Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Summary

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Abstract

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia^1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)³. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.

In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Protocol

1. Assembly of the Microfluidic Culture Chamber (MCP)

1. MCP (Figure 1) are open chambers designed for compartmented cultures of different types of cells ⁴. It typically has two compartments that are connected through the central channels (3 μm in diameter). Assembly of MCP with glass-bottomed dishes is necessary for preparing cultures and subsequent imaging analysis.
2. First, coat sterile glass-bottomed dishes with Polyornithine (Sigma-Aldrich, 1 mg/ml) dissolved in sodium tetraborate (Sigma-Aldrich, 10 mM pH 8.5) and were incubated overnight at 37 °C.
3. On the following day, the coated glass-bottomed dishes....

Results

Time-lapse imaging analysis of axon-induced GLT1 promoter activation in astrocytes

The compartmented neuron and astrocyte co-culture system allows only the neuronal processes, especially the axons, to selectively interact with astrocytes. Following the successful establishment of axon and astrocyte (or other glial cells) co-culture in the assembled MCP, different types of axon-glia interactions can be studied such as; axon-induced activation of astroglial gene promoter activation, astrocyte-induc.......

Discussion

The MCP based neuron and astrocytes co-culture system allows dissection of detailed neuron to astroglia signaling pathways by allowing only the axons pass the central channels and interacting with the astroglial cells. This co-culture system can be conveniently set up with conventional neuron and astrocyte culture procedures. We also described a practical application of this co-culture system by employing an eGFP based reporter for demonstrating axon-dependent GLT1 promoter activation in astrocytes.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fetal bovine serum	Hyclone	SH30070.03	for plating neuron for neuron cutlure medium
Fetal bovine serum	Sigma-Aldrich	F4135	for astrocyte culture medium
Glial derived nerve factor	R&D Systems	212-GD	Apply 10-20 ng/ml to neuron side of chamber
Dulbecco modified eagle medium high glucose	Sigma-Aldrich	11995
70 mm cell strainer	BD Falcon	352350
Sterile glass bottom dish	MatTek Corporation
Microfluidic culture platforms	Xona Microfluidics LLC	SND150
6 wells of the culture plate	Cellstar	657 160
Neuron culture medium Neurobasal medium 2% B27 Neurobasal supplement 2 mM glutamate by adding 1% 100x GlutaMAX 1% Penicillin-streptomysin
Neuron culture medium for plating cell Neurobasal medium 2% B27 Neurobasal supplement 2 mM glutamate by adding 1% 100x GlutaMAX 1% Penicillin-streptomysin 5% Fetal bovine serum SH30070.03
Astrocyte culture medium Dulbecco modified eagle medium high glucose 10% Fetal bovine serum F4135 1% Penicillin-streptomysin
Table 1. Materials used in the microfluidic culture platform-based neuronal axon and glia co-culture system.