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Neurostimulation is an essential technique to trigger and modulate the spatiotemporal activity of local neuronal circuits. Current stimulation methods have a trade-off relationship among aiming precision, temporal resolution, and noninvasiveness, making it difficult to stimulate and monitor a single target neuron for a long term. Here, we show that a method using two needle electrodes in combination of micropatterning techniques provides new possibilities for targeting and stimulating a single neuron selectively. Results of physiological experiments as well as analog circuit simulation reveal that two needle electrodes can stimulate a target neuron selectively by placing the two needle electrodes in proximity to and to straddle the target neuron, and that the steepness of voltage applied to two needle electrodes is important for the target neuron to fire at a low voltage. The proposed method enables a noninvasive stimulation suitable for measuring long-term activity of local neuronal circuits.

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 distance from the neuron to the electrode, the capacitive component of the needles, and the shape of voltage input pulses were changed. RESULTS AND DISSCUSSION: As shown in Fig. 1b, the neuron fires if the amplitude of the bipolar square pulse exceeds 5 Vp-p. The amplitude required for the neuron firing decreases with increasing distance from the neuron to the needle electrodes. The LTspice simulation provided the following results: (i) The positive electrode hyperpolarizes the membrane potential, whereas the negative electrode depolarizes it, indicating the membrane potential of two compartments changes to inverse direction even if a bipolar pulse was applied, as shown in Fig. 1c. (ii) The closer the electrode is to the cell, the more the membrane potential of the neuron changes and the voltage drop in the solution resistance is comparatively small. (iii) The main factor of neuronal firing is the transient potential fluctuation caused by the capacitive component of the electrical double layer and the neuronal membrane. (iv) The membrane potential of both the compartments is sensitive to transient change of tip voltage and does not change if the system reaches the steady state. This is also true if the DC voltage is continuously applied. These results indicate that applying a voltage pulse of appropriate amplitude can stimulate only cells in the vicinity of needle electrodes. Only a single neuron in neuronal culture can be stimulated by the needle electrodes by placing the target neuron between two needles. The non-invasive stimulation can also be used for long-term stimulation and monitoring. [1] E. Neher, et al., Nature 260(1976) 799. [2] C.A. Thomas Jr., et al., Exp. Cell Res. 74(1972) 61. [3] K. Deisseroth, et al., J. Neurosci. 26(2006) 10380. [4] B.D. Allen, et al., Learn. Mem. 22(2015) 232. [5] L. Bekar, et al., Nat. Med. 14(2008) 75. [6] H. Yamamoto, et al., Appl. Phys. Lett. 113(2018) 133703. [7] https://www.analog.com/en/design-center/design-tools-and-calculators/ltspice-simulator.html# Figure 1