Cryopreserved rat cortical cells develop functional neuronal networks on microelectrode arrays

Abstract

Neurons growing on microelectrode arrays (MEAs) are promising tools to investigate principal neuronal network mechanisms and network responses to pharmaceutical substances. However, broad application of these tools, e.g. in pharmaceutical substance screening, requires neuronal cells that provide stable activity on MEAs. Cryopreserved cortical neurons (CCx) from embryonic rats were cultured on MEAs and their immunocytochemical and electrophysiological properties were compared with acutely dissociated neurons (Cx). Both cell types formed neuritic networks and expressed the neuron-specific markers microtubule associated protein 2, synaptophysin, neurofilament and γ-aminobutyric acid (GABA). Spontaneous spike activity (SSA) was recorded after 9 up to 74 days in vitro (DIV) in CCx and from 5 to 30 DIV in Cx, respectively. Cx and CCx exhibited synchronized burst activity with similar spiking characteristics. Tetrodotoxin (TTX) abolished the SSA of both cell types reversibly. In CCx SSA-inhibition occurred with an IC₅₀ of 1.1 nM for TTX, 161 μM for magnesium, 18 μM for d,l-2-amino-5-phosphonovaleric acid (APV) and 1 μM for GABA. CCx cells were easy to handle and developed long living, stable and active neuronal networks on MEAs with similar characteristics as Cx. Thus, these neurochips seem to be suitable for studying neuronal network properties and screening in pharmaceutical research.

Introduction

Dissociated neurons from different regions of the central nervous system (CNS) are widely accepted in vitro models that allow the investigation of principal neurobiological mechanisms under controlled conditions. So far, dissociated neurons have been successfully used to study morphological and electrophysiological properties: Barde et al. discovered the effects of neuronal growth factors on neuronal survival and outgrowth (Barde et al., 1978, Barde, 1990). Single channel properties of excitable membranes were first described by Neher and Sakmann using their innovative patch-clamp technique (Sakmann and Neher, 1984, Hamill et al., 1981).

Beyond the investigation of single neurons, the analysis of their activity within small neuronal assemblies is a promising step forward to understand the function of networks within the CNS. Although analysis of network behavior may be achieved by multiple simultaneous patch-clamp recordings, it is at least very difficult and time-consuming.

Microelectrode arrays (MEAs) with dissociated neuronal cells or brain slices seem to be much more suitable for the investigation of neuronal networks (Gross et al., 1995, Streit et al., 2001, Jimbo et al., 2000, Egert et al., 1998, Maher et al., 1999, Potter and DeMarse, 2001, Keefer et al., 2001a, Zeck and Fromherz, 2001). These “neurochips” allow convenient monitoring of spontaneous or stimulated electrical activity of excitable cells and enable the detection of neuroactive substance effects (Pancrazio et al., 2001, O'Shaughnessy et al., 2003).

However, researchers face some difficulties when they start to utilize neuronal cells on MEAs: the serial preparation and cultivation of primary cells is labor intensive and requires highly skilled technicians beside long-term scheduling and extensive animal care. Cryopreserved neurons offer some clear advantages over freshly dissociated cells: they provide flexibility to the culturing process and represent a “cell reserve” that eliminates the need for timed pregnancy animals each time cell culture is initiated.

Here, we report on the morphological and neurophysiological development of cryopreserved rat cortical cells (CCx) on MEAs in comparison to acutely dissociated neurons from embryonic rat cortex (Cx). Both cell types developed stable spontaneously active networks with similar immunocytochemical and electrophysiological properties. We determined dose response curves of spontaneous spiking activity in CCx under application of neuroactive substances revealing a high sensitivity and reproducibility.