W.W. conducted, analyzed, and designed the experiments and wrote the manuscript. D.X. and C.P. assisted in figure preparation and conducted the experiments. This work was supported by NSFC (31320103906) and 111 Project (B16013) to T.B.

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.
<ol>
	<li>Prepare the slicing buffer (modified Gey&#39;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.</li>
	<li>Adjust the pH to 7.6 using 1 M NaOH without carbogenation.</li>
	<li>Store the buffer at 4 &#176;C. 
	NOTE: The titration clarifies the cloudy liquid. The osmolality is 305-310 mOsm. The carbogen21 contains 5% carbon dioxide and 95% oxygen.</li>
	<li>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.</li>
	<li>Add bicarbonate and glucose to reach 10 mM glucose and 26 mM NaHCO3.</li>
	<li>Carbogenate the aCSF through a perforated silicon tube with a blocked end immediately after adding the NaHCO3.</li>
	<li>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 &#176;C).</li>
</ol>
2. Preparation of Acute Hippocampal Slices
NOTE: See the Table of Materials.
<ol>
	<li>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 &#176;C.</li>
	<li>Cool the surgical instruments down to 2-4 &#176;C.</li>
	<li>Place a glass beaker filled with cold and carbogenated slicing buffer (2-4 &#176;C), maintained in an ice/water mixture, near the vibratome.</li>
	<li>Anesthetize rats or mice until the corneal reflexes disappear (30-50 s) using isoflurane by taking 500 &#181;L into a 2 L glass jar or 12.5 &#181;L into a 50 mL tube.</li>
	<li>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.</li>
	<li>Cool the brain down to 2-4 &#176;C in an ice-cold slicing buffer (for at least 2 min) before dissecting the cerebellum and separating the hemispheres.</li>
	<li>Use a cold surgical blade to dissect a piece of the dorsal cortex at a 70&#176; 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.</li>
	<li>With a cold dental cement spatula, transfer a hemisphere to a piece of filter paper to briefly dry the created surface (1-2 s).</li>
	<li>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).</li>
	<li>Place the slicing platform into the cooling chamber of a vibratome and fill that chamber with the slicing buffer (2-4 &#176;C). Carbogenate using a perforated silicon tube.</li>
	<li>Cut 350 &#181;m slices using adequate vibratome settings (e.g., blade forward speed: 1.2 mm/s, blade amplitude: 1 mm).</li>
	<li>Isolate the hippocampal formation from the subiculum and the entorhinal cortices using two injection cannulas (&#62;28 G) as scissors.</li>
	<li>Remove the bubbles from the slice-holding mesh of the previously prepared incubation chamber using a Pasteur pipette.</li>
	<li>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 &#176;C.</li>
	<li>Incubate the slices at 30 &#176;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.</li>
</ol>
3. Modifications of the Carbogenation for the aCSF Recycling of Small Reservoirs
<ol>
	<li>Add a 3-way tube connector to the outflow of the recycling system (Figure 2B).</li>
	<li>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).</li>
	<li>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).</li>
	<li>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 (&#60;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.</li>
</ol>
4. Recording Synaptic Responses in a Submersion Slice Chamber
NOTE: See the Table of Materials.
<ol>
	<li>Pre-warm the aCSF solution in a water bath to about 28-30 &#176;C (this will prevent bubble formation in the recording chamber).</li>
	<li>Start the recycling system (4-5 mL/min) and activate the inline heater to equilibrate the system for at least 1 h.</li>
	<li>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).</li>
	<li>Remove the U-shaped platinum wire.</li>
	<li>Switch off the recycling and place a slice on the lens cleaning paper.</li>
	<li>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.</li>
	<li>Fill the borosilicate micropipette (i.e., the recording electrode) with aCSF (tip resistance: 1-2 M&#937;, filled 1/3 with aCSF) and mount it into the pipette holder.</li>
	<li>Check the insulation of the metal stimulation electrode by placing it into NaHCO3 solution (&#62;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 (&#62;60x magnification).</li>
	<li>Place the tested epoxy-insulated tungsten stimulation electrode in the manipulator holder and the reference wires in the slice chamber.</li>
	<li>Add a second reference electrode to the chamber and connect it to the reference socket of the headstage of the recording electrode (Figure 4A).</li>
