Caged neuron MEA: a system for long-term investigation of cultured neural network connectivity.

Traditional techniques for investigating cultured neural networks, such as the patch clamp and multi-electrode array, are limited by: (1) the number of identified cells which can be simultaneously electrically contacted, (2) the length of time for which cells can be studied, and (3) the lack of one-to-one neuron-to-electrode specificity. Here, we present a new device - the caged neuron multi-electrode array - which overcomes these limitations. This micro-machined device consists of an array of neurocages which mechanically trap a neuron near an extracellular electrode. While the cell body is trapped, the axon and dendrites can freely grow into the surrounding area to form a network. The electrode is bi-directional, capable of both stimulating and recording action potentials. This system is non-invasive, so that all constituent neurons of a network can be studied over its lifetime with stable one-to-one neuron-to-electrode correspondence. Proof-of-concept experiments are described to illustrate that functional networks form in a neurochip system of 16 cages in a 4 x 4 array, and that suprathreshold connectivity can be fully mapped over several weeks. The neurochip opens a new domain in neurobiology for studying small cultured neural networks.

Controlling bursting in cortical cultures with closed-loop multi-electrode stimulation.

One of the major modes of activity of high-density cultures of dissociated neurons is globally synchronized bursting. Unlike in vivo, neuronal ensembles in culture maintain activity patterns dominated by global bursts for the lifetime of the culture (up to 2 years). We hypothesize that persistence of bursting is caused by a lack of input from other brain areas. To study this hypothesis, we grew small but dense monolayer cultures of cortical neurons and glia from rat embryos on multi-electrode arrays and used electrical stimulation to substitute for afferents. We quantified the burstiness of the firing of the cultures in spontaneous activity and during several stimulation protocols. Although slow stimulation through individual electrodes increased burstiness as a result of burst entrainment, rapid stimulation reduced burstiness. Distributing stimuli across several electrodes, as well as continuously fine-tuning stimulus strength with closed-loop feedback, greatly enhanced burst control. We conclude that externally applied electrical stimulation can substitute for natural inputs to cortical neuronal ensembles in transforming burst-dominated activity to dispersed spiking, more reminiscent of the awake cortex in vivo. This nonpharmacological method of controlling bursts will be a critical tool for exploring the information processing capacities of neuronal ensembles in vitro and has potential applications for the treatment of epilepsy.

An extremely rich repertoire of bursting patterns during the development of cortical cultures.

BACKGROUND: We have collected a comprehensive set of multi-unit data on dissociated cortical cultures. Previous studies of the development of the electrical activity of dissociated cultures of cortical neurons each focused on limited aspects of its dynamics, and were often based on small numbers of observed cultures. We followed 58 cultures of different densities--3000 to 50,000 neurons on areas of 30 to 75 mm2--growing on multi-electrode arrays (MEAs) during the first five weeks of their development. RESULTS: Plating density had a profound effect on development. While the aggregate spike detection rate scaled linearly with density, as expected from the number of cells in proximity to electrodes, dense cultures started to exhibit bursting behavior earlier in development than sparser cultures. Analysis of responses to electrical stimulation suggests that axonal outgrowth likewise occurred faster in dense cultures. After two weeks, the network activity was dominated by population bursts in most cultures. In contrast to previous reports, development continued with changing burst patterns throughout the observation period. Burst patterns were extremely varied, with inter-burst intervals between 1 and 300 s, different amounts of temporal clustering of bursts, and different firing rate profiles during bursts. During certain stages of development bursts were organized into tight clusters with highly conserved internal structure. CONCLUSION: Dissociated cultures of cortical cells exhibited a much richer repertoire of activity patterns than previously reported. Except for the very sparsest cultures, all cultures exhibited globally synchronized bursts, but bursting patterns changed over the course of development, and varied considerably between preparations. This emphasizes the importance of using multiple preparations--not just multiple cultures from one preparation--in any study involving neuronal cultures. These results are based on 963 half-hour-long recordings. To encourage further investigation of the rich range of behaviors exhibited by cortical cells in vitro, we are making the data available to other researchers, together with Matlab code to facilitate access.

Searching for plasticity in dissociated cortical cultures on multi-electrode arrays.

We attempted to induce functional plasticity in dense cultures of cortical cells using stimulation through extracellular electrodes embedded in the culture dish substrate (multi-electrode arrays, or MEAs). We looked for plasticity expressed in changes in spontaneous burst patterns, and in array-wide response patterns to electrical stimuli, following several induction protocols related to those used in the literature, as well as some novel ones. Experiments were performed with spontaneous culture-wide bursting suppressed by either distributed electrical stimulation or by elevated extracellular magnesium concentrations as well as with spontaneous bursting untreated. Changes concomitant with induction were no larger in magnitude than changes that occurred spontaneously, except in one novel protocol in which spontaneous bursts were quieted using elevated extracellular magnesium concentrations.

Extracellular potentials in low-density dissociated neuronal cultures.

The detection of extracellular potentials by means of multi-electrode arrays (MEA) is a useful technique for multi-site long-term monitoring of cultured neuronal activity with single-cell resolution. To optimize the geometry of the MEA it is advantageous to localize the cellular compartments that constitute the generators of these signals. For this purpose, an in vitro technique for the detection of extracellular signals with subcellular resolution has been developed. It makes use of easy-to-manufacture large-tip pipettes, monitoring of electrode-cell gap resistance for precise electrode positioning and low-density (100 cells/mm(2)) dissociated hippocampal cultures. Negative monophasic extracellular spikes, typically 60 microV, were measured over putative axonal processes and monophasic, biphasic and triphasic signals were recorded over the soma. A compartmental simulation suggests that different somatic conductance densities of Na(+) (1-10 mS/cm(2)) and K(+) (5-10 mS/cm(2)) channels can produce characteristic somatic extracellular potentials, with a variety of shapes similar to those observed experimentally.

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

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.

Moving live dissociated neurons with an optical tweezer.

The use of an optical tweezer for moving dissociated neurons was studied. The main features of the tweezers are outlined as well as the general principles of its operation. Infrared beams at 980 and 1064 nm were used, focused so as to make a trap for holding neurons and moving them. Absorption by cells at those wavelengths is very small. Experiments were done to evaluate nonsticky substrate coatings, from which neurons could be easily lifted with the tweezers. The maximum speed of cell movement as a function of laser power was determined. Detailed studies of the damage to cells as a function of beam intensity and time of exposure were made. The 980 nm beam was much less destructive, for reasons that are not understood, and could be used to safely move cells through distances of millimeters in times of seconds. An illustrative application of the use of the tweezers to load neurons without damage into plastic cages on a glass substrate was presented. The conclusion is that optical tweezers are an accessible and practical tool for helping to establish neuron cultures of cells placed in specific locations.