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. Agn&egrave;s Villers is Research Fellow at the Belgian Fund for Scientific Research.

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.
<ol>
	<li>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.</li>
	<li>Put all the components except CaCl2 in a glass graduated cylinder and bring to 2 L with distilled H2O.</li>
	<li>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.</li>
	<li>Keep 600 ml in a refrigerated rectangular glass dish and cool down to 4 &deg;C with ice around the dish. This media will be used for hippocampus dissection.</li>
	<li>Filter the remaining 1,400 ml and keep in an Erlenmeyer flask.</li>
</ol>
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 (<a href="http://www.etcsystem.com" target="_blank">www.etcsystem.com</a>) 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 &deg;C.
<ol>
	<li>Rinse all the perfusion circuit with distilled H2O for a minimum of 20 min and start the heating systems.</li>
	<li>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 &mu;m) at the outlet of the deflection ports.</li>
	<li>Drain the circuit and fill up with filtered ACSF.</li>
	<li>Put the ring which will support the slice in the holding chamber. A woven net filter with mesh openings ranging from 500 to 750 &mu;m 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.</li>
	<li>Carefully remove all the air bubbles from the circuit.</li>
	<li>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.</li>
</ol>
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).
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>Attentively check if everything is ready (instruments, temperature ...) because you will not have time afterwards.</li>
	<li>Ask someone to spray the dissection with a Pasteur pipette filled with cold ACSF.</li>
	<li>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.</li>
	<li>Dissect on the underpad as quickly as possible under cold ACSF continuous shower.</li>
</ol>
4. Brain Removal
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>For each side, cut at the caudal base of the occipital plates.</li>
	<li>Cut along the sagittal suture with spring scissors.</li>
	<li>With forceps, remove the skull halves by spreading them away from each other.</li>
	<li>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.</li>
</ol>
5. Hippocampal Dissection
<ol>
	<li>Once the brain is immerged in the dissecting dish, sever the two hemispheres from each other with a scalpel inserted in the middle.</li>
	<li>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.</li>
	<li>Sever the fornix then gently push the hippocampus out of the cortex (roll away), by inserting a spatula in the ventricle.</li>
	<li>When the hippocampus is extracted, remove excess cortex tissue and remaining blood vessels.</li>
</ol>
6. Cutting of the Slices
<ol>
	<li>With a wide mouth plastic Pasteur pipette, transfer the hippocampus in a spoon with its alvear surface upwards (convex side).</li>
	<li>Remove excess fluid with a standard plastic Pasteur pipette.</li>
	<li>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.</li>
	<li>Orientate the hippocampus and slice it transversally to 400 &mu;m with the chopper (see Figure 2C for orientation of the hippocampus relative to the razor blade). Act as quickly as possible.</li>
	<li>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.</li>
</ol>
7. Incubation of Slices in Interface
<ol>
	<li>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 &deg;C.</li>
	<li>The slice will most likely sink. Lower the fluid level to reach the slice level, and then raise it to make it float.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Keep the chamber covered with filter papers placed over the perforated lid.</li>
	<li>Let the slice rest for at least 1.5 hr at 28 &deg;C.</li>
</ol>
8. Recording of Synaptic Responses
<ol>
	<li>Recording electrode: glass capillaries are pulled to obtain a tip resistance of 2-5 M&Omega; 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 &mu;m diameter are purchased from FHC (USA). With proper care, each electrode can be used at least 30 times (Figure 1B).</li>
	<li>Position the electrodes in the stratum radiatum of the CA1 region, all the electrodes lined up (Figure 3A). Electrodes are lowered 75 to 150 &mu;m 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.</li>
	<li>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 (<a href="http://www.winltp.com">www.winltp.com</a>). 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.</li>
	<li>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.</li>
</ol>
9. Cleaning of the Setup
<ol>
	<li>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.</li>
	<li>Replace water in the water bath beneath the recording chamber.</li>
	<li>Bath the stimulating electrodes tips in alcohol for 5 min.</li>
</ol>

No conflicts of interest declared.

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...

