Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Summary

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Abstract

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.

The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

Introduction

Nowadays, there is limited understanding of how complex memories are stored and recalled at the neuronal circuit level. However, a unifying hypothesis of memory storage is available and broadly accepted: memories are stored as changes in the strength of synaptic connections between neurons in the central nervous system. On its own, research on synaptic plasticity has largely benefited from two breakthrough discoveries. (1) In a seminal experiment, Bliss and Lomo ¹, using the intact anesthetized rabbit, found that delivery of a brief high-frequency (1 sec, 100 Hz) stimulation to the perforant path of the hippocampus caused a long-lasting (several hours) increase in the related synaptic connections. This fascinating phenomenon was called "Long-Term Potentiation" or LTP by Douglas and Goddard in 1975 ². (2) Later on, it was found that a similar phenomenon could be triggered in brain slices (0.4 mm) artificially maintained alive in vitro. The most widely studied LTP was observed in vitro by delivering one or several tetani to a bundle of axons (the so-called Schaffer collaterals) while recording the resulting field excitatory synaptic potential evoked in the pyramidal neurons of the so-called CA1 region. The mechanisms of LTP induction have largely been revealed. Basically, a Ca²⁺ influx through the NMDA receptors activates enzymes with two consequences: a phosphorylation of AMPA receptors (which increases their efficiency) and an incorporation of extra AMPA receptors in the postsynaptic membrane ³. By contrast, the mechanisms of the maintenance phase of LTP are largely unknown, notably because it is experimentally much more difficult to maintain a slice healthy for many hours than for 30 to 60 min.

A lot of studies have been dedicated to the understanding of LTP mechanisms and interesting theories have been elaborated over the years ^4-11. But until now, the precise molecular mechanisms underlying the stable increase in synaptic strength have not been elucidated. This could be partly due to the difficulty to reproduce previous results in different laboratories using different techniques for the preparation and the maintenance of hippocampal slices. In their methodology paper, Sajikumar et al. ¹² stressed the importance of experimental conditions for the preparation of rat hippocampal slices and the recording of stable LTP. In this video we present all the optimization steps developed in our laboratory over the years to be able to record a very stable LTP in mouse hippocampal slices.

This optimization has been made from protocols developed and successfully used by other laboratories which study LTP mechanisms in mice ¹³ and rats ¹¹. It allows experienced researchers to induce and record a very long lasting LTP in adult mice with a high rate of success. The physiological basis of the induced LTP was carefully checked and demonstrated ¹⁴. In this methodology paper, we show that any modifications of experimental conditions, like temperature or oxygenation can have a profound impact on LTP maintenance while the dissection procedure can deeply modify slices excitability. It must also be emphasized that the precise control of all these parameters requires a training of several months for novice students.

Protocol

All animal procedures were carried out in accordance with National Institutes of Health regulations for the care and use of animals in research and with the agreement of the local ethics committee.

1. Preparation of Artificial Cerebro-spinal Fluid

The same media is used to dissect, cut and perfuse slices (1 ml/min) during the resting period and the electrophysiological recordings. This media is composed of 124 mM NaCl, 4.4 mM KCl, 26 mM NaHCO₃, 1 mM NaH₂PO₄, 2.5 mM CaCl₂, 1.3 mM MgSO₄ and 10 mM D-glucose.

1. Weigh the different components for 2 L of ACSF except MgSO₄ which is already in solution (1 M). Using a standard solution allows to be sure of the exact concentration of Mg²⁺ because MgSO₄ in powder is highly hygroscopic. Ca²⁺:Mg²⁺ ratio greatly influences LTP induction and maintenance ¹⁴ and thus their proportions must always be respected.
2. Put all the components except CaCl₂ in a glass graduated cylinder and bring to 2 L with distilled H₂O.
3. Stir vigorously. When everything is dissolved, add carbogen through an aquarium bubbler and tubing attached to the tank (95% O₂, 5% CO₂). Wait a few minutes and add calcium chloride when pH is fixed at 7.4 by the action of bicarbonate buffer.
4. Keep 600 ml in a refrigerated rectangular glass dish and cool down to 4 °C with ice around the dish. This media will be used for hippocampus dissection.
5. Filter the remaining 1,400 ml and keep in an Erlenmeyer flask.

