Developmental Neurobiology, St. Jude Children’s Research Hospital
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Smith, R., Taylor, J. P. Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction. J. Vis. Exp. (50), e2676, doi:10.3791/2676 (2011).
The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.
1. Preparation for Immunofluorescence:
2. Dissection of Larvae:
3. Removal of Tissue:
4. Immunofluorescence for Active Zones:
5. Mounting the Larval Samples on Slides:
6. Representative Results:
An example slide of dissected and immunostained larvae is shown in Figure 2. When the larvae dissections, stains, and mounting are done properly, the user can identify the larvae muscle of interest (Figure 3) in segments A2-A6. Hoanget al. provides a detailed description of muscle position as well as characterization of specific synaptic boutons (Hoang and Chiba, 2001). Using a high power objective the user can focus on a single synapse and take z-stack image. A representative maximum projection image of a portion of synapse 6/7 is shown (Figure 4). Then using imaging software, the active zones can be quantitatively evaluated for characteristics that are particular to your mutant phenotype. Examples of features that may be altered in a particular mutant phenotype include the total number, flourescence intensity, and spacing between active zones.
Immunofluorescence evaluation of the larval neuromuscular junction is a valuable system that can provide valuable insight into synaptic biology, although there are potential pitfalls that can limit the utility of this system. For example, frequently it is desirable to image the staining pattern of two or more primary antibodies in the same sample. The choice of appropriate primary antibodies, appropriate fluorophores, and appropriate negative controls is essential to success.
If imaging two antigens in your experiment, be certain that the primary antibodies are from different species so that each protein of interest can be uniquely identified with a fluorescently labeled secondary antibody. A recommended way to reduce background is to use a blocking agent containing the normal serum of the species from which the secondary antibodies were derived (e.g. use normal goat serum as a blocking agent when using goat anti-rabbit or anti-mouse secondary antibodies).
After planning the primary and secondary antibodies that you will use in your experiment, you must also consider including controls in your staining sets to ensure that the fluorescent signal that you are seeing is real and not just background. If possible, include negative control samples that do not contain the protein of interest. This is easily done in studies of exogenous transgene overexpression by including a wild type animal, but in some cases can be accomplished by using a homozygous mutant of your protein of interest. Another essential negative control, useful for measuring the background levels of your fluorescent signal, is to use the secondary antibody without including the primary. When imaging your resulting preps, it is important to understand that all of the fluorescent signal that you are seeing might not be real, and might be background from your secondary antibodies or from endogenous tissue background. You may need to normalize your images to consider background fluorescence when assaying for changes between fluorescence intensity.
Choice of fluorophore must also be made with care. Consideration of the excitation and emission spectra for each fluorophore and the ability to uniquely detect each signal is essential to avoid channel bleed through. If there is any uncertainty, a simple way to determine whether the extent of bleed through is to immunostain with a single fluorophore-labeled antibody at a time and check the image with each channel that you will be using in your final experiment. This should reveal if bleed through is occurring with your microscope filter set.
Figure 1. Flow chart of cuticle cutting and placement of pins 1-6. Click here to view a larger image
Figure 2. Placement of larvae on the final slide. Make sure that the larvae preps are exposed muscle side up and cuticle side down. Leave at least a larval body width in-between each sample.
Figure 3. When imaging, locate the muscles and segments of interest. Common neuromuscular junctions characterized innervate muscles 6/7, muscle 13, muscle 12, or muscle 4. On the opposite side of the ventral midline, there is a mirror image muscle structure of the shown hemi-segment. Use Muscle 31 as a marker to identify which segment you are imaging. Image the same synapses and segments for the rest of your samples, collecting many synaptic images per genotype that can be used for quantification.
Figure 4. A representative maximum projection image of a portion of the NMJ innervating muscle 6/7 is shown. NC82 (Bruchpilot) staining is shown in green. HRP pre-synaptic stain is shown in red. Note the active zone punctae that can be further quantified by imaging software.
For neurons, the synaptic terminal area is of critical importance, and is the bridge for proper communication between the post- and pre-synaptic cells. A powerful way to investigate the health of the neuron in disease models is to analyze proteins of the synaptic terminal by immunofluorescence. The immunofluorescence method presented here enables the researcher to examine many larvae simultaneously while limiting the environmental differences between groups. The central nervous system ofDrosophila third-instar larvae has many advantages including glutamatergic synapses, mapped and repeated synaptic terminals, accessibility, reproducibility and power of genetic manipulation. Specifically in Drosophila neuronal disease models, the levels of the active zone protein Bruchpilot has been altered. Because of the large number of larvae assayed simultaneously, the active zone analysis using the method presented enables the researcher to detect subtle differences between groups that could reflect a fundamental defect in the health of the neuron.
No conflicts of interest declared.
We thank Dr. Nael Alami and Dr. Nam Chul Kim for their helpful comments about this manuscript.
|Sylgard 184 Silicone Elastomer Base||Dow Corning||68037-59-2||After mixing allow for bubbles to rise slowly out by putting on slow rotator or allowing to sit for 30 minutes or more.|
|Stainless Steel Minutien PIns||Fine Science Tools||26002-10||Trim to approx. 3-4mm in length with regular scissors|
|Laminectomy Forceps (Blunt- Used for grasping pins)||Fine Science Tools||11223-20||Use as blunt forceps for grasping pins|
|Dissection Forceps||World Precision Instruments, Inc.||501985|
|SuperFine Vannas Scissors, 8cm long||World Precision Instruments, Inc.||501778|
|Mouse anti-Brp antibody||DSHB||NC82||Use 1:50 dilution|
|Cy3 Affinipure Goat Anti-Horseradish Peroxidase||Jackson ImmunoResearch||123-165-021||Use at 1:200 dilution|
|Alexa Fluor 488 Goat anti-Mouse IgG||Invitrogen||A11001||Use approx. 1:200 dilution|