utexas.edu

LTP Physiology Protocol

Solutions

	Standard ACSF is (in mM) NaCl, 117; KCl, 5.3; MgSO₄, 1.3; NaH₂PO₄, 1; NaHCO₃, 26; glucose, 10; CaCl₂, 2.5. Add salts in that order (calcium should be added after bicarbonate).
	ACSF can be made a few days in advance, provided it is filtered and stored at 4°. Warm it to room temp before using.
	If any drugs are to be used, they can be added on the day of the experiment. Aqueous and DMSO solutions can be made at 1000X in advance and added to the ACSF.

Electrode Placement

	Fill recording electrode with 120 mM NaCl by first backfilling the tip in a dish, then filling halfway up with a 30 gage filling needle. Place a rubber O-ring close to the tip to catch water drops. Attach to headstage.
	Turn on all rig equipment, including speaker. Be sure that stimulators, Master-8 and manipulators are on, as turning them on (and off) produces a current.
	Record temperature in room, tank temp from FHC heater, and well temp from Digisense.
	Choose a slice which is on lamella and looks to be of even thickness. Turn tank (not net or disc) so that the CA1 cell body layer is at about a 45° angle on the right to the front of the table.
	Place micromanipulators roughly by moving stands. Lower electrodes and move until their tips are all over the slice. If any electrodes cross each other or block the view of the others from either eye move the chamber and/or manipulators until this is remedied. Leave electrodes in chamber for a few minutes to warm up and wipe condensation off of stimulating electrodes with filter paper.
	Turn out room lights and manually lower all electrodes until they are within microns of the slice. Recording electrode should be located in center of CA1, about 150μm from the cell body layer (or halfway between stratum pyramidale and the hippocampal fissure) in stratum radiatum. Stimulating electrodes should be 400μm on either side of it in a line parallel to the cell bodies.
	If using a long thin tipped recording pipet, the tip must be broken to reduce resistance. Move the recording pipet away from the slice and over the net. Lower until it touches the net, then move sideways until it bends and continue until the tip breaks off. A pipet pulled at 59.5° on the Narishige puller starts with a resistance of about 8MΩ, which goes down to below 4MΩ after breaking. This is low enough to prevent most noise problems, but if the recording is noisy be suspicious of the electrode. Check for noise by placing the pipette in the bath away from the slice.
	Tighten screw on the recording manipulator to keep the electrode from drifting. Lower recording electrode first using motor (forward Z is down). Lower slowly until a response is audible from the speaker. Continue to move the electrode down about 50μm.
	Lower stimulating electrodes one at a time by giving pulses from the Master-8. Each pulse moves the electrode about 25μm. Stop as soon as a response is heard on the speaker. Watch carefully- a response may not be heard, but when the electrode hits the slice area around it looks darker.
	Lower each stimulating electrode 50μm, and recording electrode another 50μm for a final depth of 100μm. Turn off light, cover tank with a piece of grounded aluminum foil, and don’t touch anything on the air table again.
	If any of the electrodes are not placed correctly or are too deep, move on to another slice. Electrodes should not be replaced once they are in.
	Wait at least 15 minutes after electrode placement before stimulating to allow the cells to recover (somewhat) from the insult.

Standard LTP experiment

	IO: Starting at 25µA, stimulate every 30s, increasing intensity by 25µA each time. Determine response threshold and population spike threshold. Test one pathway at a time with ascending pulses, then decrease intensity back down to 25 to make sure that both curves are stable.
	Set the stimulus intensity for each pathway according to the pop spike threshold (i.e., 50%), depending on the experiment.
	Establish a baseline for at least 45min by giving test pulses every 2 minutes, stimulating one side 30s after the other.
	If the baseline is stable, chose one pathway as experimental and deliver desired stimulation.
	Theta burst stimulation consists of 10 bursts at 5Hz of 4 pulses at 100 Hz, given at 30s intervals.
	For the first minute after TBS, give a test pulse to that pathway every 10s.
	Resume baseline test pulses and continue until the end of the experiment.

Fixation

	Have fix (6% gluteraldehyde / 2% paraformaldehyde in 0.1M cacodylate buffer with 2mM Ca²⁺ and 4mM Mg²⁺) thawed and ready. The falcon tube containing the fix can be thawed in warm water if it wasn’t removed from the freezer early enough. Place water loads in the microwave and check central cold spot with neon bulb array. Invert fix tube to mix and decant 10mL into a small labeled glass jar. Leave fix in the hood until ready to use.
	Place water loads in the microwave. Three 500mL plastic bottles should be filled with water, two placed in the two front corners of the microwave and one at the back. Never run the microwave without water.
	Run microwave for a minute to warm up magnetron. Leave neon bulb array inside to confirm cold spot at the center.
	Place a glass ring in the center of a 35mm Petri dish, fill with 5mL of fix, and place in center of microwave.
	When it is time to fix the slice, stop data acquisition by the computer and retract electrodes slowly and completely, reversing the placement process.
	Using dedicated forceps, carefully lift net with slice out of the chamber and invert over glass ring in fix dish.
	Microwave at 750W or higher until the fix reaches 35-50° (the length of time should be determined in advance using 5mL of water, about 30s in the Amana).
	Record final temperature of fix and move net to glass jar of fix and leave in the hood overnight.
	Record room, tank, and well temperature and time.