Revealing Neural Circuit Topography in Multi-Color

Summary

We provide a practical guide for delivering tracers in vivo and use the spinocerebellar pathway as a model system to demonstrate essential steps for successful neuronal circuit analysis in mice. We describe in detail our versatile tracing protocol that exploits wheat germ agglutinin (WGA) conjugated to Alexa fluorophores.

Abstract

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably label the global patterning of multiple topographic projections. The cerebellum is an ideal model to study the orderly arrangement of neural circuits. For example, the compartmental organization of spinocerebellar mossy fibers has proven to be an indispensable system for studying mossy fiber patterning. We recently showed that wheat germ agglutinin (WGA) conjugated to Alexa 555 and 488 can be used for tracing spinocerebellar mossy fiber projections in developing and adult mice (Reeber et al. 2011). We found three major properties that make the WGA-Alexa tracers desirable tools for labeling neural projections. First, Alexa fluorophores are intense and their brightness allows for wholemount imaging directly after tracing. Second, WGA-Alexa tracers label the entire trajectory of developing and adult neural projections. Third, WGA-Alexa tracers are rapidly transported in both retrograde and anterograde directions. Here, we describe in detail how to prepare the tracers and other required tools, how to perform the surgery for spinocerebellar tracing and how best to image traced projections in three dimensions. In summary, we provide a step-by-step tracing protocol that will be useful for deciphering the organization and connectivity of functional maps not only in the cerebellum but also in the cortex, brainstem, and spinal cord.

Protocol

1. Delivering tracers in vivo using sterile surgery (Sections 1.1-1.16)

Throughout the procedure we advise that standard sterile surgical techniques be used. This includes using sterile gloves, gown, and facemask. Tools should either be autoclaved before use or cleaned thoroughly with water and then ethanol. During the surgery, and especially between animals, clean tissue fragments off the tools and dry sterilize instruments with a hot bead sterilizer (Steri 250 Cat. # 18000-45, Fine Science Tools). In addition, it is critical that you organize your work space such that you have areas designated for animal preparation (shaving etc),....

Discussion

We have described the surgical and technical details required for successful axonal and dendrite tracing using a novel fluorescent-based approach for rapidly labeling neural projections in developing and adult mice. Using WGA-Alexa we show how tracers and markers can be used for analyzing patterned circuit topography at high resolution and in three dimensions.

Many tracers are available for tracing neuronal circuits including Cholera toxin subunit B, Biocytin, Neurobiotin, Phaseolus vulgaris .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bead sterilizer	Fine Science Tools	Model Steri 250 Cat. # 18000-45
Cauterizer	Fine Science Tools	Cat. # 18000-00
Borosilicate glass capillaries	Harvard Apparatus	Cat. # 300056
Dual stage Glass Micropipette Puller	Narishige International	Model 001-PC-10
Micrometer syringe	Gilmont Instruments	Cat. # GS-1100
Small Animal Stereotaxic Instrument	Kopf Instruments	Model 940-A
Electrode Manipulator	Kopf Instruments	Model 960
Vetcare chamber	Harvard Apparatus	Cat. # 340508
Heating Pad	Harvard Apparatus	Cat. # 341241
Leica DFC360 FX camera	Leica Microsystems	DFC360 FX
Leica DFC490 camera	Leica Microsystems	DFC490
Leica DM5500 microscope	Leica Microsystems	DM5500
Leica DFC3000 FX camera	Leica Microsystems	DFC3000 FX
Leica MZ16 FA microscope	Leica Microsystems	MZ16 FA
CY3 Filter	Leica Microsystems	Model # 11600231
FITC Filter	Leica Microsystems	Model # 11513880
A4 DAPI/UV filter	Leica Microsystems	Model # 11504162
Wheat germ agglutinin, Alexa Fluor 488 conjugate	Invitrogen	Cat. #W11261
Wheat germ agglutinin, Alexa Fluor 555 conjugate	Invitrogen	Cat. #W32464