JoVE   
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Biology

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment

|   

JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You do not have subscription access to videos in this collection. Learn more about access.

 JoVE Biology

Loading Drosophila Nerve Terminals with Calcium Indicators

1, 1

1Department of Physiology, University of Texas Health Science Center at San Antonio (UTHSCSA)

Article
    Downloads Comments Metrics

    You must be subscribed to JoVE to access this content.

    This article is a part of   JoVE Biology. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.

    Recommend JoVE to Your Librarian

    Current Access Through Your IP Address

    You do not have access to any JoVE content through your current IP address.

    IP: 54.196.196.62, User IP: 54.196.196.62, User IP Hex: 918864958

    Current Access Through Your Registered Email Address

    You aren't signed into JoVE. If your institution subscribes to JoVE, please or create an account with your institutional email address to access this content.

     

    Summary

    Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

    Date Published: 7/30/2007, Issue 6; doi: 10.3791/250

    Cite this Article

    Rossano, A. J., Macleod, G. T. Loading Drosophila Nerve Terminals with Calcium Indicators. J. Vis. Exp. (6), e250, doi:10.3791/250 (2007).

    Abstract

    Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

    Protocol

    1. Select a clean dissection dish that has not been exposed to any fixatives.

    2. Dissect a wandering 3rd instar Drosophila larva in Schneider's Drosophila Medium containing Ca2+ and L-glutamine, (do not cut any nerves or damage muscle fibers Nos. 7, 6, 13 or 12).

    3. Select a glass filling-pipette with a 12 micron tip (internal diameter).

    4. Using a syringe and tubing (to apply negative pressure to the pipette) ensure that the pipette tip is not obstructed.

    5. Select a fine plastic filling-filament that can be inserted down the length of the glass pipette.

    6. Draw ~ 1 cm of 5 mM dextran-conjugated Ca2+-indicator into the plastic filament.

    7. Cut all segment nerves.

    8. Support the pipette on a ramp that will allow the pipette tip to approach the ventral midline of the dissected larva.

    9. Draw the cut end of a nerve to segment No.4, without pinching the nerve, into the end of the pipette (include a small amount of Schneider's medium).

    10. Remove the tubing and insert the plastic filament into the pipette until the end of the filament is within 50 microns of the cut end of the nerve (avoid touching the nerve).

    11. Eject sufficient Ca2+-indicator onto the nerve ending to increase the volume of the Schneider's medium by about 33% (final concentration should be < 2mM). Important - This must be completed within 5 minutes of cutting the nerve.

    12. Place the preparation in the dark at room temperature while the nerve loads.

    13. After 40 minutes remove the Ca2+-indicator using the filament.

    14. Leave the pipette in place and fill it completely with fresh Schneider's medium, as this will be used to apply stimulating pulses to the nerve.

    15. Allow the Ca2+-indicator to equilibrate in the nerve for at least 60 minutes, but no more than 4 hours, before commencing Ca2+-imaging.

    16. Rinse the preparation with fresh Schneider's medium every 30 minutes while it is equilibrating.

    17. 20 minutes before imaging replace Schneider's medium with Hemolymph-Like No.6 solution (HL6; Macleod et al. 2002; 2003).

    18. L-glutamic acid or glutamate can be added to HL6 solution at 7mM to desensitize postsynaptic glutamate receptors to prevent nerve-evoked muscle contraction (Macleod et al. 2004; Reiff et al. 2002; 2005).

    Subscription Required. Please recommend JoVE to your librarian.

    Disclosures

    The authors have nothing to disclose.

    Materials

    Name Type Company Catalog Number Comments
    Schneider’s Insect Medium Reagent Sigma-Aldrich S0146 must contain L-glutamine and calcium
    L-glutamic acid monosodium salt hydrate Reagent Sigma-Aldrich G1626

    References

    1. Macleod, G.T., Hegstrom-Wojtowicz, M., Charlton, M.P. & Atwood, H.L. Fast calcium signals in Drosophila motor neuron terminals. J. Neurophysiol. 88, 2659-2663 (2002).

    2. Macleod, G.T., Suster, M.L., Charlton, M.P., Atwood, H.L. Single neuron activity in the Drosophila larval CNS detected with calcium indicators. J. Neurosci. Methods. 127, 167-178 (2003).

    3. Macleod, G.T., Marin, L., Charlton, M.P., Atwood, H.L. Synaptic vesicles: test for a role in presynaptic calcium regulation. J. Neurosci. 24, 2496-2505 (2004).

    4. Reiff, D.F., Thiel, P.R., Schuster, C.M. Differential regulation of active zone density during long-term strengthening of Drosophila neuromuscular junctions. J. Neurosci. 22, 9399-9409 (2002).

    5. Reiff, D.F., Ihring, A., Guerrero, G., Isacoff, E.Y., Joesch, M., Nakai, J., Borst, A. In vivo performance of genetically encoded indicators of neural activity in flies. J. Neurosci. 25, 4766-4778 (2005).

    Comments

    6 Comments

    I would like to know what do you use as dissection pins for the larvae, and where do you get them from?
    With thanks
    Eyal Gruntman
    Cold Spring Harbor Labs, NY
    Reply

    Posted by: AnonymousMay 26, 2009, 4:10 PM

    Hello Eyal. You can purchase the entomology pins from Fine Science Tools (FST) at Foster City, CA (Cat. No.²600²-10).
    Reply

    Posted by: AnonymousMay 26, 2009, 5:41 PM

    Would you please provide me the catalogue numbers for the micro-instruments used in the larva dissection? I am starting similar dissections and have found considerable difficulty with the available intruments.
    Thanks,
    Mike Quinn
    Brock University, ON
    Reply

    Posted by: Michael Q.January 19, 2010, 4:54 PM

    Hello Mike. We use scissors from Fine Science Tools (FST) (Cat.No.15000-08) and No. 5 forceps, also from FST (Dumoxel).
    Reply

    Posted by: Greg M.January 19, 2010, 5:14 PM

    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Metrics

    Waiting
    simple hit counter