Generating Local Cornu Ammonis 1 (CA1) Gamma Oscillations Using Tetanic Stimulations in a Hippocampal Brain Slice

Protokol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Setup for Cutting Brain Slices

1. Prepare a cutting solution comprised of (mM) 125 Choline-Cl, 2.5 KCl, 0.4 CaCl₂, 6 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, 20 D-glucose saturated with carbogen gas (95% O₂–5% CO₂) and an artificial cerebrospinal fluid (aCSF) recording solution comprised of (mM) 125 NaCl, 2.5 KCl, 2 CaCl₂, 2 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, 10 D-glucose, saturated with carbogen. Place the cutting solution on ice to keep it cold.
2. Freeze approximately 400 ml of the cutting solution and blend together with 100 ml of unfrozen cutting solution to create an ice slurry. Bubble with carbogen (95 % O₂–5 % CO₂) at a flow of approximately 0.5 L/min through small caliber tubing or sintered glass to produce a steady but gentle flow of bubbles.
3. Prepare a 250 ml beaker with a raised nylon mesh insert on which the brain slices will be placed. Fill with aCSF to cover the mesh by approximately 2 cm and bubble with carbogen, ensuring that the bubbles do not directly disrupt the slice holding area. It is also important that there are no air bubbles in the nylon mesh, so if any are present remove them. This will be the holding chamber and is kept at RT (20 - 25 °C).
4. Layout the dissecting instruments including a large pair of scissors, a small pair of scissors, small and large micro spatulas, and large and small pairs of forceps and chill them on ice. Place the vibratome tissue-cutting block on ice on a square of aluminum foil. Obtain 2 pieces of 6 cm filter paper, a single-edge razor blade, and a 25 ml beaker filled with the cutting solution slurry as well.
5. Fill a second container with ice, and lay a piece of tissue paper on the ice and place a 12 cm culture dish on top. Fill the culture dish with cutting solution ice slurry and bubble with carbogen. This is the container in which the brain dissection with be performed.
6. Prepare the vibratome. Remove a fresh double-edged razor blade (use a fresh blade every time) from its wrapping and spray with 80% ethanol then deionized water. Cut a 3 ml plastic transfer pipette at the point where it starts to taper. This will be used to transfer brain slices. Bend a 27 G needle at the base by approximately 45° and attach to a 1 ml syringe. This will be used to manipulate slices during cutting.

2. Cutting Brain Slices

1. Anesthetize a mouse (P16-P18) with 2% isoflurane or a locally approved method. Following induction, decapitate the animal with a large pair of scissors and drop the head into the 12 cm culture dish that contains bubbled cutting solution slurry. The slurry must totally immerse the head for rapid cooling.
2. Hold the front of the head with one hand peeling the skin and connective tissue forward towards the nose. Using the small scissors cut the connective tissue to reveal the underlying skull. Then remove the muscles overlying the dorsal aspect of the skull and neck.
3. Remove the brain from the skull by first securing the front of the skull with the large forceps and then make two lateral cuts through the bone either side of the foramen magnum using the small scissors (Figure 1, cuts labeled A1 and A2).
   1. Make another cut between the eyes (just anterior of the bregma) (Figure 1, cut labeled B) then carefully cut along the sagittal suture anteriorly and reflect the cut skull sections to reveal the brain (Figure 1, cut labeled C).
   2. Use the small spatula to scoop out the brain and place on a culture dish. Be aware of the cranial nerves on the inferior aspect of the brain which will need to be severed, this can be done using the smaller sized micro spatula. Using the larger spatula transfer the brain to the 25 ml beaker filled with the cutting solution slurry.
4. Preparing the brain hemisphere for slicing.
   1. Place one piece of the 6 cm filter paper in the bottom of a fresh culture dish then fill with fresh cutting solution slurry and bubble. Use the larger spatula to position the brain, ventral side down, onto the filter paper. Take a new and cleaned single-edge razor blade and cut the brain to remove the cerebellum, then make a cut along the midline to separate the brain into two hemispheres.
5. Preparing the vibratome tissue block.
   1. Take the chilled vibratome tissue block, dry the surface, and place a drop of cyanoacrylate glue in the middle, and spread evenly to the approximate size of the brain. Use the small spatula to manipulate one of the brain hemispheres onto the large micro spatula so the medial side of the brain is down.
   2. Touch the edge of the spatula at the interface of the brain on the second piece of filter paper to remove as much of the solution as possible. Slide the brain off the larger spatula using the smaller spatula to guide it onto the glue.
   3. Secure the cutting block into the vibratome chamber and fill the chamber with cutting solution slurry and bubble, ensuring the brain is completely immersed. Rotate the cutting block so the ventral side of the brain is facing the blade.
6. Slicing the brain.
    NOTE: Each vibratome is unique so follow the manufacturer's instructions.
   1. Set the slice thickness to 450 µm, and ensure the blade is vibrating as it moves through the brain at a speed of approximately 0.3 mm/s. Cut entirely through the brain from the ventral side to the cortical surface, this will produce whole brain sagittal slices that can be used for electrophysiological recordings. Slices will be produced laterally to medially and typically 3 - 4 slices can be cut from each hemisphere.
   2. If the base of the slice lifts, use the bent 27 G needle to gently press the slice back down. As each slice is cut, use the transfer pipette to move it onto the platform in the holding chamber where they are viable for up to 8 hrs.

