Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Summary

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Abstract

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

Introduction

In 1951, the first-reported acute brain slice experiments were conducted¹. In 1971, after successful in vitro recordings from the piriform cortex^2,3 and the discovery that hippocampal neurons are interconnected transversely along the septotemporal axis of the hippocampus⁴, one of the first in vitro recordings of hippocampal neuronal activity was achieved⁵. The similarity of the neurophysiological or neurostructural parameters of neurons under in vivo and in vitro conditions are still the subject of some debate⁶, but in 1975, Schwartzkroin⁷ indicated that the basal properties of neurons are maintained in vitro and that high-frequency stimulation (i.e., tetanization) of afferents in the hippocampal formation induces a long-lasting facilitation of synaptic potentials⁸. Electrophysiological recording of neuronal activity in vitro greatly expanded the study of the cellular mechanisms of activity-dependent synaptic plasticity^9,10, which had been discovered in 1973 by Bliss et al.¹¹ in in vivo experiments with rabbits.

The study of neuronal activity or signaling pathways in brain slices, and especially in acute hippocampal slices, is now a standard tool. However, surprisingly, in vitro experiments have yet to be standardized, as evidenced by the multiple approaches that still exist for the preparation and maintenance of acute hippocampal slices. Reid et al. (1988)¹² reviewed the methodological challenges for the maintenance of acute brain slices in different types of slice chambers and the choices of bathing medium, pH, temperature, and oxygen level. These parameters are still difficult to control in the recording chamber due to the custom-made elements of in vitro slice-recording setups. Publications can be found that might help to overcome some of the methodological challenges and that describe new types of submersion slice chambers, such as an interstitial 3D microperfusion system¹³, a chamber with enhanced laminar flow and oxygen supply¹⁴, a system with computerized temperature control¹⁵, and a multi-chamber recording system¹⁶. Since these chambers are not easy to build, most scientists rely on commercially available slice chambers. These chambers can be mounted on a microscope system, thus allowing for the combination of electrophysiology and fluorescence imaging^17,18,19. Since these chambers keep the brain slices submerged in artificial cerebrospinal fluid (aCSF), a high flow rate of the buffer solution needs to be maintained, increasing the expense of drug application. To this end, we have incorporated a recycling perfusion system with outflow-carbogenation that provides sufficient stability for the long-term recording of field potentials in a submersion slice chamber using a relatively small aCSF volume. In addition, we summarized how the use of this experimental carbogenation/perfusion system affects the outcome of activity-dependent synaptic plasticity¹⁰ and how inhibition of eukaryotic elongation factor-2 kinase (eEF2K) modulates synaptic transmission²⁰.

Protocol

The animals were maintained in accordance with the established standards of animal care and procedures of the Institutes of Brain Science and State Key Laboratory of Medical Neurobiology of Fudan University, Shanghai, China.

1. Solution Preparation

NOTE: See the Table of Materials.

1. Prepare the slicing buffer (modified Gey's solution): 92 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 30 mM NaHCO₃, 25 mM glucose, 20 mM HEPES, 3 mM Na⁺-pyruvate, 10 mM MgSO₄, and 0.5 mM CaCl₂.
2. Adjust the pH to 7.6 using 1 M NaOH without carbogenation.
3. Store the buffer at 4 °C.
   NOTE: The titration clarifies the cloudy liquid. The osmolality is 305-310 mOsm. The carbogen²¹ contains 5% carbon dioxide and 95% oxygen.
4. Prepare the aCSF by diluting a 10x stock solution of aCSF that does not contain bicarbonate and glucose, giving a final composition of: 124 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl₂, 2 mM MgCl₂, and 1.25 mM KH₂PO₄.
5. Add bicarbonate and glucose to reach 10 mM glucose and 26 mM NaHCO₃.
6. Carbogenate the aCSF through a perforated silicon tube with a blocked end immediately after adding the NaHCO₃.
7. Measure the pH while carbogenating (pH 7.3-7.4).
   NOTE: The buffer capacity can be adjusted slightly by altering the amount of NaHCO₃. Since the resulting pH is temperature-dependent, it is necessary to check the resulting pH while carbogenating at the working temperature (e.g., 30 °C).

2. Preparation of Acute Hippocampal Slices

NOTE: See the Table of Materials.

