Extrinsic control of functional polarity in neuronal networks by microcontact-printed protein gradients.

Abstract

Guiding neuronal polarity, axonal outgrowth, and synapse formation are crucial factors in neuronal differentiation and organization of the central nervous system (CNS). This guidance allows the CNS to form functional circuits to process information or generate new circuits in case of injured nervous tissue. Unscrambling the underlying molecular and cellular mechanisms of neuronal growth could thus provide new methods of neural regeneration. Our aim is to achieve extrinsic control of functional polarity in neuronal networks. To investigate how the neuronal polarity of individual primary cortical neurons can be affected we used a previously established in vitro model system. One-dimensional protein gradients (poly-l-Lysin and laminin) were transferred onto a glass surface by microcontact-printing, and then populated with embryonic (E18) rat cortical neurons. Two growth directions can be obtained, one along the increasing gradient (+) and one in the direction of the decreasing gradient (-). A circular node was integrated in the middle of the gradient structure to control the position of the neuronal soma. After 13 to 15 days in vitro we performed double patch-clamp experiments on neighboring cells on the gradient. Due to the aligned printing of the gradients, we were able to discriminate whether the signal transmits in the (+) or (-) direction. Double patch clamp recording and the position of the pre- and post-synaptic cell with respect to each other, has shown that the signals are propagated toward the (+) direction of the underlying gradient structure (n=14, 78%). Furthermore, all synapses measured showed unidirectional signal transmission. Our studies demonstrate that microstructured protein gradients provide a tool that allows the control of not only the directionality of axon outgrowth, but also the direction of signal propagation of primary rat cortical neurons in culture. In further experiments, we will determine the synapse type by pharmacological studies using neurotransmitter antagonists