Visualizing Astrocyte Morphology Using Fluorescent Dye Iontophoresis in a Fixed Mouse Brain Slice

Abstract

Source: Moye, S. L., et.al. Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis. J. Vis. Exp. (2019).

This video demonstrates the use of fluorescent dye iontophoresis to study astrocyte morphology in fixed brain slices. It details the piercing of the astrocyte soma with an electrode to deliver an anionic, hydrophilic dye into the cytoplasm under a low-voltage current, enabling the dye to diffuse and label the soma and branches. Confocal microscopy is then used to visualize the labeled astrocytes, providing morphological insights.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Filling Astrocytes with Iontophoresis

1. Test the electrode.
   1. Place a brain slice gently into a glass bottom dish filled with 0.1 M PBS at room temperature. Hold the slice in place with a platinum harp with nylon strings.
   2. Ensure that the electrode is connected to a voltage source and place the ground electrode into the bath containing the brain slice.
   3. Move the objective to the brain region of interest.
   4. Lower the electrode into the solution. Under the bright field, move it to the center of the field of view and examine it carefully with the 40x water immersion lens to ensure that it appears clear and without debris or bubbles. If there is anything clogging the electrode tip, replace it with a new one.
      NOTE: Clogging is a concern for the 200 MΩ electrode. With centrifugation and filtration of the dye solution before every experiment, this should not be a frequent problem. However, because the tip of the electrode is small, tissue may sometimes become stuck in the opening. The authors have not found a way to prevent this, but it can be easily dealt with by simply using a new electrode.
   5. Under the bright field, slowly lower the electrode toward the slice stopping just above the surface.
2. Fill astrocyte with iontophoresis.
   1. Identify astrocytes 40−50 µm below the slice surface with the infra-red differential interference contrast (IR-DIC). Look for cells with elongated, oval-shaped somata about 10 µm in diameter. Once an astrocyte is chosen, move it to the center of the field of view.
      NOTE: A good cell for iontophoresis has clear, defined borders around it. Do not choose cells too close to the surface of the slice because they cannot be completely filled. It takes some practice over a few days to be able to routinely identify astrocytes for dye filling. Depending on the brain area used for the experiment, the number of astrocytes may vary. This may affect the time needed to identify an astrocyte before filling.
   2. Slowly lower the electrode tip into the slice, navigating through the tissue, until it is on the same plane as the cell body.
      NOTE: Move electrode slowly to avoid damaging the tissue.
   3. Once the cell body of the astrocyte is clearly visible and outlined, slowly and gently advance the electrode forward. Move electrode until the tip impales the soma of the cell. Move the focus of objective slowly up and down to note if the electrode is inside the soma.
      NOTE: The electrode tip must be inside the cell body, and a small indentation on the soma should be observed. Do not move the electrode any further to avoid the tip going through the cell.
   4. Once the electrode tip is inside the cell, turn on the stimulator at ~0.5−1 V and continuously eject current into the cell. Using the confocal microscope, watch the cell fill. Increase the digital zoom to see the details of the cell and make sure that the tip of the electrode is visible inside the cell.
      NOTE: Lower the voltage if it appears that dye is leaking out of the cell or filling other cells in the vicinity. If it appears that dye is still leaking out of the cell, pull electrode out slowly and find another cell. It is important that the dye does not leak to have a high signal/ background ratio in the final image.
   5. Wait for about 15 min until the finer branches and processes appear defined, turn off the voltage and gently withdraw the electrode tip from the cell.
3. Image the filled cell.
   NOTE: Imaging can be performed immediately after filling. An objective with a higher numerical aperture (NA) results in better resolution.
   1. Wait until the cell returns to its original form before imaging (15−20 min) with 40x objective. To image the cell, adjust the setting on the confocal to make sure that the finer branches and processes appear defined.
   2. Set up a z-stack with a step size of 0.3 µm. While imaging, move the objective until there is no signal from the cell and set that as the top. Then, move the objective down (focusing through the cell) until there is no signal, set that as the bottom.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Borosilicate glass pipettes with filament	World precision instruments	1B150F-4
Confocal laser-scanning microscope	Olympus	FV1000MPE	A similar alternative can be used
Imaris software (Version 7.6.5)	Bitplane Inc.		A similar alternative can be used
Lucifer Yellow CH dilithium salt	Sigma	L0259
Lucifer Yellow CH dipotassium salt	Sigma	L0144
Objective lens (40x)	Olympus	LUMPLFLN 40XW	A similar alternative can be used
Objective lens (60x)	Olympus	PlanAPO 60X	A similar alternative can be used
PBS tablets, 100 mL	VWR	VWRVE404	An identical alternative can be used
Pipette micromanipulator- Model ROE-200	Sutter	MP-285 / ROE-200 / MPC-200	A similar alternative can be used
Potassium Chloride	Sigma	P3911	An identical alternative can be used
Stimulator- Model Omnical 2010	World precision instruments	Omnical 2010	A similar alternative can be used