1. Place a slice-holding mesh into an incubation chamber for acute brain slices, fill the chamber with aCSF, and carbogenate (4-6 L/h) for at least 30 min²² at 28-30 °C.
2. Cool the surgical instruments down to 2-4 °C.
3. Place a glass beaker filled with cold and carbogenated slicing buffer (2-4 °C), maintained in an ice/water mixture, near the vibratome.
4. Anesthetize rats or mice until the corneal reflexes disappear (30-50 s) using isoflurane by taking 500 µL into a 2 L glass jar or 12.5 µL into a 50 mL tube.
5. Isolate the brain using a method described and visualized in detail in the publications of Mathis et al. (2011)²³ and Yuanxiang et al. (2014)²⁴.
6. Cool the brain down to 2-4 °C in an ice-cold slicing buffer (for at least 2 min) before dissecting the cerebellum and separating the hemispheres.
7. Use a cold surgical blade to dissect a piece of the dorsal cortex at a 70° angle along the dorsal edge of each hemisphere²⁵ to get transverse slices from the ventral and the intermediate part of the hippocampus, as shown in Figure 1C and E.
8. With a cold dental cement spatula, transfer a hemisphere to a piece of filter paper to briefly dry the created surface (1-2 s).
9. Mount the two hemispheres with freshly cut surfaces on the ice-cold slicing platform using fast-acting adhesive glue; have the caudal end of the hemispheres face the razor blade (Figure 1D).
10. Place the slicing platform into the cooling chamber of a vibratome and fill that chamber with the slicing buffer (2-4 °C). Carbogenate using a perforated silicon tube.
11. Cut 350 µm slices using adequate vibratome settings (e.g., blade forward speed: 1.2 mm/s, blade amplitude: 1 mm).
12. Isolate the hippocampal formation from the subiculum and the entorhinal cortices using two injection cannulas (>28 G) as scissors.
13. Remove the bubbles from the slice-holding mesh of the previously prepared incubation chamber using a Pasteur pipette.
14. Transfer the slices that have some transparency at the stratum pyramidale from the slicing platform on the mesh of the incubation chamber. Avoid any folding, stretching, overlapping, and floating by aspirating aCSF and the slice into a large-mouth Pasteur pipette.
    NOTE: The whole procedure (step 2.5-2.14) can take 5-10 min; therefore, it is important to check the temperature of the buffer solution over time to maintain it at 4 °C.
15. Incubate the slices at 30 °C for at least 1.5-2 h in the incubation chamber and provide carbogen at 4-6 L/h.
    NOTE: Adjust the flow rate of the carbogen to circulate the buffer in the incubator, but prevent the slices from floating.

3. Modifications of the Carbogenation for the aCSF Recycling of Small Reservoirs

1. Add a 3-way tube connector to the outflow of the recycling system (Figure 2B).
2. Connect the middle arm of the 3-way tube connector with a carbogen supply tube and regulate the flow rate with an airflow meter (3-4 L/h, Figure 2D and Figure 4C).
3. Add a second 3-way tube connector to the inflow of the recycling system. Connect the middle arm to the tube facing the inline heater, the upper arm to a thin silicon tube (10 cm length), and the lower arm to the inflow tube from the reservoir (low-pass filter; Figure 4C).
4. Place a tube squeezer on a thin silicon tube (OD: 2 mm) at a position along the tube where the pulsation of the aCSF in the slice chamber, generated by the peristaltic pump, is at its minimum.
   NOTE: The outflow-carbogenation (outflow-carb.) modification reduces the sensitivity of the carbogenation level to the volume of the aCSF reservoir, the aCSF recycling rate, and relative tube positions within small aCSF reservoirs (<50 mL; Figure 3). Regarding the low-pass filter, a certain amount of air in the thin silicon tube reduces the pulsation of the aCSF flow; thus, the tube should be filled mostly with air.

4. Recording Synaptic Responses in a Submersion Slice Chamber

NOTE: See the Table of Materials.

