Ex Vivo Electrophysiology

First prepare a 10x stock aCSF solution by fist add about 200 mL of ddH20 water into a clean 2L flask. Then add each of the following powders:

§NaCl: 1250 mM

§KCl: 25 mM

§NaHCO3: 250 mM

§NaH2PO4: 12.5 mM

Next, fill the flask approximately three quarters with ddH2O and using a magnetic stirrer, allow the solution to be thoroughly dissolved. The mixed solution should then be brought to a final 2L volume and stored in a 2L glass bottle and refrigerated at 4°C.

Note: the 10x stock aCSF can be used for up to a month and should be remade fresh after that time.

Prepare 1 liter of 1X aCSF (to be used as both perfusate and cutting solution):

-Fill a 2L flask with about 450 mL of ddH20.

-Add 4 mL of CaCl₂solution to the bottle.

-Add 4 mL of MgCl₂solution to the bottle.

-Add 5.02 g of glucose (final concentration of 13.93 mM)

-Add 200 mL of a 10X stock aCSF solution.

-Swirl mixture around well by hand for a few seconds.

-Fill remainder of flask to 2 L with ddH20.

-Carbogenate solution for at least 10 minutes before using.

Using continuously carbogenated 1x aCSF: §Fill a small slice holding chamber with the 1x aCSF and add 5 mM of L-glutathione at approximately 1:1000x and 1 mM of Na-Pyruvate at 1:300x .

-Set the chamber aside near a 37 °C heated water bath while the solution inside the holding chamber is continuously carbogenated.

-Additionally, decant approximately 100 mL of 1X aCSF into a small glass beaker kept cold on ice while carbogenated (to be use for perfusate)

Next, decant approximately 100 mL of the 1X solution into a 300 mL L plastic bucket and place the bucket in a −20 °C freezer for 70—80 minutes . This solution is to be used for cutting brain tissues and should be frozen over but not frozen solid. After taking the bucket out from freezer:

-Using a large spatula, break up ice and stir into a slurry.

-Add approximately 100 mL of 1X aCSF kept at room temperature.

-Mix solution with a handheld blender until forming an easily flowing slush.

-Keep the bucket of the 1x aCSF slush cutting solution on ice while being carbogenated.

-The slush solution should consist of approximately one fifths of liquid solution and be settled to the bottom of the bucket.

Overdose the mouse with a 1 mL intraperitoneallyinjection of a 150 mg/kg ketamine and 30 mg/kg xylamzine mix. While mouse is overdosing: place the slice chamber in a heated water bath set to 37 °C (water in the bath should come up the side of the chamber to approximately the same height as the aCSF). Next, after mouse is completely anesthetized:

-Transcardially perfuse mouse with the ice-cold 1x aCSF.

-Rapidly decapitate and extract brain in 1x aCSF slush.

-Using razor blade, cut brain down the midline.

-Glue brain medial side down on cutting block and place block quickly into cutting chamber of a Vibratome.

-Fill cutting chamber with aCSF slush solution, kept continuously carbogenated in chamber.

-Cut slices at 240 µm thick

-Quickly transfer each cut slice into hold chamber kept in heated bath.

-Remove holding chamber from bath 30 minutes after last slice transfer is made.

-Allow holding chamber to equilibrate to room temperature (approximately 20 minutes) before transferring slices to recording chamber of electrophysiological rig.

Using a pipette puller, glass pipettes should be pulled to ensure a pipette resistance of 3.2—3.8 MΩ . For whole-cell voltage clamp recordings, a KMeSO4 solution is used containing the following:

-KMeSO4: 135 mM

-KCl: 5 mM

-CaCl2: 0.5 mM

-HEPES: 5 mM

-EGTA-K: 5 mM

-ATP-Mg: 2 mM

-GTP-Na: 0.5 mM

-Biocytin: 0.20% (w/v in grams)

Transferred slices in recording chamber are continuously perfused with 1x aCSF that is kept at room temperature and continuously carbogenated. Neurons for recording are identified and recorded as follows:

-Pipettes are backfilled with KMeSO4 solution and inserted into headstage of amplifier.

-Pipettes are then pressurized to approximately 56 millibars .

-Pipettes area offset before cell attachment.

-Neurons are clamped at —80 mV before whole-cell access is achieved.

Stimulus generation and data acquisition are performed using an amplifier (Molecular Devices), a digitizer (Molecular Devices), and pClamp (Molecular Devices).

For current-clamp recordings

Adjust the amplifier bridge circuit to compensate for electrode resistance and subsequently monitor it. Filter the signals at 1 kHz and digitize them at 10 kHz.

KMeSO4and Na2-GTP were from ICN Biomedicals and Roche, respectively. All other reagents were obtained from Sigma-Aldrich. ₄and Na₂-GTP were from ICN Biomedicals and Roche, respectively. All other reagents were obtained from Sigma-Aldrich.

Examine the frequency-current (F-I) relationship of each cell with current-clamp recordings as follows.

-Apply a series of 500 ms current steps of n beginning at −150 pA and incremented at 25 pA for each consecutive sweep.

-A 30 second intertrial interval is used.

-The current steps are continued until depolarization block is reached.

-Monitor resting membrane potential was monitored for stability, and exclude cells that varied 20% from mean baseline from the analysis.

-Perform electrical stimulation using parallel bipolar tungsten electrodes (FHC) placed in layer 5 of the cortex.

-Adjust stimulus width and intensity via a constant current stimulator (Digitimer) to evoke a first excitatory postsynaptic current (EPSC) with an amplitude of 200–400 pA in the presence of the GABA_Areceptor antagonist SR95531 (10 µM) and CGP55845 (1 µM).

-Monitor whole-cell access was monitored with a −5 mV pulse throughout the recording. Determine off line the membrane capacitance (Cm) as Cm = Q_t* V_test, where Qt was calculated as the integral of the transient current elicited by V_test, a 10 mV voltage step.

-Take the average of the ratios of the second EPSC amplitude to the first EPSC amplitude for each recording sweep to calculate the paired-pulse ratio (PPR) for a given cell

-Exclude data if the series resistance of the patch pipette differed by > 20% between the two recordings.