Laser-guided Neuronal Tracing In Brain Explants

Summary

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

Abstract

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

Introduction

In order to define a certain neuronal (micro)circuit, one must start by finding the various participants of said circuit, and their connection pattern. Ever since Waller's publication about neurofiber tracing through lesioning¹ a large variety of neuroanatomical tracing techniques has been established. Some of these techniques can be applied in fixed tissue post mortem^2-4, others rely on the active transport of the dye in live neurons, as discovered in 1971^5-6. The latter can be further subdivided in two groups discriminating between methods taking advantage of active retrograde (from the injected area to the source of a g....

Protocol

1. Optical Genotyping

1. Optical Genotyping of Mouse Pups

1. Check for expression of the respective fluorescent marker using a laser pointer of the appropriate excitation wavelength (405 nm in the experiments described here) and corresponding filter goggles blocking the excitation wavelength but passing the emission wavelength (450 - 700 nm in the experiments described here). Point the laser pointer at the back of the head or the spinal cord of the mouse pup (see Figure 1). Avoid shining the laser into the eyes and lengthy exposure of the skin to laser light.

2. Optical Genotyping ....

Results

Figures 1 and 2 show how the laser pointer and laser goggles can be used to quickly and inexpensively genotype GFP positive animals from a litter. In cases of young mouse pups, the technique can be used to noninvasively identify GFP label in the animal's brains through the skull and overlying skin (Figure 1A - D). Emitted fluorescence can be seen through the skin and the skull of mouse pups at least up to postnatal day 3 (Figure 1C). The procedure is shown in Fig.......

Discussion

A general strength of in vitro tracer electroporation, as opposed to in vivo tracing studies, is that it gives researchers better access to the brain area of interest and hence, does not involve expensive stereotactic (and often electrophysiological) equipment. In addition, the survival period required for the brain explants spans only a few hours (1 - 4) instead of days or even weeks in the case of in vivo tracer injections (see a detailed review on the use of dextran amines and other tra.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium chloride	Sigma-Aldrich	S7653	All chemicals are from Sigma-Aldrich, unless noted otherwise.
Potassium chloride		P9333
Potassium phosphate monobasic		P5655
Sodium phosphate di-basic		S907
Magnesium chloride		M2670
Calcium chloride		C5080
Glucose		G7528
Sodium bicarbonate		S6297
Ascorbic acid		A4544
Myo-inositol		I5125
Sodium pyruvate		P2256
Bovine serum albumine		A2153	optional, for additional (immuno)histochemistry
Triton-X-100		X100	optional, for additional (immuno)histochemistry
Poly(ethylene glycol), 8000 MW		P2139	optional, for brain clearing
Formamide	Fisher Scientific	F84	optional, for brain clearing
Choleratoxin subunit-b	Molecular Probes	C-34776 (Alexa 555)
Dextrane tetramethyl-rhodamine	Molecular Probes	D-7162 (Alexa 555)
Fluorescent Nissl	Invitrogen	N-21479 (blue)	optional
Paraformaldehyde	Fisher Scientific	SF93
Agarose	Invitrogen	16520
Fluoromount-G	Southern Biotech	0100-01
Pentobarbi-tal	Vortech Pharmaceuticals	Fatal-Plus
Borosilicate glass filaments	Harvard Apparatus	G150F-10
Pipette puller	Zeitz Instruments, Germany	DMZ Universal Puller
Perfusion setup	Custom-made
Laser pointer	laserpointerpro.com, Hong Kong	HK-88007294 (405 nm)
Filter/safety goggles	Dragon Lasers, China	LSG09 (band-pass 450-700 nm)
Bionocular microscope	Wild Heerbrugg, Switzerland	Wild M3	Equipped with high-intensity illuminator (MI-150; Dolan-Jenner Inc.)
Picospritzer	Parker Instruments	Picospritzer III
PC with installed MC Stimulus software	Multi Channel Sys-tems, Germany (software)
2-channel stimulator	Multi Channel Sys-tems, Germany	STG-1002
Stimulation isolation unit	A.M.P.I., Israel	Iso-Flex
Micromanipulator	Narishige, Japan	YOU-1
Vibratome	Leica, Germany	VT1000S