Compartmentalized Neuronal Cultures.

The culturing of neurons results in formation of the layer of neurons with random extensive overlapping outgrowth. To understand specific roles of somas, axons, and dendrites in complex function of neurons and to identify molecular mechanisms of biological processes in these cellular compartments, various methods were developed. We utilized AXon Investigation System (AXIS™) manufactured by Millipore. This device provides an opportunity to orient neuronal outgrowth and spatially isolate neuronal processes from neuronal bodies. AXIS device is a slide-mounted microfluidic system, which consists of four wells. Two of the wells are connected by a channel on each side of the device. Channels are connected by microgrooves (approximately 120). The size of microgrooves (10µm in width and 5µm in height) does not permit passage of cell through while allowing extension of neurites. The microfluidic design also allows for an establishment of a hydrostatic gradient when the volume in one chamber is greater than that in the other (Park et al., Nat Protoc 1:2128-2136, 2006). This feature allows for studying of the effect of specific compounds on selected compartments of neurons.

Ligand-Induced GPR110 Activation Facilitates Axon Growth after Injury.

Recovery from axonal injury is extremely difficult, especially for adult neurons. Here, we demonstrate that the activation of G-protein coupled receptor 110 (GPR110, ADGRF1) is a mechanism to stimulate axon growth after injury. N-docosahexaenoylethanolamine (synaptamide), an endogenous ligand of GPR110 that promotes neurite outgrowth and synaptogenesis in developing neurons, and a synthetic GPR110 ligand stimulated neurite growth in axotomized cortical neurons and in retinal explant cultures. Intravitreal injection of GPR110 ligands following optic nerve crush injury promoted axon extension in adult wild-type, but not in gpr110 knockout, mice. In vitro axotomy or in vivo optic nerve injury rapidly induced the neuronal expression of gpr110. Activating the developmental mechanism of neurite outgrowth by specifically targeting GPR110 that is upregulated upon injury may provide a novel strategy for stimulating axon growth after nerve injury in adults.

A microchip for quantitative analysis of CNS axon growth under localized biomolecular treatments.

Growth capability of neurons is an essential factor in axon regeneration. To better understand how microenvironments influence axon growth, methods that allow spatial control of cellular microenvironments and easy quantification of axon growth are critically needed. Here, we present a microchip capable of physically guiding the growth directions of axons while providing physical and fluidic isolation from neuronal somata/dendrites that enables localized biomolecular treatments and linear axon growth. The microchip allows axons to grow in straight lines inside the axon compartments even after the isolation; therefore, significantly facilitating the axon length quantification process. We further developed an image processing algorithm that automatically quantifies axon growth. The effect of localized extracellular matrix components and brain-derived neurotropic factor treatments on axon growth was investigated. Results show that biomolecules may have substantially different effects on axon growth depending on where they act. For example, while chondroitin sulfate proteoglycan causes axon retraction when added to the axons, it promotes axon growth when applied to the somata. The newly developed microchip overcomes limitations of conventional axon growth research methods that lack localized control of biomolecular environments and are often performed at a significantly lower cell density for only a short period of time due to difficulty in monitoring of axonal growth. This microchip may serve as a powerful tool for investigating factors that promote axon growth and regeneration.