	<li>In the amplifier control software, click on the &#34;zero clamp mode&#34; box and proceed by double-clicking the &#34;output gain list&#34; box. Choose a gain of &#34;100,&#34; double-click the &#34;high pass filter list box (Bessel),&#34; choose &#34;0.1 Hz,&#34; and double-click the &#34;low pass filter list box (AC)&#34; to choose &#34;3 kHz.&#34;</li>
	<li>In the recording software, click the on &#34;Acquire | Open Protocol.&#34; 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.</li>
	<li>Place the stimulation and recording electrodes in line and parallel to the stratum pyramidale, for example (Figure 4B and Figure 5).</li>
	<li>Click &#34;Acquire | Edit Protocol&#34; and choose the &#34;trigger&#34; tab (in the pop-up window). Click on the &#34;trigger source&#34; box and chose &#34;space bar&#34; as the trigger source. Click the &#34;OK&#34; button. Click the &#34;record&#34; button to start the acquisition and note the pop-up window with a trigger button.</li>
	<li>In the stimulus isolator software, click on the &#34;voltage control&#34; box and enter &#34;0.&#34; Click the &#34;download&#34; button. Click on the &#34;recording software&#34; 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 &#34;voltage control&#34; box.</li>
	<li>Correlate the stimulation strength with fEPSP-slope values and determine the stimulation strength required to get 40% of the fEPSP-slope maximum. Click the &#34;voltage control box&#34; and enter the determined value. Click the &#34;download&#34; button. 
	NOTE: A stimulus isolator is used to apply brief voltage or current pulses to brain tissue. The biphasic pulses can have 100 &#181;s per phase.</li>
	<li>In the recording software, click the stop button and then the &#34;Acquire&#34; menu. Click on &#34;Edit Protocol&#34; and choose the &#34;trigger&#34; tab in the pop-up window. Click on the trigger source box and chose the &#34;internal timer&#34; as the trigger source. Click the &#34;OK&#34; button. Click the record button that starts the automatic recording of field potentials every 30 s, for example. Click the &#34;stop&#34; button after 30-60 min.</li>
	<li>Click on the &#34;Acquire&#34; menu, click on &#34;Open Protocol,&#34; choose the desired induction protocol of synaptic plasticity, and click the &#34;OK&#34; button. Click the &#34;record&#34; button to automatically activate the high-frequency stimulation and recording. Click the &#34;stop&#34; 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.</li>
	<li>In the recording software, click &#34;Acquire | Open Protocol&#34; and choose the same protocol as in step 4.12. Click the &#34;OK&#34; button and then click &#34;record.&#34; Keep automatically running the field potential recordings for 2-4 h, for example. Click the &#34;stop&#34; button to terminate the recording.</li>
	<li>In case of pharmaceutical studies, apply the compound directly to the aCSF reservoir if permanent compound administration is desired (Figure 6).</li>
</ol>
5. Cleaning the Setup and Hints
NOTE: See below for general tips.
<ol>
	<li>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.</li>
	<li>Remove precipitated salt on the surface of the objective lens or chamber surroundings with 0.1 N HCl and then water.</li>
	<li>Wash the carbogen bubbler in distilled water or 0.1 N HCl.</li>
	<li>Immerse the slice incubation chamber in 10% H2O2 for 5 min once a week and then thoroughly rinse the incubation chamber with deionized H2O.</li>
	<li>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.</li>
	<li>Reduce the resistance of commercial metal stimulation electrodes by removing the insulation at the tip through a careful swipe with sandpaper.</li>
	<li>Reduce the loss of carbogen from the aCSF while recycling through the tubes by replacing the silicon tubes with polytetrafluoroethylene tubes.</li>
	<li>Keep the silver wires of the recording electrode and reference electrode in bleach for several days to form AgCl on the wire surface.</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+the+dorsal+and+ventral+hippocampus+functionally+distinct+structures.">Are the dorsal and ventral hippocampus functionally distinct structures.</a> Neuron. 65 (1), 7-19 (2010).</li><li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+of+BDNF-gene+transcripts+with+short+3'+UTR+in+hippocampal+CA1+neurons+improves+memory+formation+and+enhances+synaptic+plasticity-relevant+signaling+pathways.">Translation of BDNF-gene transcripts with short 3' UTR in hippocampal CA1 neurons improves memory formation and enhances synaptic plasticity-relevant signaling pathways.</a> Neurobiol Learn Mem. , (2016).</li></ol>