2. Preparation of the Electrophysiological Rig and the Brain Slice Recording Chamber

The perfusion circuit is made of 2 peristaltic pumps, one pumping fresh fluid from a reserve tank and the other pumping used fluid from the recording chamber to a bin. Fresh fluid is added to a home-made perfusion system where it is re-oxygenated and warmed before reaching the interface recording chamber by gravity (Figure 1A). The interface recording chamber was designed by FST (Fine Science Tools, Vancouver, Canada, Figure 1B). Tissue slices are placed on woven net filter attached to a removable well ring and can be continuously maintained under interface conditions by adjusting the height of the suction well needle. A home-made plastic tube blocked at its end and perforated laterally (0.5 mm) is fixed to the suction needle to increase level stability in the chamber. The recording head is mounted over a water bath containing a gas dispersion stone which helps maintain a humid environment by passing moist air through deflection ports in the recording head (to decrease water droplet formation). Bath and medium temperature are controlled by continuously varying the level of DC current through a silicone rubber-embedded heater element in the water bath. Thermistors in the water bath and recording wells allow the chamber temperature to be set and precisely monitored at these sites.

The heating system of the chamber is completed by a global heating system controlling the temperature of the entire rig. The software developed by researchers at the University of Edinburgh (www.etcsystem.com) monitors and equalizes the temperature of the oxygenated air, the brain slice, the electrodes and the remaining rig providing a stable environment for long-term recordings (Figure 1C). The temperature used for mouse hippocampal slices is 28 °C.

1. Rinse all the perfusion circuit with distilled H₂O for a minimum of 20 min and start the heating systems.
2. Start the carbogen bubbling in the circuit. Carbogen arrives in the home-made perfusing tubes through filter candles and in the water bath below the recording chamber. The flow rate in the water bath is controlled by a flow meter. If the flow rate is too slow, tissue could become hypoxic. Conversely, if the flow rate is too high, the gas might not be sufficiently warmed and moisturized leading to drying of the tissue. High gas flow rate could also increase the chance of water spraying over the tissue from the water bath below leading to osmotic shock. This can be prevented by adding bits of nylon mesh (100 μm) at the outlet of the deflection ports.
3. Drain the circuit and fill up with filtered ACSF.
4. Put the ring which will support the slice in the holding chamber. A woven net filter with mesh openings ranging from 500 to 750 μm is stretched and attached to the ring with two-part resin epoxy glue. The woven net filter is made of 87% polyamide and 13% elastane (panty 15 den). Glue must be applied carefully to avoid the formation of reliefs at the surface of the ring.
5. Carefully remove all the air bubbles from the circuit.
6. Adjust the level of ACSF in the recording chamber with the screw of the suction needle. The speed of the peristaltic pumps is adjusted to 1 ml/min for the inlet pump and to 5 ml/min for the outlet pump.

The perfusion system is placed on a rigid vibration-resistant table and surrounded by a Faraday cage.

3. Preparation of the Dissection Area

Surgical instruments: a scalpel with a #11 blade, small standard dissecting scissors, spring scissors, curved forceps, two Heidemann spatulae and a spoon.

Supplementary material: a guillotine, an underpad, a McIlwain tissue chopper with a razor blade, filter paper, a glass Pasteur pipette with a rubber teat, 2 plastic Pasteur pipettes one of which with a wide mouth, a Petri dish, a metallic cylinder of 7 mm diameter (Figure 2A).

