Effective parameters for stimulation of dissociated cultures using multi-electrode arrays
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.
References (44)
- et al.
A multielectrode array for intrafascicular recording and stimulation in sciatic nerve of cats
Brain Res. Bull
(2000) - et al.
Activity-dependent development of spontaneous bioelectric activity in organotypic cultures of rat occipital cortex
Dev. Brain Res
(2000) - et al.
A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays
Brain Res Protocols
(1998) - et al.
Stimulation of monolayer networks in culture through thin-film indium-tin oxide recording electrodes
J. Neurosci. Methods
(1993) - et al.
Multi-electrode stimulation and recording in the isolated retina
J. Neurosci. Methods
(2000) Investigating dynamic aspects of brain-function in slice preparations—spatiotemporal stimulus patterns generated with an easy-to-build multielectrode array
J. Neurosci. Methods
(1995)- et al.
A three-dimensional multi-electrode array for multi-site stimulation and recording in acute brain slices
J. Neurosci. Methods
(2002) - et al.
Electrical stimulation and recording from cultured neurons using a planar electrode array
Bioelectrochem. Bioenerg
(1992) - et al.
Simultaneous induction of pathway-specific potentiation and depression in networks of cortical neurons
Biophys. J
(1999) - et al.
The neurochip: a new multi-electrode device for stimulating and recording from cultured neurons
J. Neurosci. Methods
(1999)
The linear and non-linear electrical properties of the electrode–electrolyte interface
Biosens. Bioelectr
Excitation of central nervous system neurons by nonuniform electric fields
Biophys. J
Functional synapses in synchronized bursting of neocortical neurons in culture
Brain Res
Multisite hippocampal slice recording and stimulation using a 32 element microelectrode array
J Neurosci Methods
Recording action potentials from cultured neurons with extracellular microcircuit electrodes
J. Neurosci. Methods
A new approach to neural cell culture for long-term studies
J. Neurosci. Methods
The basic mechanism for the electrical stimulation of the nervous system
Neuroscience
Modeling by shortest data description
Automatica
A miniature microelectrode array to monitor the bioelectric activity of cultured cells
Exp. Cell. Res
Real-time multi-channel stimulus artifact suppression by local curve fitting
J. Neurosci. Methods
Single-unit neural recording with active microelectrode arrays
IEEE Trans. Biomed. Eng
Measurement of sealing resistance of cell-electrode interfaces in neuronal cultures using impedance spectroscopy
Med. Biol. Eng. Comput
Cited by (256)
The technology, opportunities, and challenges of Synthetic Biological Intelligence
2023, Biotechnology AdvancesNeuronal network-based biomimetic chip for long-term detection of olfactory dysfunction model in early-stage Alzheimer's disease
2022, Biosensors and BioelectronicsCharacterization of neuronal viability and network activity under microfluidic flow
2021, Journal of Neuroscience MethodsA novel approach for removing micro-stimulation artifacts and reconstruction of broad-band neuronal signals
2020, Journal of Neuroscience MethodsCitation Excerpt :The impact of these electrically evoked spikes can then be directly correlated to changes in perception and behavior or reveal anatomical connections between neurons at the stimulation site and other brain regions (Histed et al., 2009; Parvizi et al., 2012; Tolias et al., 2005). On the other hand, simultaneous recording of neuronal activity, while electric pulses are applied, is problematic because these pulses affect data acquisition by superimposing artifacts onto the actual neuronal signal (Schiff et al., 1994; Wagenaar et al., 2004). These artifacts are often characterized by large initial transients, which subsequently decay rather slowly until reaching the pre-stimulation level of the neuronal signal (Wagenaar and Potter, 2002).