic acid by rat CYP2C11, 2C13, and 2C23, and these P450s were expressed in PC12 cells. An inhibitor of these P450s, ketoconazole, inhibited neurite outgrowth, whereas inhibition of soluble epoxide hydrolase, which hydrolyzes EETs to their corresponding diols enhanced neurite outgrowth. To determine the mechanism of neurite formation enhancement by arachidonic acid metabolites, we focused on transient receptor potential (TRP) channels expressed in PC12 cells. The TRPV4 inhibitor HC067047, but not the TRPV1 inhibitor capsazepine, inhibited the effects of 14,15-EET on neurite outgrowth of PC12. Furthermore, 14,15-EET increased the cytosolic calcium ion concentration and this increase was inhibited by HC067047. 14,15-EET also enhanced neurite outgrowth of primary cultured neuron from rat hippocampus. This study suggests that arachidonic acid metabolites produced by P450 contribute to neurite outgrowth through calcium influx.

Analysis of the developing neural system using an in vitro model by Raman spectroscopy.

We developed an in vitro model of early neural cell development. The maturation of a normal neural cell was studied in vitro using Raman spectroscopy for 120 days. The Raman spectra datasets were analyzed by principal component analysis (PCA) to investigate the relationship between maturation stages and molecular composition changes in neural cells. According to the PCA, the Raman spectra datasets can be classified into four larger groups. Previous electrophysiological studies have suggested that a normal neural cell goes through three maturation states. The groups we observed by Raman analysis showed good agreement with the electrophysiological results, except with the addition of a fourth state. The results demonstrated that Raman analysis was powerful to investigate the daily changes in molecular composition of the growing neural cell. This in vitro model system may be useful for future studies of the effects of endocrine disrupters in the developing early neural system.

Relationship between inter-stimulus-intervals and intervals of autonomous activities in a neuronal network.

To investigate relationships between neuronal network activity and electrical stimulus, we analyzed autonomous activity before and after electrical stimulus. Recordings of autonomous activity were performed using dissociated culture of rat hippocampal neurons on a multi-electrodes array (MEA) dish. Single stimulus and pared stimuli were applied to a cultured neuronal network. Single stimulus was applied every 1 min, and paired stimuli was performed by two sequential stimuli every 1 min. As a result, the patterns of synchronized activities of a neuronal network were changed after stimulus. Especially, long range synchronous activities were induced by paired stimuli. When 1 s inter-stimulus-intervals (ISI) and 1.5 s ISI paired stimuli are applied to a neuronal network, relatively long range synchronous activities expressed in case of 1.5 s ISI. Temporal synchronous activity of neuronal network is changed according to inter-stimulus-intervals (ISI) of electrical stimulus. In other words, dissociated neuronal network can maintain given information in temporal pattern and a certain type of an information maintenance mechanism was considered to be implemented in a semi-artificial dissociated neuronal network. The result is useful toward manipulation technology of neuronal activity in a brain system.

Fundamental short-term memory of semi-artificial neuronal network.

Spatiotemporal pattern of neuronal network activity is a key component of brain information processing. Cultured rat hippocampal neurons on the multielectrodes array dish are suitable for analyzing and manipulating network dynamics and its developmental changes. We applied paired electrical inputs at various inter-stimulus intervals (ISi) and analyzed the spatio-temporal pattern of evoked responses. We found that the pattern of evoked electrical activity was affected by existence of a prior input in the case that ISi of paired stimuli was within 2 s. These results suggest that a semi-artificial neuronal network on a culture dish has a fundamental component of short-term memory, and the origin of this hysteresis is transition among the internal states of the network, undertaken by synaptic transmissions.

Neuronal cell patterning on a multi-electrode array for a network analysis platform.