1. Pre-warm the aCSF solution in a water bath to about 28-30 °C (this will prevent bubble formation in the recording chamber).
2. Start the recycling system (4-5 mL/min) and activate the inline heater to equilibrate the system for at least 1 h.
3. Presoak a small piece of lens cleaning paper and a nylon mesh fixed on a U-shaped platinum wire in the submersion slice chamber for a few minutes (Figure 4).
4. Remove the U-shaped platinum wire.
5. Switch off the recycling and place a slice on the lens cleaning paper.
6. Immediately place the slice-holding mesh on top of the slice, switch on the pump, and let the slice equilibrate for 30 min without dipping the objective into the aCSF.
7. Fill the borosilicate micropipette (i.e., the recording electrode) with aCSF (tip resistance: 1-2 MΩ, filled 1/3 with aCSF) and mount it into the pipette holder.
8. Check the insulation of the metal stimulation electrode by placing it into NaHCO₃ solution (>40 mM). Connect the electrode to the negative pole of a DC voltage generator (1-2 V, positive pole: silver or platinum wire), and observe the formation of bubbles at the tip under a dissection microscope (>60x magnification).
9. Place the tested epoxy-insulated tungsten stimulation electrode in the manipulator holder and the reference wires in the slice chamber.
10. Add a second reference electrode to the chamber and connect it to the reference socket of the headstage of the recording electrode (Figure 4A).
11. In the amplifier control software, click on the "zero clamp mode" box and proceed by double-clicking the "output gain list" box. Choose a gain of "100," double-click the "high pass filter list box (Bessel)," choose "0.1 Hz," and double-click the "low pass filter list box (AC)" to choose "3 kHz."
12. In the recording software, click the on "Acquire | Open Protocol." Choose a protocol that has settings allowing for episodic stimulations and the digitalization of amplified potentials at 10-20 kHz for 50-100 ms and that automatically triggers a stimulus isolator 10 ms after the start of an episodic recording.
13. Place the stimulation and recording electrodes in line and parallel to the stratum pyramidale, for example (Figure 4B and Figure 5).
14. Click "Acquire | Edit Protocol" and choose the "trigger" tab (in the pop-up window). Click on the "trigger source" box and chose "space bar" as the trigger source. Click the "OK" button. Click the "record" button to start the acquisition and note the pop-up window with a trigger button.
15. In the stimulus isolator software, click on the "voltage control" box and enter "0." Click the "download" button. Click on the "recording software" window and press the space bar. Repeat this cycle by sequentially entering values from 1 to 8, for example, at 1 mV steps in the "voltage control" box.
16. Correlate the stimulation strength with fEPSP-slope values and determine the stimulation strength required to get 40% of the fEPSP-slope maximum. Click the "voltage control box" and enter the determined value. Click the "download" button.
    NOTE: A stimulus isolator is used to apply brief voltage or current pulses to brain tissue. The biphasic pulses can have 100 µs per phase.
17. In the recording software, click the stop button and then the "Acquire" menu. Click on "Edit Protocol" and choose the "trigger" tab in the pop-up window. Click on the trigger source box and chose the "internal timer" as the trigger source. Click the "OK" button. Click the record button that starts the automatic recording of field potentials every 30 s, for example. Click the "stop" button after 30-60 min.
18. Click on the "Acquire" menu, click on "Open Protocol," choose the desired induction protocol of synaptic plasticity, and click the "OK" button. Click the "record" button to automatically activate the high-frequency stimulation and recording. Click the "stop" button.
    NOTE: In case of studies relating to synaptic plasticity, apply one of the standard induction paradigms for long-term potentiation or long-term depression after prolonged baseline recording. Representative examples of the resulting modulation of the synaptic transmission are depicted in Figure 7 and Figure 8.
19. In the recording software, click "Acquire | Open Protocol" and choose the same protocol as in step 4.12. Click the "OK" button and then click "record." Keep automatically running the field potential recordings for 2-4 h, for example. Click the "stop" button to terminate the recording.
20. In case of pharmaceutical studies, apply the compound directly to the aCSF reservoir if permanent compound administration is desired (Figure 6).

5. Cleaning the Setup and Hints

NOTE: See below for general tips.

1. Clean the system weekly by recycling 10% H₂O₂ for no more than 20 min, followed by washing with deionized H₂O.
   NOTE: Ethanol is not recommended for cleaning. It is likewise not recommended to immerse the reference wires and objective in H₂O₂.
2. Remove precipitated salt on the surface of the objective lens or chamber surroundings with 0.1 N HCl and then water.
3. Wash the carbogen bubbler in distilled water or 0.1 N HCl.
4. Immerse the slice incubation chamber in 10% H₂O₂ for 5 min once a week and then thoroughly rinse the incubation chamber with deionized H₂O.
5. In case of metal stimulation electrodes, rinse the stimulating electrode tip with deionized water after each experiment and gently wipe the stimulating electrode tip with a cotton ball soaked with ethanol to remove residual tissue.
6. Reduce the resistance of commercial metal stimulation electrodes by removing the insulation at the tip through a careful swipe with sandpaper.
7. Reduce the loss of carbogen from the aCSF while recycling through the tubes by replacing the silicon tubes with polytetrafluoroethylene tubes.
8. Keep the silver wires of the recording electrode and reference electrode in bleach for several days to form AgCl on the wire surface.

