TECHNICAL NOTE |
Correspondence to: Kazuhiro E. Fujimori, Dept. of Anatomy, Fukui Medical School, Matsuoka, Fukui 910-11, Japan.
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Summary |
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We have devised a simple method that combines retrograde labeling of projecting neurons and in situ hybridization histochemistry to examine mRNA expression in the retrogradely labeled neurons. First, projecting neurons were retrogradely labeled in vivo by injection of the lipophilic neuronal tracer DiI. The fluorescence of the labeled neurons in the brain slices was photoconverted into stable DAB precipitate by green light illumination. The slices were cut into thinner sections and processed for detection of specific mRNA by in situ hybridization. Using this highly sensitive method, we demonstrate here that the corticospinal tract neurons in newborn rats express mRNA for the cell adhesion molecule L1. TAG-1 mRNA was not detected in these neurons. Therefore, the present method provides an important tool to study the molecular expression of projection neurons during the development of neuronal circuitry. (J Histochem Cytochem 45:455-459, 1997)
Key Words: double labeling, in situ hybridization, L1, photoconversion, pyramidal neuron, rat, retrograde labeling, TAG-1
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Introduction |
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The mammalian central nervous system has complex but highly organized neuronal circuits composed of a tremendous number of neurons with a variety of types and subtypes. Different types of neurons typically show different patterns of axonal projection and select different target neurons for synapse formation. To identify specific types of neurons with specific axonal projection patterns, various neuronal tracers, especially retrograde tracers, have been used (
For combining in situ hybridization histochemistry with retrograde labeling, fluorescent tracers were used in most cases to label neuronal somata (
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Materials and Methods |
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In Vivo Retrograde Labeling of DiI
Sprague-Dawley rats staged at postnatal Day 1 (P1; day of birth = PO) were deeply anesthetized by hypothermia. The dorsal surface of the spinal cord was exposed at cervical level 2-3 by removing vertebral arches, adhesive muscles, and adipose tissue. A small crystal of DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; Molecular Probes, Eugene, OR) was injected into the median plane of the spinal cord (
Photoconversion
The brains were cut into slices 200µm thick with a microslicer. The slices were immersed in 3,3'-diaminobenzidine (DAB: 1 mg/ml in 0.1 M Tris, pH 7.4) solution and irradiated with green light to photoconvert the DiI fluorescence into DAB precipitation (
In Situ Hybridization
We essentially followed a standard protocol (
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Results |
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Two days after DiI injection into the spinal cord (Figure 1A), fluorescence was detected in the neocortex (Figure 1B). The laminar position of the labeled neurons in the section indicated that they were Layer V pyramidal neurons. DiI labeling was observed as granules in the cell somata and dendrites (
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The laminar expression patterns of TAG-1 and L1 mRNA were visualized by in situ hybridization in the P3 rat neocortex (Figure 2B and Figure 2C). Comparison of the laminar position of the retrogradely labeled neurons (Figure 2A) with laminar distribution of the mRNAs (Figure 2B and Figure 2C) in adjacent sections suggests that the Layer V pyramidal cells express L1 but not TAG-1. However, with such a comparison it is not possible to determine whether or not the retrogradely labeled Layer V pyramidal neurons express L1 and TAG-1.
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Using the combination of retrograde labeling and in situ hybridization methods, we demonstrated that L1 mRNA was expressed by the retrogradely labeled pyramidal neurons. Silver grains of hybridization signals of L1 mRNA overlapped the areas marked by brown DAB precipitation, as shown in Figure 3A. However, intense signals of TAG-1 mRNA were not observed in the retrogradely labeled pyramidal neurons.
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In the same section, we compared the in situ hybridization signals between areas with and without photoconversion but could not detect any differences between them. In addition, DAB precipitation did not appear to decrease after the in situ hybridization procedure (Figure 3A-D). Nonspecific binding of cRNA sense probes to the areas of DAB precipitation was not detected (Figure 3B and Figure 3D).
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Discussion |
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The method presented here combines DiI labeling of cortical pyramidal neurons and in situ hybridization histochemistry. One concern in combining the methods was the possibility that DiI labeling and/or DAB photoconversion might cause problems in the following in situ hybridization steps. Another concern was the possibility that the in situ hybridization method might cause disappearance of the retrogradely transported DiI. Although the photoconversion of fluorescent labeling into stable DAB precipitate was carried out before in situ hybridization, it involved only a single incubation in the DEPC-treated DAB solution. Messenger RNA is resistant to illumination with visible light, as demonstrated in the photobiotinylation of RNA (
DiI is an excellent anterograde and retrograde tracer with high sensitivity in both fixed and unfixed tissues. Therefore, DiI is suitable for investigation of neuronal circuits not only in embryos but also in the adult central nervous system (
In this technique, there are several problems that should be overcome with repeated trials. First, single DiI injection into the spinal cord reveals only a fraction of the corticospinal projection neurons in the neocortex. The area of illumination through a x 20 objective lens was so small that only a few DiI-labeled pyramidal neurons were illuminated at one time. Therefore, to collect enough of double-labeled samples and to draw a reliable conclusion, many sections should be processed for in situ hybridization with each cRNA probe. Second, in autoradiography, the emulsion-dipped sections should not be exposed for too long. If the silver grains cover the DAB precipitation, it may be hard to detect overlapping of the two signals under brightfield microscopy.
To demonstrate the usefulness of the double-labeling method, we used it to reveal that the corticospinal projecting Layer V neurons express L1 mRNA but not TAG-1 mRNA in newborn rats. Recent immunohistochemical study revealed that the pyramidal tract in developing mouse has TAG-1 immunoreactivity (
Because the present method is applicable to a wide variety of molecules involved in the control of axonal projection, it offers an important tool for the study of molecular mechanisms in formation of the neuronal circuitry.
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Acknowledgments |
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Supported by grant-in-aid #08271215 for Scientific Research on Priority Areas on "Functional Development of Neural Circuits," Ministry of Education, Science and Culture (to NT).
Received for publication June 10, 1996; accepted November 13, 1996.
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