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%.
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.
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.
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.
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.
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).
4. Brain Removal
5. Hippocampal Dissection
6. Cutting of the Slices
7. Incubation of Slices in Interface
8. Recording of Synaptic Responses
9. Cleaning of the Setup
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 hippocampus was too slow or too harmful, slices excitability increased and polysynaptic responses could be observed after LTP induction (Figure 3B). In this case, LTP induction was much less effective and potentiation was not maintained.
We would like to emphasize the fact that, even in slices apparently healthy, technical conditions have a great influence on the duration of the LTP induced by a single train of stimulation. In previous publications, our group showed that a short-lasting LTP can be transformed into a long-lasting one by modifying the recovery conditions of the slice 15. Over years of daily practice, we observed that successive improvements in our technique have led to a progressive increase in the duration of the LTP induced by a single train in standard conditions. Indeed, recordings made in 2005 showed a short-lasting LTP going back to baseline within 3 hr (Figure 3C, filled circles). Modifications in the dissection procedure reducing tissue stretching have made it possible to obtain an LTP sustained for 6 hr 16 (Figure 3C, filled squares). And finally, improvements in the interface oxygenation and the temperature control, combined with electrode standardization have led to an LTP stable for more than 8 hr (Figure 3C, open circles). The difference between the three groups is significant (one-way ANOVA, F(2,20) = 49,5, P < 0.001).
Moreover, any modification of temperature or oxygenation induced an increase or a decrease of the synaptic response (Figure 4). The control of these parameters was checked by always using two stimulating electrodes. LTP was induced on one bundle of Schaffer collaterals while another bundle was used as an internal control of synaptic strength stability.
Figure 1. Electrophysiological rig and brain slice recording chamber. (A) Perfusion circuit with home-made gravity perfusion system alimented by a peristaltic pump, isolated stimulation units (two per electrode), surgical microscope and recording chamber. (B) Interface recording chamber with stimulating and recording electrodes. Filter papers have been removed from the lid to see the ring supporting the slice. (C) Control unit with 2 stimulators, oscilloscope, A/D converter, temperature control software and WinLTP software.
Figure 2. Tools and material used for hippocampus slicing. (A) Dissection dish containing refrigerated ACSF and surgical microscope. Scalpel mounted with a #11 blade, small standard dissecting scissors, spring scissors, curved forceps, two Heidemann spatulae, spoon, 2 plastic Pasteur pipettes one of which with a wide mouth, Petri dish, metallic cylinder of 7 mm diameter and ring which supports the slice in the recording chamber. (B) McIlwain tissue chopper. (C) Drawing and photograph of the left hippocampus in position for cutting. The orientation of the razor blade is indicated by the red dashed line. Scale of the reticle: 1 mm. Click here to view larger figure.
Figure 3. Induction of LTP in hippocampal slices. (A) Sketch showing the two independent synaptic inputs S1 and S2 to the same neuronal population. In each slice, two stimulating electrodes (S1 and S2) were put in place. S1 pathway was used to induce LTP, while S2 pathway acted as a control. (B) Sample fEPSP traces from individual experiments in a perfectly healthy slice (left) and in a slice presenting a high level of excitability (right). They were recorded just before the LTP induction (red traces) and one hour after LTP induction (blue traces). When slices are not perfectly healthy (right), polysynaptic responses are observed and fEPSP slope potentiation is reduced. (C) Comparison of the time courses of the fEPSP slope after LTP induced by a single train of high-frequency stimulation (100 Hz, 1 sec) recorded in 2005, 2010 and 2011 in our laboratory. First experiments were recorded in 2005 (filled circles, n = 11). Subsequent recordings (Villers, et al. 2010) benefited from an improved dissection procedure (filled squares, n = 6). Current results arise from optimization of interface oxygenation and temperature control along with electrode standardization (open circles, n = 6). Click here to view larger figure.
Figure 4. Variation of fEPSP slope as a function of temperature and oxygen flow rate. (A) Increasing oxygen flow rate in water bath beneath the recording chamber from 0.15 to 0.25 L/min (blue curve) induces a decrease in fEPSP slope of 20% (pink curve). (B) Increasing the temperature from 28 to 29 °C (green curve) induces a more than 50% increase in fEPSP slope (pink curve). Click here to view larger figure.
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 slices could necessitate some adaptations. For example, most of the studies performed in rats use a temperature of 32 °C instead of 28 °C. Mathis et al. 20 proposed another method for the preparation of hippocampal slices from aging mice or rats.
Material and methods
C57BL/6J mice come from JANVIER SAS (France) but same results are obtained with C57BL/6J mice from Charles River France.
The hippocampus is rapidly isolated while the tissue is submerged in cold ACSF to reduce cell damage. This allows a reduction in excitotoxicity but is known to induce synapse proliferation 21,22 which can mask further structural synaptic plasticity.
Then we use a tissue chopper for slicing instead of vibratome. A tissue chopper is particularly well adapted to the sectioning of small pieces of tissue like mouse hippocampus. On the tissue chopper, the hippocampus is always orientated in the same way according to the procedure of Alger et al. 23. They placed the striations on the alvear surface, visible with oblique lighting, parallel to the razor blade. This method provides hippocampal slices of the dorsal part cut with an angle of 15° from the transverse axis (See Figure 2C). Subsequently, slices are manipulated with a minimum of direct contact.