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.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent/Material&lt;/strong&gt;</td></tr><tr><td>NaCl</td><td>Sigma - Aldrich</td><td>S7653</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma - Aldrich</td><td>S8875</td><td></td></tr><tr><td>KCl</td><td>Sigma - Aldrich</td><td>P9333</td><td></td></tr><tr><td>D-glucose</td><td>Sigma - Aldrich</td><td>G7528</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma - Aldrich</td><td>S9638</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; 1M</td><td>Sigma - Aldrich</td><td>63126</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma - Aldrich</td><td>C4901</td><td></td></tr><tr><td>Carbogen</td><td>Air Liquide (Belgium)</td><td></td><td></td></tr><tr><td>Capillaries</td><td>WPI, Inc. (UK)</td><td>TW150-4</td><td></td></tr><tr><td>Stimulating Electrodes</td><td>FHC (USA)</td><td>CE2B30</td><td></td></tr><tr><td>Surgical tools</td><td>FST (Germany)</td><td></td><td></td></tr><tr><td>Filter paper 84 g/m&lt;sup&gt;2&lt;/sup&gt;</td><td>Sartorius</td><td>FT-3-105-110</td><td></td></tr><tr><td>Mesh</td><td>Lycra</td><td>15 den</td><td></td></tr><tr><td>Glue</td><td>UHU</td><td>plus endfest300</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Instrument&lt;/strong&gt;</td></tr><tr><td>Amplifier</td><td>WPI, Inc. (UK)</td><td>ISO-80</td><td></td></tr><tr><td>Interface recording chamber</td><td>FST (Germany)</td><td></td><td></td></tr><tr><td>Peristaltic pumps</td><td>Gilson (USA)</td><td>Minipuls 3</td><td></td></tr><tr><td>Temperature controller</td><td>University of Edinburgh</td><td>&lt;a href=&quot;http://www.etcsystem.com&quot; target=&quot;_blank&quot;&gt;www.etcsystem.com&lt;/a&gt;</td><td></td></tr><tr><td>Tissue Chopper</td><td>Mcllwain</td><td></td><td></td></tr><tr><td>Stimulators</td><td>Grass (USA)</td><td>S88X + SIU-V</td><td></td></tr><tr><td>Program analysis</td><td>WinLTP</td><td>&lt;a href=&quot;http://www.winltp.com&quot; target=&quot;_blank&quot;&gt;www.winltp.com&lt;/a&gt;</td><td></td></tr><tr><td>Micromanipulators</td><td>Narishige</td><td>MM-3 and MMO-220A</td><td></td></tr><tr><td>Surgical microscope</td><td>Leica Microsystem</td><td></td><td></td></tr><tr><td>A/D converter</td><td>National Instruments</td><td>NIPCI-6229 M-series</td><td></td></tr></tbody></table>

improved preparation and preservation of hippocampal mouse slices for a very stable and reproducible recording of long-term potentiation

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.

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 ...

Watch this Scientific Journal Video about Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation at JoVE.com

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

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 ...

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Universidad de Cartagena UDEC

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26.7K Views.  University of Mons. 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%.

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

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

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

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.

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.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Investigating Long-term Synaptic Plasticity in Interlamellar Hippocampus CA1 by Electrophysiological Field Recording

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the longitudinal interlamellar CA1-CA1 network. Recently however, an associational connection between CA1-CA1 pyramidal neurons has been shown. Therefore, there is the need to investigate whether the longitudinal interlamellar CA1-CA1 network of the hippocampus supports synaptic plasticity.
We designed a protocol to investigate the presence or absence of long-term synaptic plasticity in the interlamellar hippocampal CA1 network using electrophysiological field recordings both in vivo and in vitro. For in vivo extracellular field recordings, the recording and stimulation electrodes were placed in a septal-temporal axis of the dorsal hippocampus at a longitudinal angle, to evoke field excitatory postsynaptic potentials. For in vitro extracellular field recordings, hippocampal longitudinal slices were cut parallel to the septal-temporal plane. Recording and stimulation electrodes were placed in the stratum oriens (S.O) and the stratum radiatum (S.R) of the hippocampus along the longitudinal axis. This enabled us to investigate the directional and layer specificity of evoked excitatory postsynaptic potentials. Already established protocols were used to induce long-term potentiation (LTP) and long-term depression (LTD) both in vivo and in vitro. Our results demonstrated that the longitudinal interlamellar CA1 network supports N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) with no directional or layer specificity. The interlamellar network, however, in contrast to the transverse lamellar network, did not present with any significant long-term depression (LTD).

We used recording and stimulation electrodes in longitudinal hippocampal brain slices and longitudinally positioned recording and stimulation electrodes in the dorsal hippocampus in vivo to evoke extracellular postsynaptic potentials and demonstrate long-term synaptic plasticity along the longitudinal interlamellar CA1.

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

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

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Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Horizontal Hippocampal Slices of the Mouse Brain