We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.
Part 1: Tissue Preparation
Part 2: Neuronal Bouton Labeling with fluorescently labeled primary antibody against HRP.
Part 3: Perisynaptic space labeling with dextran flurophore conjugate
Part 4: Mounting the tissue for visualization (with confocal microscopy).
If you do not need to perfuse your prep (short observations) or you want to keep the volume of bathing HL-6 small, use the double bridged slide method
If you want to perfuse your prep, try using a perfusion chamber. We used a modified Chamber from Warner Instruments.
Details for both follow.
Mounting prep on a double-bridged slide.
Part 5: Representative Results:
This procedure permits long- term imaging of live labeled proteins and cell processes. The in situ tissue prep we described has an intact and functioning CNS, PNS and reflex circuits. This tissue prep has advantages over standard larval fly muscle protocols, where the larval body wall muscle is stretched (when it is pinned out). Stretching can distort synaptic morphology and trigger reflex based contractions. Our inside out prep was mechanically stable, and had exceptionally good optics that facilitated high resolution analysis of cellular changes in real time. In addition the inside out preparation survived for up to an hour and allowed visualization of synaptic changes over a long time course.
During dissection, take care to avoid damaging the PNS or CNS. Also avoid tearing or pulling on the tracheae as this damages the muscle cells. Keep the body wall muscle as relaxed as possible. Chilling the HL-6 and the tissue relaxes the body wall muscle and reduces trans-body wall tension. Calcium (2mM) in the in the artificial hemolymph is essential for the health and morphology of the glial cells.
Imaging must be performed on stable, non-contracting muscle tissue. While 5 mM Glutamate efficiently blocks neurally evoked muscle contractions, the Calcium channel blocker Nifedipine may be a useful alternative. Nifedipine at 10 micromolar blocks most (but not all) nerve evoked muscle contraction. Nifedipine blocks voltage gated Calcium channels on the muscle cell membrane more effectively than channels associated with neurotransmitter release, thus blocking muscle contraction while permitting normal NMJ synaptic function (Morrales et al 1999).
Using the antibody labeling procedure, we robustly labeled the extracellular carbohydrate residues expressed on neuron terminals, which are recognized by the anti-HRP primary antibody. We obtained excellent labeling using the anti-HRP antibody from Jackson labs, available pre-conjugated to a variety of fluorophores (Cy5- in our studies). This approach may be adapted for use with any primary antibody that recognizes an extracellular epitope, for example labeling extracellular matrix molecules.
The in situ “inside-out” tissue preparation, coupled with a modification of the protocol used by Stork et al 2008, is useful for probing cellular diffusion barriers. Fluorescent dextran dye diffusion might also be useful for timing and visualizing perfusate and drug entry into inter-cellular spaces, such as synaptic clefts.
With fluorescent dextran dyes, it is critical to use a small volume perfusion chamber. If there is too “thick” a boundary layer of dye solution covering the structure you seek to image, the dye solution may obscure the feature of interest, because of lack of contrast between dye infused spaces and the surrounding structures.
Finally, our structural labeling techniques require a high resolution imaging system suitable for live tissue. We used spinning disc confocal microscopes (Quorum technologies and Perkin- Elmer systems) in conjunction with Volocity software (Improvision); a long working length, high numerical aperture 63X water immersion lens; and lasers and filters appropriate for visualizing GFP, dsRed and far red fluorophores. For imaging we used custom formulated microscope objective oil from Cargill labs to match the refractive index of the artificial hemolymph, HL-6, in conjunction with the 63X water immersion lens. Without this oil, refractive error was significant and resulted in distorted images. Our in situ preparation might be used with other 3-D imaging systems, such as the API DeltaVision Scanning Restoration system. However, care must be taken to overcome the high levels of light scatter from the thick, live tissue preparation.
This project was funded by the CIHR and NSERC. We would like to acknowledge Barb Jusiak for contributing to the creation of the fly strains expressing dsRed labeled SSR (BJ line), and the UBC Bio-imaging Facility.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
HL-6: Artifical Drosophila hemolymph, with 5 mM L-glutamate added, and 2 mM Calcium. | Reagent | NA | NA | 5 mM L-glutamate blocked muscle contractions. We used Molecular grade L-Glutamate (Sigma). 2 mM Calcium is close to physiological Calcium levels in natural larval hemolymph. References: Macleod et al 2002 and Macleod 2004 |
Dextran, Alex Fluor 680; 10,000 MW, anionic, fixable | Reagent | Molecular Probes/Invitrogen | D34680 | Use a small volume perfusion chamber to keep the total volume of dye low |
Anti-HRP-CY5 conjugate (goat) | Reagent | Jackson ImmunoResearc | 123-175-021 | Dilute 2.0 mg into 1 ml ddH2O; aliquot into 4 microliter aliquots. Freeze at –20C. Dilute one aliquot into 100 microliters of HL-6 |
Alexa 647 antibody labeling kit | Reagent | Molecular Probes/Invitrogen | A10475 | We prepared a total of 80 micro liters of conjugated primary antibody, and stored as 2 microliter aliquots. We diluted each aliquot into 100 microliter of HL-6 for labeling. |
Custom Formulated Objective Oil, refractive index 1.3379 | Reagent | Cargill Labs | Custom Formulated | |
Ultra Fine Forceps | Tool | Fine Science Tolls | 11252-23 or 11295-20 | |
Spring scissors | Tool | Fine Science Tools | 91500-09 | |
Ultra fine clipper scissors | Tool | Fine Science Tools | 15200-00 | |
Perfusion Chamber RC 20 Series | Tool | Warner Instruments | 64-02222 | |
Spinning Disc confocal | Microscope | Quorum | Quorum Wave FX | Mounted on a Leica DMI6000 Inverted Microscope |