Effective parameters for stimulation of dissociated cultures using multi-electrode arrays

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Abstract

Electrical stimulation through multi-electrode arrays is used to evoke activity in dissociated cultures of cortical neurons. We study the efficacies of a variety of pulse shapes under voltage control as well as current control, and determine useful parameter ranges that optimize efficacy while preventing damage through electrochemistry. For any pulse shape, stimulation is found to be mediated by negative currents. We find that positive-then-negative biphasic voltage-controlled pulses are more effective than any of the other pulse shapes tested, when compared at the same peak voltage. These results suggest that voltage-control, with its inherent control over limiting electrochemistry, may be advantageous in a wide variety of stimulation scenarios, possibly extending to in-vivo experiments.

Introduction

Multi-electrode arrays (MEAs) Gross, 1979, Heuschkel et al., 2002, Pine, 1980, Potter, 2001, Thomas et al., 1972 have been used to record from a wide variety of neuronal preparations. Electrical stimulation through MEAs has been used to elicit spiking activity in dissociated cultures Gross et al., 1993, Jimbo and Kawana, 1992, Jimbo et al., 1999, Maher et al., 1999b, Regehr et al., 1988, as well as brain slices Echevarria and Albus, 2000, Egert et al., 1998, Harsch and Robinson, 2000, Heck, 1995, Novak and Wheeler, 1988, Tscherter et al., 2001 and isolated retina Branner and Normann, 2000, Grumet et al., 2000. MEAs and related technology for multi-site extracellular stimulation and recording, such as silicon probes Bai and Wise, 2001, Wise and Angell, 1975 and multi-wire probes (Nicolelis et al., 1998) have gained popularity because the relatively non-invasive nature of the technology allows for long-term interaction with healthy cells, and because they scale well to a large number of recording and stimulation channels.

When designing stimulation paradigms, researchers have to make many choices even after they have decided on electrode material: whether to use bipolar stimuli (between two electrodes) or monopolar stimuli (between one electrode and a large and usually distant ground electrode), whether to use voltage or current control, what pulse shape to use (monophasic, biphasic, perhaps even multi-phasic or asymmetric). Compromises have to be found between efficacy of stimuli, harm to electrodes or cells, and stimulation artifacts that hamper recording of responses. For long-term experiments, it is crucial to prevent damaging electrodes and killing cells. Cell damage can result from high charge injection or high charge densities (McCreery et al., 1990), but our MEA electrodes cannot inject dangerous amounts of charge before exceeding electrolysis limits (Weiland et al., 2002). Electrolysis, which starts to play a role when electrode voltages exceed about 1 V, directly damages electrodes, and is also harmful to cells. (This harm can be much reduced by employing charge-balanced stimuli, making such stimuli preferable for long-term or in-vivo work when large voltages cannot be avoided Lilly et al., 1955, Shepherd et al., 1991.) A secondary constraint can be the width of stimulus pulses: since recording is generally impossible for the duration of the stimulus pulse, short pulses are often desirable.

Here, we study electrical stimuli intended to evoke activity in dissociated cortical cultures on MEAs, with the aim of establishing robust two-way communication between such cultures and a computer system. Knowing a set of stimuli that are reliably and consistently effective is essential for long-term experiments on the development of functional networks, as well as for research on learning in-vitro DeMarse et al., 2001, Shahaf and Marom, 2001. While the electrical properties of MEA electrodes have been described in the literature Buitenweg et al., 1998, Kovacs, 1994, McAdams et al., 1995, McIntyre and Grill, 2001, the published knowledge base on what kinds of stimuli are most effective at evoking responses is remarkably slim. A full quantitative understanding would require a detailed model of the electric fields that current pulses induce along axonal and somatic membranes, but in high density cultures, the arrangement of neurons and glia is too complex to construct such a model. In this paper we hope to provide new practical information by identifying a range of stimuli that are effective, unharmful, and produce minimal artifacts. The results in this paper were obtained from dense neocortical cultures grown on MEAs with 30 μm titanium nitride electrodes. Qualitatively, the results should extend to other dissociated neuronal cultures, and to electrodes of different sizes and construction.

Section snippets

Cell culture

Neocortex was dissected from rat embryos (E18) under sterile conditions. Cortices were cut into pieces of about 1 mm3, prior to dissociation using papain and trituration. Cells were plated at 5000 cells/mm2 on multi-electrode arrays (MultiChannel Systems, Reutlingen, Germany) coated with poly-ethylene-imine (PEI) and laminin. Cultures were maintained for 2–3 weeks prior to recording, in a medium adapted from Jimbo et al. (1999): high glucose DMEM (Irvine Scientific cat. no. 9024) with 10% Horse

Results

All electrodes tested could be used to evoke responses, given sufficiently strong stimuli (Fig. 2). Responses to individual stimuli could be differentiated into three parts:
Direct responses   Any response that does not depend on glutamatergic synapses. These occur in the first 10–20 ms post-stimulus, have less than 0.25 ms temporal jitter and can be close to 100% reliable (i.e. observed in close to 100% of trials). Direct responses most likely result from antidromic excitation through an axon

Discussion

From the great variety of possible stimulation pulse shapes that can be applied to MEA electrodes, we studied eight important families: monophasic and biphasic rectangular current pulses of either polarity, and monophasic and biphasic rectangular voltage pulses of either polarity. We found that the efficacy of stimuli in any of these families can be attributed to the generation of negative electrode currents.

To explain why negative current pulses are effective stimuli while positive currents

Acknowledgements

This work was partially supported by grants NS044134 and NS38628 from NIH-NINDS, and EB000786 from NIH-NIBIB, and by the Burroughs-Wellcome Fund and the Whitaker Foundation. We thank our cell culture technician Sheri McKinney for very helpful assistance.

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