We studied neuronal cell patterning on a commercial multi-electrode array (MEA). We investigated the surface chemical modification of MEA in order to immobilize Poly-D-lysine (PDL) and then to pattern PDL with a photolithographic method using vacuum ultraviolet light (VUV). We have clarified that the PDL layer was not fully decomposed but was partially fragmented by short-time irradiation with VUV, resulting in a change in the cell adhesiveness of the PDL. We succeeded in patterning primary rat cortex cells without manipulating the cells on MEA more than two months. This cell-adhesiveness change induced by VUV can be applied to any immobilized PDL on other kinds of MEA and culturing substrate. We conducted electrophysiological measurements and found that the patterned neuronal cells were sufficiently matured and developed neural networks, demonstrating that our patterning method is useful for a neuronal network analysis platform.

Cell patterning using a template of microstructured organosilane layer fabricated by vacuum ultraviolet light lithography.

Micropatterning techniques have become increasingly important in cellular biology. Cell patterning is achieved by various methods. Photolithography is one of the most popular methods, and several light sources (e.g., excimer lasers and mercury lamps) are used for that purpose. Vacuum ultraviolet (VUV) light that can be produced by an excimer lamp is advantageous for fabricating material patterns, since it can decompose organic materials directly and efficiently without photoresist or photosensitive materials. Despite the advantages, applications of VUV light to pattern biological materials are few. We have investigated cell patterning by using a template of a microstructured organosilane layer fabricated by VUV lithography. We first made a template of a microstructured organosilane layer by VUV lithography. Cell adhesive materials (poly(d-lysine) and polyethyleneimine) were chemically immobilized on the organosilane template, producing a cell adhesive material pattern. Primary rat cardiac and neuronal cells were successfully patterned by culturing them on the pattern substrate. Long-term culturing was attained for up to two weeks for cardiac cells and two months for cortex cells. We have discussed the reproducibility of cell patterning and made suggestions to improve it.

Resynchronization in neuronal network divided by femtosecond laser processing.

We demonstrated scission of a living neuronal network on multielectrode arrays (MEAs) using a focused femtosecond laser and evaluated the resynchronization of spontaneous electrical activity within the network. By an irradiation of femtosecond laser into hippocampal neurons cultured on a multielectrode array dish, neurites were cut at the focal point. After the irradiation, synchronization of neuronal activity within the network drastically decreased over the divided area, indicating diminished functional connections between neurons. Cross-correlation analysis revealed that spontaneous activity between the divided areas gradually resynchronized within 10 days. These findings indicate that hippocampal neurons have the potential to regenerate functional connections and to reconstruct a network by self-assembly.

A simple exploratory algorithm for the accurate and fast detection of spontaneous synaptic events.

We have developed a program for the fast and accurate detection of spontaneous synaptic events. The algorithm identifies each event of which the slope and amplitude which meet criteria. The significant feature of this algorithm is its stepwise and exploratory search for the onset and the peak points. During the first step, the program employing the algorithm makes a rough estimate of the candidate for a synaptic event, and determines a 'temporary' onset data point. The next step is the detection of the true onset data point and 'temporary' peak data point, which probably exist several points after the temporary onset data point. The third step is a backward search to detect the true peak data point. The final step is to check whether the amplitude of the detected event exceeds the threshold. This stepwise and shuttlewise search allows for the accurate detection of the peak points. Using this program, we succeeded in detecting an increased frequency and amplitude of spontaneous excitatory postsynaptic currents in chick cerebral neurons following the application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In addition, we demonstrated that the program employing the algorithm was able to be used for the detection of extracellular action potentials.

A synaptic potentiation by a protein factor distinct from those induced by neurotrophins.

We report a protein factor(s) contained in the conditioned medium (CM) of the Mg(2+)-free treatment induced the synaptic potentiation. This type of potentiation shared a different pathway from those induced by neurotrophins. Neurotrophins were confirmed to induce a synaptic potentiation in the dissociated chick neurons. Furthermore, K252a, an inhibitor of tyrosine kinase, abolished this potentiation. Nevertheless, the potentiation induced by the CM was not blocked by K252a. In addition, the CM prepared from the chick neurons induced a similar potentiation in rat and mouse neurons. These results suggest that the protein factor is a novel protein molecule for inducing the potentiation and it plays a critical role in the common mechanism for the potentiation between avian and mammal.