Abstract
In this protocol, we introduce an effective method for voltage-sensitive dye (VSD) loading and imaging of leech ganglia as used in Tomina and Wagenaar (2017). Dissection and dye loading procedures are the most critical steps toward successful whole-ganglion VSD imaging. The former entails the removal of the sheath that covers neurons in the segmental ganglion of the leech, which is required for successful dye loading. The latter entails gently flowing a new generation VSD, VF2.1(OMe).H, onto both sides of the ganglion simultaneously using a pair of peristaltic pumps. We expect the described techniques to translate broadly to wide-field VSD imaging in other thin and relatively transparent nervous systems.
Highlights
As part of the dissection process, removing the sheath from the surface of the target ganglion is necessary to ensure that the dye can reach the neurons
During voltage-sensitive dye (VSD) imaging, the nervous system must be strictly immobilized in order to suppress motion artifact
All those steps are critical for successful whole-ganglion VSD imaging using a double-sided microscope
Summary
Refer to Tomina and Wagenaar (2017) for details on building a double-sided microscope. Glass capillary (standard-wall 1.20 x 0.68 mm, 4”, A-M Systems, catalog number: 627000) b. Coarse dissection scissors (e.g., Heritage, model: Electricians Scissors 103C) for cutting body wall tissue 3. Perfusion pump head (Easy-Load II Head, Masterflex L/S, Cole-Parmer Instrument, catalog number: EW-77201-60) 2. Perfusion pump driver (Economy drive, Masterflex L/S, Cole-Parmer Instrument, catalog number: 07554-80) 3. Glass cutting ceramic tile (Sutter Instrument, catalog number: CTS) 5. Instrument for drilling a hole in dye-loading/recording dishes (e.g., a lathe such as: Jet Tools, model: BDB-1340A, catalog number: 321102AK). Upright fluorescent microscope (Olympus, model: BX51) 2. Water-immersion 20x objective lens for top microscope (Olympus, model: XLUMPlanFLN20XW) 4. 5x objective lens top microscope (Olympus, model: MPlan FLN) 5. 20x objective lens for bottom microscope (Olympus, model: UCPlanFLN) 6.
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