Results

In the protocol section, we described the preparation of acute hippocampal slices from the ventral and intermediate part of the hippocampal formation (Figure 1) of male C57BL/6 mice and male Wistar rats (5-8 weeks). The position of the hemispheres on the slicer platform helps to keep them stable and removes the need of stabilization with agar or agarose. The perfusion system itself is based on a peristaltic pump operated on high rotation to give the required ...

Discussion

Although interface slice chambers exhibit more robust synaptic responses^25,26,27,28, submersion chambers provide additional convenience for patch-clamp recording and fluorescence imaging. Thus, we have described several aspects of field potential recordings in acute hippocampal slices using a commercial submersion slice chamber that can easily be extended to the imaging of fluorescence probes i...

Materials

Name	Company	Catalog Number	Comments
Reagents required
NaCl	Sinopharm Chemical Reagent, China	10019318
KCl	Sinopharm Chemical Reagent, China	10016318
KH2PO4	Sinopharm Chemical Reagent, China	10017618
MgCl2·6H2O	Sinopharm Chemical Reagent, China	10012818
CaCl2	Sinopharm Chemical Reagent, China	10005861
NaHCO3	Sinopharm Chemical Reagent, China	10018960
Glucose	Sinopharm Chemical Reagent, China	10010518
NaH2PO4	Sinopharm Chemical Reagent, China	20040718
HEPES	Sigma	H3375
Sodium pyruvate	Sigma	A4043
MgSO4	Sinopharm Chemical Reagent, China	20025118
NaOH	Sinopharm Chemical Reagent, China	10019718
Tools and materials for dissection
Decapitators	Harvard apparatus	55-0012	for rat decapitation
Bandage Scissors	SCHREIBER	12-4227	for mouse decapitation
double-edge blade	Flying Eagle, China	74-C
IRIS Scissors	RWD, China	S12003-09
Bone Rongeurs	RWD, China	S22002-14
Spoon	Hammacher	HSN 152-13
dental cement spatula	Hammacher	HSN 016-15
dental double end excavator	Blacksmith Surgical, USA	BS-415-017
Vibrating Microtome	Leica, Germany	VT1200S
surgical blade	RWD, China	S31023-02
surgical holder	RWD, China	S32007-14
Electrophysiology equipment and materials
Vertical Pipette Puller	Narishige, Japan	PC-10
Vibration isolation table	Meirits, Japan	ADZ-A0806
submerged type recording chamber	Warner Instruments	RC-26GLP
4 Axis Micromanipulator	Sutter, USA	MP-285, MP-225
Platinum Wire	World Precision Instruments	PTP406
Amplifier	Molecular Devices, USA	Multiclamp 700B
Data Acquisition System	Molecular Devices, USA	Digidata 1440A
Anaysis software	Molecular Devices, USA	Clampex 10.2
Fluorescence Microscope	Nikon, Japan	FN1
LED light source	Lumen Dynamics Group, Canada	X-cite 120LED
micropipettes	Harvard apparatus	GC150TF	extracelluar recording
borosilicate micropipettes	Sutter, USA	BF150-86	patch clamp
tungsten electrode	A-M Systems, USA	575500
peristaltic pump	Longer, China	BT00-300T
tubes for peristaltic pump	ISMATEC, Wertheim, Germany	SC0309	1x inflow, ID: 1.02 mm
tubes for peristaltic pump	ISMATEC, Wertheim, Germany	SC0319	2x tubes for outflow, ID: 2.79 mm
CCD camera	PCO, Germany	pco.edge sCMOS
lens cleaning paper	Kodak
50 mL conical centrifuge tube	Thermo scientific	339652
Prechamber	Warner Instruments	BSC-PC
Inline heater	Warner Instruments	SF-28
Temperature Controller	Warner Instruments	TC-324B