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.

To investigate comprehensive synaptic connectivity, we examined Ca(2+) responses with quantitative electric current stimulation by indium-tin-oxide (ITO) glass electrode with transparent and high electro-conductivity. The number of neurons with Ca(2+) 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.

Electrical stimulation of cultured neurons using a simply patterned indium-tin-oxide (ITO) glass electrode.

BACKGROUND: Indium-tin-oxide (ITO) glass electrodes possess the properties of optical transparency and high electrical conductivity, which enables the electrical stimulation of cultured cells to be performed whilst also measuring the responses with fluorescent imaging techniques. However, the quantitative relationship between the intensity of the stimulating current and the cell response is unclear when using conventional methods that employ a separated configuration of counter and stimulation electrodes. NEW METHOD: A quantitative electrical current stimulation device without the use of a counter electrode was fabricated. RESULTS: Nerve growth factor (NGF)-induced differentiated PC12 cells were cultured on an ITO single glass electrode, and the Ca(2+) response to electrical stimuli was measured using fluorescent Ca(2+) imaging. ITO electrode devices with a width less than 0.1mm were found to evoke a Ca(2+) response in the PC12 cells. Subsequent variation in the length of the device in the range of 2-10mm was found to have little influence on the efficiency of the electric stimulus. We found that the stimulation of the cells was dependent on the electrical current, when greater than 60 µA, rather than on the Joule heat, regardless of the width and length of the conductive area. COMPARISON WITH EXISTING METHOD(S): Because of the cells directly in contact with the electrode, our device enables to stimulate the cells specifically, comparing with previous devices with the counter electrode. CONCLUSIONS: The ITO device without the use of a counter electrode is a useful tool for evaluating the quantitative neural excitability of cultured neurons.

Odorant-induced membrane potential depolarization of AIY interneuron in Caenorhabditis elegans.

Although some interneurons in C. elegans have been shown to have unusual region-specific Ca(2+) dynamics, the region-specific Ca(2+) and membrane potential response properties of these neurons are largely unknown due to technical limitations. In this report, we focused on one of these neurons, AIY interneuron, where Ca(2+) dynamics have been detected only in neurites, and not the soma, during odor and temperature stimulation to determine whether membrane potential and Ca(2+) are region-specific dynamics and distinct from one another. To visualize voltage change both in the soma and neurites of AIY, we used voltage-sensitive fluorescent protein (VSFP) 2.42. First, we confirmed that the sensor protein worked correctly in C. elegans by depolarizing AIY interneuron with high concentrations of KCl. Next, we observed membrane potential depolarization during odor (isoamyl alcohol) stimulation in both neurites and the soma. Additionally, depolarization of membrane potential with direct application of high KCl induced a Ca(2+) increase in the soma. From these results, we conclude that membrane potential behavior and Ca(2+) dynamics in AIY differ in its subcellular regions and that VSFP2.42 can be a useful tool for studying information processing in single neurons.