Ai Kiyohara

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.

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.

Biomodeling System – Interaction Between Living Neuronal Networks and the Outer World

Rat hippocampal neurons reorganized into complexnetworks in a culture dish with 64 planar micro-electrodes and the electrical activity of neurons wererecorded from individual sites. Multi-site recordingsystem for extracellular action potentials was used forrecording the activity of living neuronal networks andfor applying input from the outer world to the net-work. The living neuronal network was able to dis-tinguish among patterns of evoked action potentialsbased on different input, suggesting that the livingneuronal network can express several pattern inde-pendently, meaning that it has fundamental mecha-nisms for intelligent information processing. We aredeveloping a “biomodeling system,” in which a livingneuronal network is connected to a moving robot withpremised control rules corresponding to a geneticallyprovided interface of neuronal networks to peripheralsystems. Premised rules are described in fuzzy logicandthe robot cangenerateinstinctive behavior,avoid-ing collision. Sensor input from the robot body wassenttoaneuronalnetwork,andtherobotmovedbasedon commands from the living neuronal network. Thisis a good modeling system to analyze interaction be-tween biological information processing and electricaldevices.Keywords: neuron, dissociated culture system, multi-electrode array (MED), fuzzy logic, moving robot

Vitroid – the robot system with an interface between a living neuronal network and outer world

We have developed a neuro-robot-hybrid system using a living neuronal network and a miniature moving robot. The living network of rat hippocampal neurons can distinguish patterns of action potentials evoked by different inputs, suggesting that a cultured neuronal network can represent particular states as symbols. We used a Khepera II robot and a robot made using a LEGO mindstorm NXT kit to interface with a living neuronal network and the outer world. We call the system ‘vitroid’. Vitroid has living neurons, a robot body, and direct coupling controllers to interface the neurons with the robot. Vitroid was able to perform obstacle avoidance behaviour with premised control rule sets.

Optical trapping of synaptic vesicles in neurons

We demonstrate intracellular manipulation of synaptic vesicles in living neurons by optical trapping. When an infrared trapping laser is focused on synapses of a neuronal cell labeled with a fluorescent endocytic marker, fluorescence is observed at the focal spot. The fluorescence spectrum is attributed to fluorescent dye in the synaptic vesicles, indicating excitation by two-photon absorption of the trapping laser. The fluorescence intensity increases gradually within ∼100 s of laser irradiation, suggesting that trapping force causes vesicles assembly at the focus. Our method can be applied to manipulate synaptic transmission of a particular neuron in a neuronal network.

Fuzzy bio-interface: Indicating logicality from living neuronal network and learning control of bio-robot

Recently, many attractive brain-computer interface and brain-machine interface have been proposed. The outer computer and machine are controlled by brain action potentials detected through a device such as near-infrared spectroscopy (NIRS) and electroencephalograph (EEG), and some discriminant model determines a control process. In this paper, we introduce a fuzzy bio-interface between a culture dish of rat hippocampal neurons and the khepera robot. We propose a model to analyze logic of signals and connectivity of electrodes in a culture dish, and show the bio-robot hybrid we developed. We believe that the framework of fuzzy system is essential for BCI and BMI, thus name this technology “fuzzy bio-interface”. We show the usefulness of a fuzzy bio-interface through some examples.

The effects of the current stimulation on electrical activity in dissociated neurons

Rat hippocampal neurons were cultured on a dish with 64 micro planer electrodes. We found that the silent and reproducible period lasting for 1 sec immediately after the activity evoked in advance. In addition, the repetitive stimuli suppress the spontaneously occurring bursting activity in frequency. These results suggest that distinct internal state of the neuronal circuit was triggered by an electrical stimulation, and these dynamics of network activity may contribute to information processing. In previous study, we developed the neuro-robot system in which the neurons were connected to a robot body and interacted with external world. By utilizing the dynamics of a living neuronal network, it can be designed that, so to speak, the robot, which behaves according to the history of the sensor inputs which the neuronal network has memorized. We are verifying whether the dynamics intrinsic in a neural network can contribute to the action determination of a creature, by moving such a model system.

Vitroid - a robot with link between living neuronal network in vitro and robot body

Rat hippocampal neurons organized complex networks on a culture dish which has 64 planar microelectrodes and the spontaneous action potentials were frequently observed. The living neuronal network was able to distinguish patterns of action potentials evoked by different inputs, suggesting that a cultured neuronal network can represent and process particular states as symbols. We use a Khepera II robot and a robot made by LEGO mindstorm NXT kit as a robot body for interfacing with a living neuronal network and the outer world. We call the system ldquoVitroidrdquo. Vitroid has the living neurons, robot body, and direct coupling type of controllers to interface neurons with the robot. We succeeded in performing obstacle avoidance behavior with premised control rule sets. Using self-tuning fuzzy reasoning, we associated a distinct spatial pattern of electrical activity with a particular phenomenon in the outside of the culture dish.

Self-organization of the cultured neuronal network and dynamics of the evoked activity

Long-term potentiation (LTP) and long-term depression (LTD) are considered as cellular models for memory. LTP/LTD are characterized by temporal phases (early and late phase). Early-LTP/LTD, lasting less than 2 h can be dissociated from lateLTP/LTD by blockers of protein synthesis. It was hypothesized that a synaptic tag enables capture of newly synthesized plasticity-related proteins. Previously, we found that neuropsin involved in formation of the synaptic tag in apical CA1 dendrites. Here, we show that in addition to apical CA1 dendrites, neuropsin-dependent synaptic tagging also takes place within basal CA1 dendritic compartments after LTP induction. Furthermore our findings demonstrate that neuropsin is involved in the LTP process-specific synaptic tag, whereas it is not involved in LTD tag.

Developmental change of heterogeneous distribution of the functional synaptic connections in a rat hippocampal dissociated neurons

Perineuronal nets (PNNs) consisting of chondroitin sulfate proteoglycans (CSPGs) and hyaluronic acids are known to be associated with distinct neuronal population in mammalian brain. In present study, we examined the function of neuroprotective action of PNNs against amyloid protein (A ) in cultured cortical neurons, and the effect of chondroitinase ABC (ChABC) on Fos expression in GABAergic interneuron in vivo. In vitro study, the application of A to cultured neurons induced apoptosis on PNNs-negative neurons, however, PNNs-positive neurons survived. In vivo study, immobilization stress caused Fos expression in the primary somatosensory cortex (S1). There was no significant difference of total number of Fos-positive neurons in the S1 after ChABC injection, however, double labeling study of Fos and GABAergic interneuron revealed that the percentage of Fos-positive neurons in GABAergic interneurons was fewer than control. These results indicate that PNNs are concerned with neuroprotection against A and neural activity of GABAergic interneuron.