3. Extracellular Electrophysiology Recordings

1. Mounting the slice in the recording chamber.
   1. Using the transfer pipette, place a brain slice into a submerged recording chamber perfused with aCSF flowing at 1 - 2 ml/min and heated to 32 °C. The aCSF used for recordings differs from that in the holding chamber as the Mg²⁺ concentration is increased from 2 mM to 4 mM.
   2. Secure the slice with a "harp" (semi-circular stainless steel with nylon strands stretched across at 2 - 3 mm spacing). Place the harp so that the strands run parallel to CA1. Increase the speed of the perfusion to 8 - 10 ml/min at 32 °C.
2. Under a dissecting microscope place a stimulating electrode and a recording electrode (glass electrode filled with the aCSF recording solution) on the surface of the stratum radiatum of the CA1 (Figure 2A). Place the stimulating electrode first, then place the recording electrode.
3. Stimulate the Schaffer collaterals with a 120 - 150 µA amplitude and 0.1 msec duration test pulse and observe the resulting field excitatory post synaptic potential (fEPSP) waveform to determine slice health (Figure 2B). The stimulating and recording electrodes may need to be moved into the slice approximately 50 - 100 µm from the slice surface to get a fEPSP recording with a small fiber volley and large amplitude. The Schaffer collateral evoked fEPSPs are stereotypical and provide a good indicator of slice health. Healthy slices typically show a fiber volley to fEPSP amplitude ratio of less than 0.3.
4. Repositioning the electrodes.
   1. Using a dissecting microscope, move the stimulating electrode to the middle of the stratum oriens and move the recording electrode to the pyramidal cell layer as close to the recording electrode as possible, as shown in Figure 3A. The stimulating and recording electrodes may need to be pushed into the slice approximately 50 - 100 µm so that the fEPSP response amplitude to the 120 - 150 µA test pulse is approximately 1 mV.
5. Generating γ oscillations.
   1. To generate γ oscillations, stimulate the tissue with a train of 20 x 0.1 msec pulses delivered at 200 Hz. This tetanic stimulus can yield reproducible responses when delivered every 5 min.
6. Use the following recording parameters.
   1. Scale the gain of the output signal to match the input voltage range of the analog-to-digital converter. Make sure that the maximum expected signal excursion uses a minimum of 30% of the input voltage range. Be careful when setting gains too high, as signals might clip. The typical bandwidth needed for field recordings is 500 Hz with AC coupling at 0.1 Hz to remove baseline drift.
   2. Digitize at least 4 - 5 times faster than the corner frequency of the low pass filter to avoid signal aliasing.

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Sonuçlar

Figure 1. Schematic of the skull and overlying skin showing the location of cuts to reveal the brain. A1, A2, B and C mark the locations of cuts that need to be made to open up the skull for the removal of the brain.

Figure 2. Placement of the stimulating and recording electrodes in the CA1 Schaffer collaterals to test slice health. (A) Shows the placement of the stimulating electrode (Stim) and the recording electrode (Rec). (B) A representative example of a fEPSP evoked by a 120 µA stimulation (fiber volley marked by *).

Figure 3. Configuration of recording and stimulation electrodes used to evoke oscillation. (A) Location of the stimulating electrode (Stim) in the stratum oriens and recording electrode (Rec) in the stratum pyramidale of the CA1. (B) A representative example of the γ oscillations induced by tetanic stimulation (artifact marked by "Stim"). Representative trace showing quantifiable outputs. ISI inter-spike interval.

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Malzemeler

Name	Company	Catalog Number	Comments
4-(N-Ethyl-N-phenylamino)-1,2 dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288)	Sigma-Aldrich	Z3777
Biuculline	Sigma-Aldrich	14340
6-cyano-7-nitroquinoxa- line-2,3 dione (CNQX)	Sigma-Aldrich	C127
Nickel	Sigma-Aldrich	266965
Carbamazepine	Sigma-Aldrich	C4024
(2R)-amino-5-phosphonopentano ate (APV)	Tocris Bioscience	105
Retigabine	ChemPacific	150812-12-7
Choline-Cl	Sigma-Aldrich	C1879-5KG
KCl	Sigma-Aldrich	P9333-500G
NaH2PO4	Sigma-Aldrich	S9638-250G
NaHCO3	Sigma-Aldrich	S6297-250G
NaCl	Sigma-Aldrich	S7653-5KG
Glucose	Sigma-Aldrich	G8270-1KG
CaCl2.2H2O	Sigma-Aldrich	223506-500G
MgCl2.6H2O	Sigma-Aldrich	M2670-500G
Electrode glass	Harvard Apparatus	GC150F-10
Concentric bipolar stimulating metal electrode	FHC	CBBPF75
Digital Isolator	Getting Instruments	Model BJN8-9V1
Model 1800 amplifier	A-M systems	Model 1800 amplifier
Digitizer	National Intruments	NI USB-6211
Vibrotome	Leica	VT1200s