Microcontact printing of patterns, containing circles for neuron soma attachment and narrow lines for neuron process growth, directed development of neural networks of hippocampal neurons. Aligned printing of these patterns to MEAs resulted in regular placement of neurons on electrodes. The number of neurons associated with each electrode was dependent on initial neuron plating density. Spontaneous electrical activity was observed within two weeks of plating when cell densities of 200 cells/mm2 or higher were used.
Action potentials were recorded from approximately one third of the electrode sites on each MEA even though neurons were observed on nearly all the electrode sites. This is consistent with previous studies (Chang et al., 2001
). This inefficiency for functional recording sites may be an obstacle for optimal MEA use. One possible contributing factor for this observation is low neuron density. We observed no spontaneous activity when cells were plated at 100 cells/mm2
. There are several possible explanations for this observation. Low synaptic input and/or insufficient trophic interactions may be major contributors. At lower densities neurons may not receive sufficient synaptic input to develop into functional networks. Neurons in vitro
may have far fewer synapses when compared to neurons in vivo
. There are several experimental strategies that might overcome this problem. One is to provide electrical stimulation during network development (Nam et al., 2003
). This might provide greater neurotransmitter release and promote more synapse formation (Grubb et al., 2004
). Another strategy would be to include an NMDA (N-methyl-D-aspartate) receptor antagonist in the culture media during network development. This procedure has been shown to increase the total number of synapses and spontaneous neuronal activity (Corner et al., 2002
; Martinoia et al., 2005
). Trophic signals could also be increased by using more cells in the neuronal network, by applying specific trophic factors, or providing feeder cells. The first is not consistent with efforts to develop neural networks with limited elements, but does increase spontaneous electrical activity in network (unpublished observation). Application of brain-derived neurotrophic factor has also been reported to increase spontaneous activity in neuronal cultures (Legrand et al., 2005
). This approach is direct, but also requires knowledge of the necessary trophic factors for optimal effects. Close association of feeder layers of neurons and/or astrocytes might provide the necessary trophic factors without increasing neuron numbers on the MEAs and without specific knowledge of the precise factors required. Feeder layers of astrocytes could be used, since these cells are critical for neuron development and function in the brain. Such feeder cultures could be included using opposing cultures (Banker and Goslin, 1997) or by including cells constrained to regions outside the recording regions of the MEAs. We are exploring these possibilities.
Our pharmacological experiments demonstrated that excitatory glutamate receptors, AMPA receptors, are critical to network activity in more mature cultures, e.g. after 21 days. More complete pharmacological studies will indicate the importance of other types of glutamate receptors and the possible role of other neurotransmitters. For instance, the synaptic signals occurring between hippocampal neurons during early developmental stages are mediated by GABAergic synapses before the maturation of glutamatergic transmission (Ma et al., 1998
). Consistent with this we have observed suppression of spontaneous activity by bicuculline methiodide, a GABAA
receptor antagonist, at 7 days in culture (unpublished observation).
Our morphological analysis of these neuronal networks demonstrated that neuronal somas are usually located on electrode sites providing close association with electrode sites. Pre-synaptic proteins were observed near neuronal somas, along MAP-2 positive processes and in regions of larger bundles of neuronal processes. SEM imaging () clearly demonstrates that the neuronal organization of these cultures is more complex than the cytochemistry suggests. Tightly compacted bundles of neuronal processes were observed along lines and coursing over electrode sites. Thus the signals observed at a particular electrode site may be initiated by neurons at different locations.
Additional evidence of the complexity of these networks can be derived from the electrophysiological recordings. The conduction velocities recorded from specific electrode sites was 0.03 m/sec (see ), which is significantly slower than reported conduction velocities of 0.2–0.35 m/s, expected of single unmyelinated axons (Andersen et al., 1978
). Thus many of connections we are observing may be part of more circuitous multi-synaptic pathways. Simultaneous recording from the entire network and tracing of individual neurons will be needed to understand the contributions of each neuron to the network activity.
These data demonstrate that low-density neuronal networks can be used to advance our knowledge of neuron networks on MEAs, and demonstrate that these methods will enable more precise investigations of network function. MEA-based systems show great promise for the study of nervous system functions, including memory, learning, and development of biosensors for pharmacology and other applications.