TECHNICAL NOTE |
Correspondence to: N. Mark Tyrer, Dept. of Optometry and Neuroscience, U. of Manchester Institute of Science and Technology, Manchester, UK M60 1QD. E-mail: mark.tyrer@umist.ac.uk
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Summary |
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We describe staining protocols for serial semithin sections of Drosophila central ganglia that allow visualization of gene expression in particular neurons with counterstaining to display the ganglion architecture. Green fluorescent protein (GFP), expressed in a subset of sensory neurons from a selected enhancer trap line, is visualized by conventional immunohistochemistry with a peroxidase-linked antibody, and neural architecture is revealed by reduced silver staining. This makes visible in histological sections the same GFP-labeled cells seen with confocal microscopy, but with the especial advantage that neuropil structures are also revealed at the level of individual cells and neuron processes. Not only does this allow the physical relationships among intracellularly labeled neurons to be determined by reference to specific features in the neuropil but it also enables a function to be ascribed provisionally to particular regions of neuropil. These methods have particular utility for mapping morphological information on specific neurons in the context of central nervous system architecture, both in adult Drosophila and during development.
(J Histochem Cytochem 48:15751581, 2000)
Key Words: Drosophila, central nervous system, semithin sections, serial sectioning, gene expression immunohistochemistry, silver staining, database, three-dimensional reconstruction
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Introduction |
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Molecular genetic techniques, such as enhancer trapping, have given enormous impetus to the study of the brain in Drosophila because they allow noninvasive, selective labeling of potentially every neuron in the central nervous system (CNS). The use of a GAL4-responsive reporter with a bovine tau cDNA under the control of a yeast upstream activation sequence (UAS) is particularly powerful, revealing individual neurons in detail comparable with that of Golgi impregnation (
The next crucial analytical step is to relate such images of individual neurons to the detailed architecture of the central ganglia, to define their precise spatial relationships with each other and with features such as fiber tracts and commissures. When neurons are universally stained to reveal ganglion architecture, it is necessary to cut sections. Confocal microscopy is less successful when many features are stained, and only histological sections are accessible to the necessary staining techniques. The strategy currently favored for matching histological datasets from physical sectioning with optical datasets from confocal microscopy is to create a database of anatomic information such as the one provided by "Flybrain" (
There are three particular difficulties with this strategy. First, there are few features in central ganglia universally revealed by both methodologies that could act as suitable landmarks for matching datasets. Features such as ganglion contours, cell body positions, and neuropil boundaries are too approximate, and too inherently variable, to supply accurate reference points. Second, there are separate and different problems with 3D reconstruction from both methodologies that make image matching difficult. In confocal microscopy, image quality deteriorates at greater depths in the tissue, which introduces artifacts into the final reconstruction. Different artifacts from reconstruction from histological sections arise because alignment is less accurate than that of confocal sections, because of the physical distortions microtomy produces. Third, a "standard" ganglion ignores individual variations in size, shape and proportion of CNS structures. This can arise from experience (
All these difficulties could be resolved by a method that reveals a molecular genetic label in the same preparation as ganglion architecture staining. If individually labeled neurons could actually be visualized in the context of their cellular environment, it would resolve doubts and ambiguities in reconciling images from each methodology. Here we describe just such a method. We have devised staining protocols for serial semithin sections (
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Materials and Methods |
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The thoraco-abdominal ganglion complex was dissected from adult fruit flies, Drosophila melanogaster. The GAL4-enhancer trap line C42 was identified from a large screen of GAL4 lines performed in the Southampton laboratory. C42 was selected for reporter gene expression in a subset of sensory neurons from chordotonal organs in the femur, the wings, the halteres, and a multiscolophorous organ in the abdomen (
Immunolabeling
The GAL4 lines selected had been crossed to a UASGFP-tau reporter line using standard genetics. Staining in whole ganglia was done with an antibody to GFP and DAB staining, in a similar manner for making permanent preparations of Lucifer Yellow-labeled neurons (
Sectioning
The ganglia were dehydrated in an ethanol series, infiltrated with celloidin (Fisher Scientific; Leicester, UK) in etherethanol, and embedded according to the celloidin-wax sandwich technique (
Reduced Silver Staining
After dewaxing sections in xylol, slides were first washed in a 1:1 mixture of 100% ethanol and chloroform instead of the usual 100% ethanol, because the celloidin embedding medium is soluble in pure ethanol. They were then progressively hydrated in 95%, 70%, and 50% ethanol and then to distilled water.
The basic procedure followed was the silver staining method described by
For all preparations, the following protocol was used. The soak was for 3 hr in 20% silver nitrate in the dark. The impregnating solution was made as follows: 150 ml distilled water, 12 ml borax buffer at pH 7.0, 12 ml 1% silver nitrate, 6 ml lutidine. Slides were incubated in this for 24 hr at 50C, after which they were rinsed, first in distilled water for 5 min, then in 2% sodium sulfite for 2 min before returning to distilled water.
The developer solution was made as follows: 170 ml 9% sodium sulfite, 5 ml 5% silver nitrate, 11.25 ml 0.5% hy-droquinone. The slides were developed in the dark, stirring continuously. Development time was the only variable changed and was determined precisely by a stopwatch. Initially, 22 preparations were developed at five different times: 4 min, 4.5 min, 5 min, 5.5 min, and 6 min. The shorter development times produced lighter staining of a reddish color, whereas longer times produced more intense staining of a blue-black color, although both color and intensity of staining varied slightly even with the same development time. Although developing for 5.5 min gave good definition of neuropil features, in some regions the immunolabel became less distinct. The heavier staining in six preparations that were developed for 6 min tended to obscure much of the immunolabel and were therefore excluded, leaving 16 preparations in the final analysis.
