TITLE: Ex Vivo Electrophysiology
AUTHORS: Harry Xenias, Savio Chan, Loukia Parisiadou

[DESCRIPTION]
This protocol describes the preparation of brain slices, setup of the electrophysiology rig, and solutions for collecting whole-cell recordings.

[STEPS]
SECTION: Setup
1. 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
SECTION: Setup
2. 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.
SECTION: Setup
3. 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.
SECTION: Setup
4. 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)
SECTION: Setup
5. Next, decant approximately 100 mL of the 1X solution into a 300 mL L plastic bucket and place the bucket in a −20 °Cfreezer 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. 
SECTION: Setup
6. Overdose the mouse with a 1 mLintraperitoneallyinjection 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 µmthick
-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.
SECTION: Whole-cell Recordings
7. 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)
SECTION: Whole-cell Recordings
8. 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.
SECTION: Whole-cell Recordings
9. Stimulus generation and data acquisition are performed using an amplifier (Molecular Devices), a digitizer (Molecular Devices), and pClamp (Molecular Devices). 
SECTION: Whole-cell Recordings
10. 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.
SECTION: Excitability of SPNs:
11. 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. 
SECTION: Corticostriatal responses recorded in voltage-clamp
12. -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 GABAAreceptor 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 = Qt * Vtest, where Qt was calculated as the integral of the transient current elicited by Vtest, 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.