1. In the dissecting dish filled with cold ACSF (prepared in Step 1), immerse a refrigerated glass lid from a staining dish, wrapped in filter paper. Carefully remove air bubbles and oxygenate the ACSF through an aquarium bubbler (Figure 2A).
2. Lay out instruments in order of use. Add three layers of filter paper on the tissue chopper plate and a new razor blade cleaned with distilled water and ether (Figure 2B). The razor blade must be horizontal when it touches the paper. Blade force is adjusted to half of maximal value and speed to approximately one third of maximal value.
3. Attentively check if everything is ready (instruments, temperature ...) because you will not have time afterwards.
4. Ask someone to spray the dissection with a Pasteur pipette filled with cold ACSF.
5. Anesthetize the mouse with Nembutal IP (100 mg/kg) before decapitation. Halothane anesthesia can also be used. We have compared both methods and obtained same results. Decapitation must be performed under anesthesia but cervical dislocation can also be used. However cervical dislocation needs a very good practice to avoid animal suffering.
6. Dissect on the underpad as quickly as possible under cold ACSF continuous shower.

4. Brain Removal

1. While holding still the head with the index finger and the thumb of one hand on the muzzle, make an incision with the dissecting scissors along the middle of the top of the head starting at the edge of the guillotine cut and running rostrally to the frontal bone.
2. Cut through the cutaneous muscle on each side of the head to fully expose the skull plates and remove the muscles at the caudal side of the head.
3. With the dissecting scissors, cut the temporalis muscle on each side, along the temporalis plate, then cut the frontal plates in the middle, transversally. Then, make a little cut on the occipital bone, between the two plates.
4. For each side, cut at the caudal base of the occipital plates.
5. Cut along the sagittal suture with spring scissors.
6. With forceps, remove the skull halves by spreading them away from each other.
7. With the scalpel, cut transversally just after the olfactory bulb and just before the cerebellum then plunge the extracted brain into the dissecting dish with cold ACSF.

5. Hippocampal Dissection

1. Once the brain is immerged in the dissecting dish, sever the two hemispheres from each other with a scalpel inserted in the middle.
2. The dissection of hippocampus is performed with spatulae under visual control through a binocular surgical microscope (X25, Figure 2A). On one hemisphere, carefully spread the structures to see the lateral ventricle. Remove the brain stem and diencephalon. This is done by applying the spatulae on the frontal cortex on one side and on the diencephalon on the other side. Take care to not touch the hippocampus with the spatulae and to not stretch it during tissue sectioning.
3. Sever the fornix then gently push the hippocampus out of the cortex (roll away), by inserting a spatula in the ventricle.
4. When the hippocampus is extracted, remove excess cortex tissue and remaining blood vessels.

6. Cutting of the Slices

1. With a wide mouth plastic Pasteur pipette, transfer the hippocampus in a spoon with its alvear surface upwards (convex side).
2. Remove excess fluid with a standard plastic Pasteur pipette.
3. Tip up the spoon vertically nearly touching the filter paper of the chopper (Figure 2B) and drop off the hippocampus from the spoon, by rapidly touching the filter paper then move back the spoon.
4. Orientate the hippocampus and slice it transversally to 400 μm with the chopper (see Figure 2C for orientation of the hippocampus relative to the razor blade). Act as quickly as possible.
5. Remove the filter paper with the sliced hippocampus and wrap it around the metallic cylinder to spread the slices a little. Then, free the slices with sprays of ACSF using a standard plastic Pasteur pipette and collect them in a Petri dish filled with cold ACSF.

7. Incubation of Slices in Interface

1. Select slices with a standard plastic Pasteur pipette and rapidly place them in the recording chamber. Slices recover from the dissection trauma directly in the recording chamber, in interface at 28 °C.
2. The slice will most likely sink. Lower the fluid level to reach the slice level, and then raise it to make it float.
3. Turn the slice in the correct position while lowering the level. Slices are always orientated in the same way to facilitate the positioning of electrodes (2 stimulating and one recording electrodes) in the CA1 region.
4. Stop when the fluid is in interface. A "meniscus" of media around the slice is indicative of a sufficient level of media. The net filter must be saturated with media but not completely submerged.
5. Keep the chamber covered with filter papers placed over the perforated lid.
6. Let the slice rest for at least 1.5 hr at 28 °C.

