Department of Biological Sciences, Florida Atlantic University
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Boerner, J., Godenschwege, T. A. Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection. J. Vis. Exp. (52), e3080, doi:10.3791/3080 (2011).
To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective (CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the intact head. In this preparation most nerves of the VNC should remain attached to their muscles.
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.
1. Dissection of the ventral nerve cord
Dissection and dye filling of the GF can be performed at room temperature. For all dissection steps cold (4°C) saline should be used to slow down cell metabolism and keep the neurons alive longer.
The dissection can be performed within 5 to 10 minutes. By using fresh, cold saline the dissected, undamaged nervous system will remain alive for at least one hour.
2. Intracellular GF fill
It is recommended to perform the dye filling as soon as possible after the dissection.
3. Representative Results:
A representative dye fill for one GF with Lucifer Yellow is shown in Figure 4A. The image was acquired as an image stack in Nikon Elements software using a spot camera mounted on the microscope. In this sample current was passed through the electrode for about 2 minutes. The GF is thoroughly filled and the large GF terminal in the thorax is well labeled (Figure 4A, ellipse). No postsynaptic neurons are visible in this case, although Lucifer Yellow is small enough to pass through gap junctions. If the dye is not injected long enough into the GF then trans-synaptic fills of the postsynaptic neurons may not been seen because the signal is too weak to be detected. Neurons postsynaptic to the GFs (Figure 4B, arrow heads) can be visualized more reliably using even smaller neuronal tracers, such as Neurobiotin. Injecting a 7% Neurobiotin in 2 M potassium acetate solution with a depolarizing current for 2 minutes is sufficient to label postsynaptic neurons strongly. Neurobiotin can be visualized for confocal microscopy using Streptavidin coupled to dyes of different wavelengths. Because Neurobiotin is not a fluorescent dye, co-injection of Rhodamin-Dextran or Alexa hydrazides in the same solution, can be used to monitor a successful injection into the GF (Figure 4C, from Boerner and Godenschwege, 20105).
Figure 1. Schematic drawing of a fly viewed laterally. The thorax is displayed with a longitudinal incision to visualize the internal structures. During the dissection, the dorsal longitudinal flight muscles (DLMs, blue) were cut without damaging the ventral nerve cord (VNC, deep red), which lies ventral to the flight muscles. The gut (green) lies above the VNC.
Figure 2. Dorsal view of a dissected fly. Two pins are placed behind the head to position the fly. The pins affixed through the flight muscles (DLM) hold the thorax open. Internal organs were removed to expose the ventral nerve cord (VNC).
Figure 3. Dye fill setup.
Figure 4. A) Z-projection of the posterior part of the VNC showing one GF filled with Lucifer Yellow. The image was acquired immediately after dye filling with a spot camera. In wild-type animals the terminal (ellipse) in the thorax is clearly visible. B) Confocal image of a GF dye fill with Neurobiotin in a wild-type fly. Both GFs (arrows) and neurons postsynaptic to the GFs (arrow heads) are labeled. Neurobiotin was visualized with Streptavidin coupled to Cy3. C) Confocal image of wild-type GFs filled with Alexa555.
The nervous system can be dissected without extracting it from the fly body. This has two advantages, first, the dissection causes little damage to the nervous system, and second, most nerves stay attached to the muscles and sensory organs. Performing the dissection, as described, prepares the sample for straight forward dye-labeling of the GFs. Conveniently, the motoneurons postsynaptic to the GFs stay attached to the muscles. Axons, therefore, are undamaged, keeping the neurons alive longer. In addition, damage to the nervous system is likely to disrupt gap junction function and thus is likely to affect the efficacy of trans-synaptic fills.
Lucifer Yellow, Rhodamine dextran, and Alexa Fluor hydrazide sodium salt (Invitrogen) can be used for instant visualization of the GF morphology under a fluorescent microscope, however signal amplification using secondary fluorescent antibodies are recommended for high resolution imaging with a confocal microscope5. Both, Lucifer Yellow as well as Neurobiotin label the postsynaptic cells via trans-synaptic fills, with Neurobiotin being much more reliable. However, Neurobiotin is non-fluorescent and requires immunohistochemistry following injection as well as the co-injection of a second fluorescent dye in order to monitor a successful injection.
This technique has been routinely used to compare the morphology of the GF terminals between individuals of the same genotype or between individuals of different genotypes5,6,7,8. In addition, the morphology of the synaptic terminal can also be correlated with its function by obtaining electrophysiological recordings from the circuit prior to dissection by standard procedures9,10.
Finally, although our demonstrated dissection method only exposed the VNC for dye-injection and imaging of the GF terminal, a subsequent dissection of the brain from the head capsule after dye injection would allow for imaging of the GF soma and dendrites in the brain as well.
No conflicts of interest declared.
The project described was supported by Grant Number R01HD050725 from the National Institute of Child Health and Human Development to T.A.G. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Child Health and Human Development or the National Institutes of Health. We thank members of the Godenschwege lab as well as Barbara Schreader for their input to the manuscript and video.
|Sylgard 184 Silicone Elastomer Kit||Dow Corning|
|Vannas Scissors Superfine||Academic Instruments||VS1023|
|Dumont Forceps Dumontstar 55||Fine Science Tools||11295-51|
|Austerlitz Insect pins||Fine Science Tools||26002-10||Ã˜ 0.1mm|
|Borosilicate Glass Electrodes||World Precision Instruments, Inc.||1B100F-4|
|Vertical Pipette Puller 700c||David Kopf Instruments||Model 700C|
|Lucifer Yellow CH dilithium salt||Sigma-Aldrich||L0259||1% in H2O|
|Fixed Stage Upright Microscope & Camera||Nikon Instruments||FN-1 & DS-U2 Camera|
|Plan Fluor 10X Air Objective||Nikon Instruments||CFI Plan Fluor 10X NA 0.3 WD 16mm|
|Fluor 40X water dipping Objective||Nikon Instruments||CFI Fluor 40X W NA 0.80 WD 2.0mm|
|3D hydraulic Micromanipulator||Narishige International||Model MW0-3|
|Amplifier||Getting Instruments, Inc.||Model 5A|