AMPA-type glutamate receptor (AMPAR) is one of the major neurotransmitter receptors at excitatory synapses. The initial assembling states of AMPARs at cell surface are essential for synaptic transmission, which is related with learning and memory in living neural systems. To realize artificial control of synaptic transmission, we demonstrate to modulate the initial assembling states of quantum-dot conjugated AMPARs (QD-AMPARs) with optical trapping. The optical trapping dynamics of QD-AMPARs on living neurons was evaluated with fluorescence imaging and fluorescence correlation spectroscopy (FCS). The transit time at laser focus of QD-AMPARs on neurons estimated from FCS analysis increased with the culturing days and addition of neurotransmitter, which suggests that QD-AMPARs are confined at the focal spot due to optical trapping.
Molecular dynamics of glutamate receptor, which is major neurotransmitter receptor at excitatory synapse located on neuron, is essential for synaptic plasticity in the complex neuronal networks. Here we studied molecular dynamics in an optical trap of AMPA-type glutamate receptor (AMPAR) labeled with quantum-dot (QD) on living neuronal cells with fluorescence imaging and fluorescence correlation spectroscopy (FCS). When a 1064-nm laser beam for optical trapping was focused on QD-AMPARs located on neuronal cells, the fluorescence intensity of QD-AMPARs gradually increased at the focal spot. Using single-particle tracking of QD-AMPARs on neurons, the average diffusion coefficient decreased in an optical trap. Moreover, the decay time obtained from FCS analysis increased with the laser power and the initial assembling state of AMPARs depended on culturing day, suggesting that the motion of QD-AMPAR was constrained in an optical trap.
Molecular dynamics at synaptic terminals in neuronal cells is essential for synaptic plasticity and subsequent modulation
of cellular functions in a neuronal network. For realizing artificial control of living neuronal network, we demonstrate
laser-induced perturbation into molecular dynamics in the neuronal cells. The optical trapping of cellular molecules such
as synaptic vesicles or neural cell adhesion molecules labeled with quantum dots was evaluated by fluorescence imaging
and fluorescence correlation spectroscopy. The trapping and assembling dynamics was revealed that the molecular
motion was constrained at the focal spot of a focused laser beam due to optical trapping force. Our method has a
potential to manipulate synaptic transmission at single synapse level.
The combination of confocal micro-Raman spectroscopy and multivariate analysis is carried out for analysis of
maturation of neurons. This study suggests that Raman data reflects the stages of neural maturation which relates with
the expression of new neural function such as spontaneous activity. Neurons obtained from a hippocampus of rat
embryos are cultured in a dish with quartz bottom. According to the previous electrophysiological study, matured neural
cell network showed regulated pulsation with interval of several seconds without any stimulation. It suggested that
alterations in the molecular composition took place in the cell. The Raman measurements are carried out to observe this
alteration along with the maturing process of neurons. The spectra of live neural cells measured after 2, 8, 15, 30, 45, 60,
75, 90, 105 and 120 days of culturing are analyzed by principal component analysis (PCA). The result shows several
groups suggesting the maturation scheme which is observed by the electrophysiological studies. It demonstrates that the
maturation process of neural cells can be monitored by Raman spectroscopy.
Neurons form complex networks and it seems that the living neuronal network can perform certain type of information processing. We are interested in intelligence autonomously formed in vitro. The most important features of the two-dimensional culture neural network are that it is a system in which the information processing is autonomously carries out. We reported previously that the functional connections were dynamically modified by synaptic potentiation and the process may be required for reorganization of the functional group of neurons. Such neuron assemblies are critical for information processing in brain. Certain types of feedback stimulation caused suppression of spontaneous network electrical activities and drastic re-organization of functional connections between neurons, when these activities are initially almost synchronized. The result suggests that neurons in dissociated culture autonomously re-organized their functional neuronal networks interacted with their environment. The spatio-temporal pattern of activity in the networks may be a reflection of their external environment. We also interfaced the cultured neuronal network with moving robot. The planar microelectrodes can be used for detecting neuronal electrical signals from the living neuronal network cultured on a 2-dimensional electrode array. The speed of actuators of moving robot was determined by these detected signals. Our goal is reconstruction of the neural network, which can process "thinking" in the dissociated culture system.
The neurons in dissociation culture autonomously re-organized their functional neuronal networks, after the process for elongating neurites and establishing synaptic connections. The spatio-temporal patterns of activity in the networks might be a reflection of functional neuron assemblies. The functional connections were dynamically modified by synaptic potentiation and the process may be required for reorganization of the functional group of neurons. Such neuron assemblies are critical for information processing in brain. To visualize the functional connections between neurons, we have analyzed the autonomous activity of synaptically induced action potentials in the living neuronal networks on a multi-electrode array, using "connection map analysis" that we developed for this purpose. Moreover, we designed aan original wide area covering electrode array and succeeded in recording spontaneous action potentials from wider area than commercial multi electrode arrays.