Induction of Long-Term Depression in Cerebellar Purkinje Neurons in a Mouse Brain Slice

プロトコル

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

1. Brain Slicing

1. Orient the sample such that the dorsal side of the cerebellum is on the front side. Pour ice-cold cutting ACSF (Artificial cerebrospinal fluid), containing 1 µM TTX(Tetrodotoxin), sufficient to immerse the cerebellum completely. Place a gas tube into the ACSF and start bubbling with the O₂/CO₂ gas mixture.
2. Remove the arachnoid mater using a fine tweezer under binoculars. Cut the cerebellar peduncle with a blade, and remove the brainstem and agar block. Rotate the tray 180°, so that the dorsal surface of the cerebellum faces a razorblade.
3. Set the blade, and adjust the first cutting location. Set the vibratome slicing parameters to the following: amplitude to 5.5, frequency to 85 Hz, speed to 3–4, and slice thickness to 300 µm.
4. Transfer the cerebellar slice on a nylon net into an acrylic incubator and immerse the slice completely in the oxygenated ACSF. The incubator should be placed in a water bath that maintains a temperature of 26 °C.
5. Store the slices for at least 1 h to allow recovery from the damage during slicing.

2. Whole-cell Patch-clamp Recording

NOTE: A patch-clamp recording requires the following equipment: an upright microscope with infrared differential interference contrast (IR-DIC) optics, a patch-clamp amplifier, data digitizer, digital stimulator, isolator, computer, software for data acquisition and analysis, motorized manipulator, microscope platform, vibration isolation table, Faraday cage, solution heating system, peristaltic pumps, and electrode puller.

1. Add picrotoxin (0.1 mM) to ACSF and resolve it using ultra-sonication for 3 min.
2. Perfuse a recording chamber with picrotoxin-containing, O2-CO2-saturated ACSF at rate of 2 mL/min. Maintain the temperature of the recording chamber at around 30 °C.
3. Make a recording electrode by pulling a borosilicate glass capillary with filament (outer diameter = 1.5 mm) using a puller with 4 steps. The tip-diameter should be around 1 µm.
4. Make a stimulating electrode by pulling the same capillary using the puller with 2 steps, then break to produce a fine tip by striking the tip against an iron block under a binocular microscope. The final diameter should be 3–5 µm.
5. Transfer the cerebellar slice to the recording chamber and fix it with a Pt-weight with nylon threads. Fill a stimulating electrode with ACSF.
6. For stimulation of the PFs, place the stimulating electrode on the surface of the molecular layer, around 50 µm away from the Purkinje cell layer.
7. For stimulation of the CF, place the stimulating electrode at the bottom of the Purkinje cell layer (steps 3.3, 3.4).
8. Filter K⁺-based or Cs⁺-based internal solution with a 0.45 µm filter. Use a micro-loader to fill a recording electrode with 8 µL of internal solution.
9. Apply a weak positive pressure to the recording electrode before immersing it into the ACSF. Its resistance should be 2–4 MΩ and the liquid junctional potential should be corrected.
10. Approach the healthy, bright cell body of the PC with the recording electrode. Push the surface of Purkinje cell slightly, stop applying positive pressure, next apply negative pressure until forming a giga-ohm seal. Then establish the whole-cell configuration using negative pressure.
11. Hold the membrane potential at -70 mV, and apply -2 mV pulse (duration, 100 ms) at 0.1 Hz to monitor input resistance, series resistance and input capacitance, continuously. Do not use the series resistance compensation. Discard data when the series resistance varies by more than 15%.

3. Induction of LTD (Long-term depression)

1. Stimulate the molecular layer with a pulse (duration, 0.1 ms). Identify the PF(Parallel fibers)-excitatory postsynaptic currents (EPSC) by applying a double pulse stimulus (interspike interval (ISI) of 50 ms). The PF-EPSC should show paired-pulse facilitation and gradual increase in amplitude relative to increase in stimulation intensity.
2. Record the test response of the PF-EPSC by applying a single pulse at 0.1 Hz. Adjust the intensity of the stimulus so that the evoked EPSC amplitude is around 200 pA. Avoid contamination of current through the voltage-dependent ionic channel.
3. Stimulate the CF at the bottom of the Purkinje cell layer, and identify the EPSC elicited by the CF activation (by applying a double pulse stimulus). The CF-EPSC should show paired-pulse depression and an all-or-none manner according to the increase in stimulation intensity. For LTD induction, a single stimulus should be used.
4. LTD-inducing protocol 1​
   1. Using an electrode containing K⁺-based internal solution under current-clamp conditions, apply a single PF-stimulus and a single CF-stimulus simultaneously at 1 Hz for 5 min (300 pulses) (Figure 1A).
5. LTD-inducing protocol 2
   1. Using an electrode-containing K⁺-based internal solution under current-clamp conditions, apply double PF-stimuli (ISI of 50 ms) and single CF-stimulus as the second PF-stimulus is coincident with CF-stimuli at 1 Hz for 5 min (Figure 1B).
6. LTD-inducing protocol 3
   1. Using an electrode containing Cs⁺-based internal solution under voltage-clamp conditions, apply a double PF-stimulus (ISI of 50 ms) and a single depolarizing voltage-step (-70 to 0 mV, 50 ms) to the soma at 1 Hz for 3 min, so that the second PF-stimulus is equivalent to the beginning of the depolarizing voltage step (Figure 1C).
7. LTD-inducing protocol-4​
   1. Using an electrode containing Cs⁺-based internal solution under voltage-clamp conditions, apply the PF-stimuli (5x at 100 Hz) and a single depolarizing voltage-step (-70 to 0 mV, 50 ms) to the soma at 0.5 Hz for 3 min, simultaneously (Figure 1D).

結果

Figure 1: Schematic illustration of protocols to induce LTD in PF-PC synapse. (A) Protocol 1 Cj. 1 PF and 1 CF stimuli are applied simultaneously 300 times at 1 Hz (5 min) under current-clamp conditions. Electrode for whole-cell recording contains K⁺-based internal solution. (B) Protocol 2 Cj. 2 PF and 1 CF stimuli are applied simultaneously 300 times ...

資料

Name	Company	Catalog Number	Comments
Amplifier	Molecular Devices-Axon	Multiclamp 700B
Borosilicate glass capillary	Sutter	BF150-110-10
Digitizer	Molecular Devices-Axon	Digidata1322A
Electrode puller	Sutter	Model P-97
Isoflurane	FUJIFILM Wako Pure Chemical	26675-46-7
Isolator	A.M.P.I.	ISOflex
Linear slicer	Dosaka EM	PRO7N
Microscope	NIKON	Eclipse E600FN
Peristaltic pump	Gilson	MP1 Single Channel Pump
Picrotoxin	Sigma-Aldrich	P1675
Pure water maker	Merck-Millipore	MilliQ 7000
Software for experiment	Molecular probe-Axon	pClamp 10
Software for statistics	KyensLab	KyPlot 5.0
Stimulator	WPI	DS8000
Temperature controller	Warner	TC-324B
Tetrodotoxin	Tocris	1078