8. Recording of Synaptic Responses

1. Recording electrode: glass capillaries are pulled to obtain a tip resistance of 2-5 MΩ when filled with ACSF. A small piece of filter paper is placed around the tip of the electrode and bone wax is applied at the extremity to reduce condensation. Stimulating electrodes: platinum-iridium bipolar cluster electrodes with a 12.5 μm diameter are purchased from FHC (USA). With proper care, each electrode can be used at least 30 times (Figure 1B).
2. Position the electrodes in the stratum radiatum of the CA1 region, all the electrodes lined up (Figure 3A). Electrodes are lowered 75 to 150 μm under the surface of the slice with Narishige micromanipulators. The two stimulating electrodes are placed to stimulate two distinct bundles of Schaffer collaterals. When the electrodes are lowered onto the slice, filter papers are carefully placed all around to close the chamber.
3. Biphasic stimulation (0.08 msec pulse duration per half-wave) is performed at constant voltage with a Grass stimulator connected to SIU-V isolation units. Maximal response is checked by increasing stimulus intensity from 2 V to maximum 12 V. Field EPSPs are amplified 1,000 times with a WPI ISO-80 amplifier and filtered at 10 Hz and 10 kHz. The signal is then sent to a PC through a National Instrument A/D converter. Stimulation, data acquisition and analysis are performed using the WinLTP program (www.winltp.com). Field EPSPs are recorded at 40% of the maximum amplitude obtained in an input-output curve. For each slice, the fEPSP slopes are normalized against the average slope over the 30 min preceding LTP induction.
4. LTP is triggered by applying a single train of stimulation (100 Hz) at test strength on one pathway while the second pathway serves as a control.

9. Cleaning of the Setup

1. Rinse all the circuit with a 3% solution of hydrogen peroxide (H₂O₂) for at least 10 min, then drain. The circuit will be carefully rinsed with distilled water before starting any experiment. The use of H₂O₂ is not absolutely required as other labs use only distilled water for cleaning but we have noticed the deposition of dark residues in the tubing system when using only water.
2. Replace water in the water bath beneath the recording chamber.
3. Bath the stimulating electrodes tips in alcohol for 5 min.

Results

This methodology has been used to analyze the properties of long-lasting long-term potentiation induced in acute hippocampal slices from adult C57Bl/6J mice (JANVIER SAS, France) ¹⁴. Surprisingly, improvement of the experimental conditions has led to a new way of looking at LTP. We showed that long-lasting increase in synaptic strength did not require the synthesis of new proteins.

Here, we show that LTP induction depends on slices viability and excitability. When dissection of the ...

Discussion

We have developed in our laboratory a protocol resulting from the combination of methods developed and used by other laboratories having a big expertise in LTP recordings ^11,17. This protocol is adapted to adult mouse hippocampus and can be used in animals of any age and any background genotype. It also allows the analysis of LTP in transgenic mice developing neurodegenerative diseases like Alzheimer's disease ^18,19.

Utilization of this protocol for rat hippocampal slice...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent/Material
NaCl	Sigma - Aldrich	S7653
NaHCO3	Sigma - Aldrich	S8875
KCl	Sigma - Aldrich	P9333
D-glucose	Sigma - Aldrich	G7528
NaH2PO4	Sigma - Aldrich	S9638
MgSO4 1M	Sigma - Aldrich	63126
CaCl2	Sigma - Aldrich	C4901
Carbogen	Air Liquide (Belgium)
Capillaries	WPI, Inc. (UK)	TW150-4
Stimulating Electrodes	FHC (USA)	CE2B30
Surgical tools	FST (Germany)
Filter paper 84 g/m2	Sartorius	FT-3-105-110
Mesh	Lycra	15 den
Glue	UHU	plus endfest300
Instrument
Amplifier	WPI, Inc. (UK)	ISO-80
Interface recording chamber	FST (Germany)
Peristaltic pumps	Gilson (USA)	Minipuls 3
Temperature controller	University of Edinburgh	www.etcsystem.com
Tissue Chopper	Mcllwain
Stimulators	Grass (USA)	S88X + SIU-V
Program analysis	WinLTP	www.winltp.com
Micromanipulators	Narishige	MM-3 and MMO-220A
Surgical microscope	Leica Microsystem
A/D converter	National Instruments	NIPCI-6229 M-series