Slices are maintained in interface at 28 °C directly after dissection. This method has been shown to reduce polysynaptic activity and epileptogenicity in mouse and rat slices 24.
External parameters are carefully controlled to obtain reproducible results. Bipolar cluster stimulating electrodes (FTC) are used instead of home-made electrodes in order to increase reproducibility in induction, seeing that the amplitude of LTP induction really depends on the electrodes quality.
ETC system from the University of Edinburgh 11 maintains a constant and uniform temperature inside the whole experimental rig and carbogen consumption is stabilized with a flow meter. Interface level is controlled visually with the help of a binocular surgical microscope and is maintained very stable with a peristaltic pump aspiration system.
Results
This protocol allows the induction of a very stable LTP which remains dependent on external conditions like temperature and oxygenation. We have previously demonstrated that LTP induced in these conditions was dependent on NMDA receptors, α-CaMKII autophosphorylation and PI3-kinase activation which are the classical molecular pathways involved in LTP 14. By contrast, we were unable to reproduce the dependency of the maintenance phase of LTP on new protein synthesis. The ability of protein-synthesis inhibitors, and of anisomycin in particular, to prevent the development of the late phase of L-LTP when they were applied around the induction has been repeatedly reported 4,25. This has led to the commonly held hypothesis that stabilizing synaptic plasticity for longer than about 2-3 hr required the triggering by the LTP-inductive stimulus of a transitory synthesis of new proteins 26. However, recent experiments have suggested that things were more complicated 27-32. LTP induced in our experimental conditions was maintained even in the presence of protein synthesis inhibitors suggesting that other mechanisms than new proteins synthesis could be involved in the lasting phase of LTP.
Nevertheless, in vitro experimentation always raises the question of the physiological relevance of the observed phenomenon. Slicing induces modifications in the phosphorylation state of proteins involved in activity-dependent forms of synaptic plasticity 33 and alteration in the metabolic state of the tissue 34. The rate of protein synthesis is reduced to 10 to 15% of that observed in vivo 35 and balance between mRNA and proteins is disturbed 36. For all these reasons, we must be cautious in the interpretation of the results.
By definition LTP is a rapidly induced long-lasting (days, weeks) enhancement of an excitatory synapse, which probably plays an important role in long-term memory. LTP, in vivo, can last for several weeks and similar changes in synaptic strength occur during learning 37,38. In addition, most of the time, when long-term LTP is prevented by mutations, long-term memory is impaired 39.
More problematic is the parallel between short-term memory and short-lasting long-term potentiation. The first problem is that the duration of each phenomenon is unknown. Short-term memory has been studied 1 hr to 1 day after learning procedure whereas short-term LTP is supposed to last less than 5 hr. The second question is whether short-term memory (or short-lasting LTP) is merely a step forward long-term memory (or long-lasting LTP) or a separate entity with distinct molecular mechanisms 40. Thirdly, we don’t know if short-term LTP is induced by a weak stimulus that falls short of causing long-lasting LTP or if it is induced by the same stimulus but appears only when mechanisms underlying long-lasting LTP fail.
The long-lasting stability of LTP under our experimental conditions could be viewed as a short-term LTP equivalent to short-term memory lasting between 10 hr and 24 hr. In this form of synaptic plasticity, protein synthesis, which is highly reduced in slices, is not needed and LTP can be induced by a single train of stimulation. This long-lasting increase in synaptic strength could be due to a stable increase in the number of AMPA receptors in the post-synaptic membrane without spine remodelling.
According to this hypothesis, protein synthesis could be needed for spine enlargement and structural modifications of the synapses leading to long-term memory lasting several weeks. In this case, the decremental phase of LTP could only be observed 24 hr after induction. Unfortunately, until now, acute slices can only be maintained alive during approximately 16 hr.
This hypothesis could be tested by using Tc1 mouse model of Down syndrome. Morice et al. 41 have shown that, in these mice, short-term LTP in vivo and short-term memory are impaired whereas long-term synaptic plasticity and memory are preserved.
Discrepancies between different studies performed in different laboratories could be due to the duration of short-lasting LTP due to variation in the enzymatic activity, in the metabolic state of the slices or in the rate of protein degradation 42. Protein synthesis could be viewed as a permissive replenishment process involving reposition of enzymes that have been used by the learning process, or of other constitutive cell elements 43.
These results highlight the importance of experimental conditions on LTP stability. A stable LTP independant of protein synthesis could help to study the role of long-lasting post-traductional modifications of synaptic proteins and the mechanisms underlying stable increase of the number of AMPA receptors in the post-synaptic membrane in spite of receptors turnover.
This video shows one detailed protocol which should hopefully help to obtain reproducible results in different laboratories.
The authors have nothing to disclose.
We thank Bernard Foucart for technical assistance. This work was supported by the Belgian Fund for Scientific Research (F.R.S.-FNRS) and by the Queen Elisabeth Fund for Medical Research. Agnès Villers is Research Fellow at the Belgian Fund for Scientific Research.
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 |