Characterizing Extracellular Space in a Brain Slice Using Ion-Selective and Iontophoresis Microelectrodes

Protocol

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

1. Real-time Iontophoresis in Brain Slices

1. Place a 400 µm-thick brain slice in the recording chamber, ensuring that it is fully submerged in the flowing artificial cerebrospinal fluid (ACSF). Position the slice using a watercolor paintbrush and gently secure it with a grid.
2. Move both the iontophoresis microelectrode and the ISM above the field of interest on the brain slice. Submerge both in the flowing ACSF but above the slice.
3. Offset the voltage for both the reference and the ion-sensing channels to "0" mV. Wait for the voltage in both channels to stabilize. On the chart recorder, mark the voltage measured on the ion sensing channel of the ISM. Use this to calculate the baseline V parameter in Wanda.
4. Place the ISM and iontophoresis microelectrode 200 µm deep in the slice and 120 µm away from each other. Wait for the stabilization of the signal after moving the microelectrode into the brain slice.
   NOTE: The bias current applied to the iontophoresis microelectrode causes a small accumulation of tetramethylammonium (TMA). It is a common mistake to take a recording too soon and underestimate the signal buildup.
5. On the chart recorder, mark the stabilized voltage measured in the brain slice on the ion-sensing channel of the ISM. Calculate the voltage difference between the TMA signal measured in step 8.3 and step 8.4 and input this value into the "Baseline V (mV)" field in the Measuring Electrode box of the Wanda GUI.
6. On the left side of the GUI, ensure that all experimental parameters are correctly recorded/entered. Set "Medium" to "Brain," "Transport number" to the average value calculated for the iontophoresis microelectrode in step 7.4, and "Temperature" to the temperature of the bath containing the slice.
   NOTE: V must be recorded for each set of measurements. The baseline V will be converted by Wanda into the baseline C (mM) parameter (i.e., the concentration of TMA in the brain tissue).
7. Start the recording by clicking "Acquire" and allow it to take a full recording. Wait until the TMA signal returns to baseline before acquiring a new recording.
8. Take two to three successive recordings before removing the microelectrodes from the chosen brain location. Input the temperature measured into the Wanda software immediately before each recording.
9. Move both microelectrodes diagonally back to the surface of the slice. Raise both to at least 50 µm above the slice. Using the chart recorder, determine any change between the V measured now and its measurement from step 8.3.
10. Center the tips of the ISM and the iontophoretic microelectrodes relative to each other in the x-, y-, and z-axes. Obtain spacing changes, if any, from the display of the micromanipulator control box.

Materials

Name	Company	Catalog Number	Comments
amplifier for ISM	Dagan	Model IX2-700 Dual Intracellular Preamplifier	ion and reference voltage amplifier with N=0.1 (for reference barrel) and N=0.001 (for ion barrel) headstages
biological compound miscroscope (with 4x and 10x objective)			for chipping the microelectrode tips and inspecting microelectrodes; various suppliers, e.g. AmScope
borosilicate theta capillary glass tubing	Harvard Apparatus	Warner Instruments model TG200-4; order #64-0811	double-barreled glass tubing for ion-selective microelectrodes and iontophoretic microelectrodes; O.D. 2.0 mm, I.D. 1.4 mm, septum 0.2 mm, length 10 cm
chart recorder			to record continuously voltages on ion-selective microelectrode during calibration in tetramethylammonium standards and during RTI experiment; e.g. Kipp & Zonen type BD112 dual-cannel chart recorded, available refurbished
Commercial Software	The MathWorks	MATLAB, Data acquisition toolbox	for data acquisition and analysis using Wanda and Walter programs. Note that an academic license is available.
eye protective goggles	Fisher
fixed-stage compound microscope	Olympus	BX51WI	can use other compound microscopes with fixed stages
forceps	Fine Science Tools	#11251-10	to chip glass capillary; Dumond #5, preferably used and no longer needed for fine work
iontophoretic unit	Dagan	ION-100 and PS-100	ION-100 is a single channel iontophoresis unit +/- 130 V compliance; PS-100 is an external power supply; alternatives: e.g. Axoprobe-1A made by Axon Instruments (now Molecular Devices), out of production, check for availability of refurbished units (eBay and other sites)
microelectrode holder	WPI	M3301EH	to hold ion-selective microeletrode prefabricate for silanization and filling the tip of ion-selective barrel with liquid ion exchanger; WPI sells two versions of this holder, clear M3301EH and black M3301EH. In our experience, the clear M3301EH appears to be sturdier then the black M3301EH.
micromanipulator	Narishige	MM-3	to position ion-selective microelectrode prefabricate during silanization and filling the tip of ion-selective barrel with liquid ion exchanger; can be substituted with any three-axis micromanipulator in good working condition
objective 5x dry	Olympus	MPlan N
objective 10x water immersion	Olympus	UMPlan FL N	10x objective is water immersion, numerical aperture is 0.3, working distance is 3.3 mm
platform and x-y translation stage for fixed-stage microscope	EXFO	Gibraltar Burleigh	platform holds slice chamber, micromanipulators and accesorries, x-y translational stage moves microscope without compromising recording stability
robotic micromanipulator with precise x,y,z positioning	Sutter Instruments	MP-285	two mircomanipulators are needed to hold separately ion-selective microelectrode and iontophoretic microelectrode. Also possible to glue micropipettes in a spaced array (see text).
signal conditioning unit with low-pass filter	Axon Instruments	CyberAmp 320 or 380	no longer available from the manufacturer but may be available from E-Bay; alternatives: e.g. FLA-01 Filter/Amplifier from Cygnus Technology. This is a single channel instrument with a minimum cutoff at 10 Hz using a multipole Bessel filter but the company may be willing to modify it for a lower cutoff frequency (2 Hz) if needed.
silver wire	A-M Systems	#7830	diameter 0.015", bare (no coating)
slice chamber	Harvard Apparatus	Warner Model RC-27L	this is submersion slice chamber; do not use interface slice chamber
stereomicroscope			for silanization and filling the tip of ion-selective barrel with liquid ion exchanger; horizontally mounted; various suppliers
tetramethyammonium (TMA) chloride	Sigma-Aldrich	T-3411	5 M solution; CAUTION: acute toxicity (oral, dermal, inhalation), carcinogenicity, hazardous to the aquatic environment, see Sigma-Aldrich Safety Information for full description