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Although interface slice chambers exhibit more robust synaptic responses25,26,27,28, 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...

In 1951, the first-reported acute brain slice experiments were conducted1. In 1971, after successful in vitro recordings from the piriform cortex2,3 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 plasticity9,10, 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 imaging17,18,19. 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.

<table><tbody><tr><td>&lt;strong&gt;Reagents required&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sinopharm Chemical Reagent, China</td><td>10019318</td><td></td></tr><tr><td>KCl</td><td>Sinopharm Chemical Reagent, China</td><td>10016318</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent, China</td><td>10017618</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm Chemical Reagent, China</td><td>10012818</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent, China</td><td>10005861</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent, China</td><td>10018960</td><td></td></tr><tr><td>Glucose</td><td>Sinopharm Chemical Reagent, China</td><td>10010518</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent, China</td><td>20040718</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma</td><td>A4043</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent, China</td><td>20025118</td><td></td></tr><tr><td>NaOH</td><td>Sinopharm Chemical Reagent, China</td><td>10019718</td><td></td></tr><tr><td>&lt;strong&gt;Tools and materials for dissection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Decapitators</td><td>Harvard apparatus</td><td>55-0012</td><td>for rat decapitation</td></tr><tr><td>Bandage Scissors</td><td>SCHREIBER</td><td>12-4227</td><td>for mouse decapitation</td></tr><tr><td>double-edge blade</td><td>Flying Eagle, China</td><td>74-C</td><td></td></tr><tr><td>IRIS Scissors</td><td>RWD, China</td><td>S12003-09</td><td></td></tr><tr><td>Bone Rongeurs</td><td>RWD, China</td><td>S22002-14</td><td></td></tr><tr><td>Spoon</td><td>Hammacher&amp;nbsp;</td><td>HSN 152-13</td><td></td></tr><tr><td>dental cement spatula</td><td>Hammacher&amp;nbsp;</td><td>HSN 016-15</td><td></td></tr><tr><td>dental double end excavator</td><td>Blacksmith Surgical, USA</td><td>BS-415-017</td><td></td></tr><tr><td>Vibrating Microtome</td><td>Leica, Germany</td><td>VT1200S</td><td></td></tr><tr><td>surgical blade&amp;nbsp;</td><td>RWD, China</td><td>S31023-02</td><td></td></tr><tr><td>surgical holder</td><td>RWD, China</td><td>S32007-14</td><td></td></tr><tr><td>&lt;strong&gt;Electrophysiology equipment and materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Vertical Pipette Puller</td><td>Narishige, Japan</td><td>PC-10</td><td></td></tr><tr><td>Vibration isolation table</td><td>Meirits, Japan</td><td>ADZ-A0806</td><td></td></tr><tr><td>submerged type recording chamber</td><td>Warner Instruments</td><td>RC-26GLP</td><td></td></tr><tr><td>4 Axis Micromanipulator</td><td>Sutter, USA</td><td>MP-285, MP-225</td><td></td></tr><tr><td>Platinum Wire</td><td>World Precision Instruments</td><td>PTP406</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices, USA</td><td>Multiclamp 700B</td><td></td></tr><tr><td>Data Acquisition System</td><td>Molecular Devices, USA</td><td>Digidata 1440A</td><td></td></tr><tr><td>Anaysis software</td><td>Molecular Devices, USA</td><td>Clampex 10.2</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Nikon, Japan</td><td>FN1</td><td></td></tr><tr><td>LED light source</td><td>Lumen Dynamics Group, Canada</td><td>X-cite 120LED</td><td></td></tr><tr><td>micropipettes</td><td>Harvard apparatus</td><td>GC150TF</td><td>extracelluar recording</td></tr><tr><td>borosilicate micropipettes</td><td>Sutter, USA</td><td>BF150-86</td><td>patch clamp</td></tr><tr><td>tungsten electrode</td><td>A-M Systems, USA</td><td>575500</td><td></td></tr><tr><td>peristaltic pump</td><td>Longer, China</td><td>BT00-300T</td><td></td></tr><tr><td>tubes for peristaltic pump</td><td>ISMATEC, Wertheim, Germany</td><td>SC0309</td><td>1x inflow, ID: 1.02 mm</td></tr><tr><td>tubes for peristaltic pump</td><td>ISMATEC, Wertheim, Germany</td><td>SC0319</td><td>2x tubes for outflow, ID: 2.79 mm</td></tr><tr><td>CCD camera</td><td>PCO, Germany</td><td>pco.edge sCMOS</td><td></td></tr><tr><td>lens cleaning paper</td><td>Kodak</td><td></td><td></td></tr><tr><td>50 mL conical centrifuge tube</td><td>Thermo scientific</td><td>339652</td><td></td></tr><tr><td>Prechamber</td><td>Warner Instruments</td><td>BSC-PC</td><td></td></tr><tr><td>Inline heater</td><td>Warner Instruments</td><td>SF-28</td><td></td></tr><tr><td>Temperature Controller</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr></tbody></table>

