Multielectrode arrays (MEAs) are used for analysis of neuronal activity. Here we report two variations on commonly accepted techniques that increase the precision of extracellular electrical stimulation: (i) the use of a low-amplitude recorded spontaneous synaptic signal as a stimulus waveform and (ii) the use of a specific electrode within the array adjacent to the stimulus electrode as a hard-grounded stimulus signal return path. Both modifications remained compatible with manipulation of neuronal networks. In addition, localized stimulation with the low-amplitude synaptic signal allowed selective stimulation or inhibition of otherwise spontaneous signals. These findings indicate that minimizing the area of the culture impacted by external stimulation allows modulation of signaling patterns within subpopulations of neurons in culture. The simple modifications described herein may be useful for precise monitoring and manipulation of neuronal networks.

Commerically available MEAs generally consist of a culture chamber with electrodes etched into the base. MEAs are often used for the analysis of neuronal signaling during formation and manipulation of networks. In addition to monitoring and capturing spontaneous activity, any of the array electrodes can be used to stimulate a neuronal network by applying an external signal. For example, a biphasic square pulse of ±700 millivolts (mV) amplitude and 800 milliseconds (msec) duration is often applied to a single electrode (“monopolar stimulation”) or to two electrodes (“bipolar stimulation”) within the MEA (1,–11). More recently, region-specific excitation has been attempted by employing these types of signals at multiple electrodes, referred to as “multipolar stimulation” (12). Results are generally reported as the total number of spikes or as changes in the inter-spike or inter-burst interval as a consequence of stimulation. The latter reporting method relies upon statistics of modulated spike timing as the determinant of an invoked response.

In addition to the 59 array electrodes, MEAs also have a single so-called “bath ground” electrode etched onto the glass substrate that is required to keep electrical noise throughout the culture sufficiently low enough to record signals reproducibly above background. The bath ground electrode is relatively large, is situated distant from the 59 electrode array, and is often utilized as the reference to complete the stimulus circuit. This configuration is commonly referred to as “monopolar” (7, 13). Unfortunately, stimulation via an array electrode and coupled with the bath ground electrode results in dispersion of the applied electric field throughout a large area of the culture (Fig. 1A) and consequently can impact a large portion of the neuronal network.

Figure 1. The applied signal field and response signals induced by different stimulation patterns. (Click to enlarge)

Related factors that complicate the use of this relatively large electrical field include stimulator driving impedances and the effects of displacement current densities along the insulated conductive traces required to connect the electrodes to external electronics. These traces, also etched onto the glass, transfer time-varying charges to and from electrodes during stimulation that radiate electromagnetic energy across the electrical insulator and into the culture (14). The problem becomes more complicated in the presence of a monolayer of cells, since the effective propagation medium becomes non-uniform and generates inflections in the field (15). These factors can collectively exert non-uniform effects on neurons within the stimulated region, which can compromise stimulation and confound interpretation of results. Herein, we examined the impact of using one of the array electrodes, rather than the bath electrode, as a ground to complete the stimulus circuit, to form a localized electrical field.

Along with minimizing electrical input, we also considered the potential benefit of stimulation with a low amplitude signal. The rationale for use of a biphasic square pulse of large amplitude as a neuronal stimulus was to bring the amplifier out of saturation as quickly as possible following a monophasic stimulus event of the same magnitude (7, 12). It has further been suggested that the first half of a biphasic pulse serves as a pre-pulse, lowering thethreshold for activation of response signaling (16). We examined herein the relative efficacy of utilizing a synaptic burst, recorded from a neuronal network maintained on an MEA for >1 month. We also examined the effect of stimulation with a square wave and a recorded synaptic signal using a localized stimulation circuit, which allowed us to monitor the effects of stimulation on selected regions within the network.

Materials and methods

Cultures

Cortical neurons harvested from day 15 C57BL/6 mouse embryos were dissociated and plated at 100 cells/mm² in Laminin-coated, 250mm² glass multielectrode arrays containing 59 titanium nitride electrodes in a 1.4mm² area on a 200μm grid at the center of the chamber from Multi Channel Systems (Reutlingen, Germany) and maintained in B27-supplemented Neurobasal medium (Invitrogen, Carlsbad, CA) as described (17). Sacrifice of the pregnant female was carried out under procedures approved by our Institutional Animal Care and Use Committee. Spontaneous neuronal signals were discernable within ten days of plating. However, cultures were maintained for ≥1 month prior to recording in order to allow maturation and stabilization of the neuronal network (17,–19).