Extracellular Stimulation of a Target Neuron in Micro-Patterned Neuronal Circuits Using a Pair of Needle Electrodes

BACKGROUND: Electrical stimulation of neurons is essential to control neuronal activities. Thus far, direct stimulation by whole-cell patch-clamp methods [1] and extracellular stimulation using multi-electrode arrays (MEAs) [2] have been performed commonly. While the whole-cell patch-clamp method merits from the precise control of stimulation, the use of MEAs takes advantage of maintaining neuronal activity over a long period owing to its non-invasiveness. An optogenetic technique [3] has a targeting precision of a single-cell level, but this has difficulties in applying fast stimulation patterns due to the slow photoresponse of opsin [4]. Alternative non-invasive methods capable of targeting a single neuron with arbitrary stimulation patterns open new possibility of controlling and monitoring neuronal activities over a long duration. In this presentation, we propose a novel method to combine cell-patterning techniques [5] with electrical stimulation of a target cell with a pair of needle electrodes [6]. We show that the firing of a single neuron cultured on the micropattern can be controlled by the proposed method. We also show the controllability of the stimulation by analogue circuit simulation of a neuron stimulated electrically by a pair of needle electrodes via culture medium. EXPERIMENTAL METHODS: Neurons were cultured on a micropatterned glass substrates in isolation. A pair of tungsten needles except for their tip apexes were coated with an insulating polymer and mounted on manipulators. Two needles were immersed in the medium so that the target neuron is placed in-between. The distance from the neuron to the needle tip was controlled to be several tens to 100 μm. A bipolar square pulse was applied between electrodes, and the neuronal activity was visualized by Ca imaging. SIMULATION: The membrane potential response of a neuron to extracellular stimulation was analysed using LTspice simulator [7]. The circuit model is shown in Fig. 1a. The integrate-and-fire neuron was connected electrically with two needle electrodes via the electric double layers and solution resistances. The key of the circuit simulation is to divide the neuron into two compartments, each is stimulated with either of two needle electrodes. We conjecture that the neuron fires if the membrane potential either of two compartments exceeds the threshold upon the electrical stimulation. We investigated the membrane potential response when the resistance corresponding to the relative