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.

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 ...

Watch this Scientific Journal Video about Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System at JoVE.com

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

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 ...

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11.5K Views.  Fudan University. Dit protocol beschrijft de stabilisatie van het zuurstofgehalte in een klein volume van gerecycled buffer en methodologische aspecten van de opname van de activiteit-afhankelijke synaptische plasticiteit in verzonken acute hippocampal plakjes.

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

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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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 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 ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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 ...

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 ...

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 ...

Recording of Local Field Potential in Mouse Hippocampal-Entorhinal Cortex Slices

Source: Whitebirch, A. C. <a href="https://app.jove.com/v/61704">Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity.</a> J. Vis. Exp. (2020)
The video demonstrates a procedure to record the local field potential (LFP) in mouse hippocampal-entorhinal cortex slices. Electrical stimulation is provided at the CA3 stratum radiatum of the hippocampus, which causes changes in the postsynaptic potential at the CA1 stratum pyramidale. The combined change in the membrane potential of CA1 neurons, termed the local field potential, is recorded.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Transcardial perfusion

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Neurophysiology

Electrophysiology Techniques

Recording Neuronal Field Excitatory Postsynaptic Potentials in Acute Hippocampal Slices

Source: Weng, W., et al. <a href="https://app.jove.com/v/55936">Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System.</a> J. Vis. Exp. (2018).
This video demonstrates the procedure for setting up a brain slice in a submersion chamber to record synaptic activity. The process involves securing the slice on lens paper, maintaining a continuous flow of artificial cerebrospinal fluid (aCSF) to ensure neuronal viability, and accurately positioning the stimulation and recording electrodes relative to a specific brain region of interest. This setup allows for precise monitoring of baseline synaptic activity and the effects of electrical stimulation.

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

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