Abstract
The delivery of tracers into populations of neurons is essential to visualize their anatomy and analyze their function. In some model systems genetically-targeted expression of fluorescent proteins is the method of choice; however, these genetic tools are not available for most organisms and alternative labeling methods are very limited. Here we describe a new method for neuronal labelling by electrophoretic dye delivery from a suction electrode directly through the neuronal sheath of nerves and ganglia in insects. Polar tracer molecules were delivered into the locust auditory nerve without destroying its function, simultaneously staining peripheral sensory structures and central axonal projections. Local neuron populations could be labelled directly through the surface of the brain, and in-vivo optical imaging of sound-evoked activity was achieved through the electrophoretic delivery of calcium indicators. The method provides a new tool for studying how stimuli are processed in peripheral and central sensory pathways and is a significant advance for the study of nervous systems in non-model organisms.
Highlights
After conventional antibody staining against Neurobiotin[10] which served to enhance tracer detection, we observed numerous stained fibers reaching the entire length of the CNS (~20 mm), with the cell body and main dendrites of the cercal medial giant interneuron (MGI)[11] clearly identifiable in the terminal ganglion as well as its putative axonal arborizations in the brain
All functional imaging presented in this study was recorded about 6 hours after calcium indicator delivery, as this minimum time was required to allow a proper spreading of the dye
Neuronal activity in the neuropils could be imaged 24 hours after dye delivery in the auditory nerve typically resulting in a lower ΔF/F, possibly due to the indicator not being retained within the neurons
Summary
Current injection times of 15 seconds were sufficient to deliver the calcium indicators Fluo-6 (Fig. 3) and Oregon Green (Supplementary Fig. 3) into the afferents; longer injection times overloaded the neurons with dye and reduced their viability With this protocol the calcium indicators spread within 6 hours into the metathoracic auditory neuropils. To the best of our knowledge our method of dye delivery is currently the only one that allows simultaneous anterograde and retrograde labelling of fiber populations in insect peripheral nerves and that allows the labelling of local interneuron populations in fully-intact ganglia and brains This confirms that this technique of electrophoretic dye loading can be successfully used for functional labelling of populations of neurons in various locations in the insect CNS. Plasmids encoding fluorescent proteins for cell labelling would require longer survival times but would overcome the issue of local extracellular dye buildup at the injection site, as well as advance the possibility of genetic modification in more systems
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