After developing, the slides were rinsed in distilled water (5 min), running tapwater to remove all traces of developer (5 min), and distilled water again (5 min). They were then immersed for 5 min each in (a) 0.2% gold chloride (acidified with a few drops of glacial acetic acid), (b) 2% oxalic acid, and (c) 5% sodium thiosulfate, each solution interspersed with a 5- min wash in distilled water. After the sodium thiosulfate, the slides were washed for 20 min in running tapwater, dehydrated in an ethanol series, transferred to xylol, and coverslipped with Canada balsam.
Microscopy and Photography
Confocal Microscopy.
Labeled neurons were imaged with a Biorad MRC600 confocal microscope The ganglion was imaged with a x40 objective (NA 0.75) at an emission wavelength of 488 nm, and the fluorophore was imaged using a 560-nm dichroic and 522-nm emission filter. Optical sections were cut at consecutive intervals of 2 µm and saved as a Z-series. To obtain a 2D image of the labeled neurons, the Z-series was merged using the confocal software.
Light Microscopy. Stained sections were examined on a Leitz Dialux microscope and photographed with Kodak Elitochrome T and Kodak Gold 200 film using x63 (NA 1.25) and x100 (NA 1.32) oil immersion objectives.
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Results |
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GFP Expression in the C42 Line
The reporter gene expression in enhancer trap line C42 reveals a subset of chordotonal sensory neurons. Axons from these neurons enter the thoracic ganglion through each of the three pairs of leg nerves, forming a club-like projection with a "shaft" in each nerve root and a knob-like cluster of endings close to the midline. Smaller components enter from the wings through the anterior dorsal mesothoracic nerves and from the halteres through the haltere nerves (Fig 1A). A prominent bundle of axons from the abdominal multscolophorous organ enters by the first abdominal nerve and runs forward to contribute to the clusters of endings close to the midline (Fig 1A). A small population of motor neuron cell bodies is also labeled but the label does not extend to the branches of these neurons and so they do not interfere with the analysis of the sensory projections.
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Double Staining with Antibody and Reduced Silver
Reduced silver staining techniques are the traditional methods used for neuropil staining [
In our hands, with this type of semithin section, the Rowell method (1963) normally produces a predominantly red-purple color, with some large axon profiles having a gray color. The slightly red tinge after gold toning is apparently caused when the gold/silver particles are small compared with the wavelength of light. Lengthening development time shifts the color more to blue-black, which enhances contrast between neuropil staining and the orange-brown color of the peroxidase-labeled neuron profiles. However, the intensity of the silver staining also increases and, because the immunoreactive profiles also have argyrophilic elements, this can obscure the immunolabel. Preparations developed for 55.5 min showed best the basic architecture of tracts and commissures identified by other workers (e.g.
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More problematic is the interpretation in regions lightly labeled by antibody staining, which have a pale orange color. In some cases this orange label is located in well-defined structures, such as cell bodies (Fig 1E), and it is plain that these are cell components showing weak expression of the gene product. In other areas, however, the orange color appears less confined (e.g., in the endings in the "club"the sensory terminal clusters in central regions of neuropil seen in Fig 1B and Fig 1D). In these cases it is difficult to decide if the reaction product is located in profiles below the resolution of the microscope or if it has diffused from more heavily stained regions. This is a question that must be resolved by electron microscopy. Even so, definition at this level of resolution is clearly sufficient to map neuronal projections accurately to within at least a few micrometers in the context of neuropil structure.
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Discussion |
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We present here a technique that is easy, inexpensive, and which we believe has far-reaching implications for integrating neuroanatomic data for Drosophila, both in the adult CNS and at different developmental stages. First, it is versatile. The methodology is not confined simply to the definition of neuron relationships. It can be used with any antibody, whether to a reporter gene, a transmitter, a receptor, or to a substance such as intracellularly injected Lucifer Yellow. Consequently, it will be possible to plot the precise positions of, e.g., glial cells, to assign chemical identity to particular neurons, and to relate intracellular recordings to their anatomic context.
Second, the method offers the prospect of assignment of function to neuropil features with names that hitherto have had to be confined to their position or appearance. This promise has been implicit from the beginning of the neuroanatomic use of gene expression patterns (
Finally, the method provides a solution to the profound problem of integrating gene expression data with 3-D information on the anatomy of pathways and identified neurons in a database such as Flybrain. The combination of techniques we describe here provides a strategy that will allow, for the first time, the visualization of individual molecularly labeled neurons in the context of well-defined neuropil staining. Using neuropil features as landmarks for reference, it then becomes possible to relate specific neurons labeled in different preparations to each other with a precision that cannot be achieved by techniques based on the averaging of virtual images (
Such a map is an important step not only for developmental studies of individual neurons and their interactions but also for determining how neurons are connected to each other. Of course, neuronal connectivity cannot be decided from a database of information collected from light microscopy alone, but studies at the light microscopic level have a vital role. Establishing proximity of identified neurons is the logical first step in determining connectivity, and then this knowledge can be used to direct higher resolution and functional studies. As ultrastructural information and data from physiological and pharmacological experiments become available, they can be integrated with the database assembled from light microscopic techniques such as those we describe here.
Received for publication December 6, 1999; accepted May 24, 2000.
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