We thank Mandy Marbler-P&#246;tter (Charite-Unversit&#228;tsmedizin, Berlin) for excellent technical assistance. P.F. was funded by the German Research Foundation (DFG, Deutsche Forschungsgemeinschaft) under Germany&#180;s Excellence Strategy-EXC-2049-390688087. This work has been supported by the QUEST Center for Transforming Biomedical Research at the Berlin Institute of Health.

Patients must give informed written consent prior to operation, and necessary ethical agreements must be in place prior to the experiment. Concerning the representative results, all studies involving human participants were reviewed and approved by Charit&#233;-Universit&#228;tsmedizin, Berlin (EA2/111/14).
1. Preparation of 10x solutions
NOTE: Due to difficulties in planning access to human brain tissue, it is recommended to prepare 10x solutions as described here. Alternatively, final 1x solutions can be prepared freshly by adding individual substances in final concentration to double-distilled water (ddH2O).
<ol>
	<li>For individual 10x solutions, add substances to ddH2O according to Table 1 and stir until dissolved.</li>
	<li>Use 10x solutions up to 1 month after preparation (up to 1 year for frozen 10x choline aCSF).</li>
	<li>For 10x choline aCSF, prepare 50 mL aliquots of 10x 1.1 choline aCSF (Table 1) and freeze at -20 &#176;C or -80 &#176;C until further use. 
	NOTE: Do not add glucose and CaCl2 to 10x 1.1 choline aCSF to prevent contamination with bacteria and precipitation of calcium carbonate.</li>
	<li>10x solution 2 can be used for all 1x final solutions, whereas 10x solutions 1.1&#8211;1.4 are customized and named accordingly (Table 1).</li>
</ol>
2. Preparation of 1x final solutions
NOTE: Final 1x solutions should be prepared fresh or earliest&#160;as possible on the day before use. All final solutions should be carbogenated with 5% CO2 and 95% O2 using a glass gas disperser to enrich solutions with oxygen, and adjust the pH to 7.4 (max = 7.4 &#177; 0.2).
<ol>
	<li>Choline aCSF for transport and preparation
	<ol>
		<li>For the final 500 mL solution, thaw a 50 mL aliquot of the 10x solution 1.1 aliquot for choline aCSF in 37 &#176;C water bath.</li>
		<li>Add the thawed 50 mL aliquot of the 10x solution 1.1 and 50 mL of 10x solution 2 to approximately 300 mL of ddH2O.</li>
		<li>Add final concentrations of glucose and CaCl2,&#160;then stir until dissolved (Table 1, solution 1.1).</li>
		<li>Add ddH2O to a final volume of 500 mL and measure osmolarity (300 mOsm &#177; 10 mOsm).</li>
		<li>Optionally, use a filter to sterilize the solution (see discussion on prolonged slice viability under sterile conditions).</li>
		<li>Fill a separate bottle with approximately 100 mL of 1x choline aCSF for transport from the operation room to laboratory.</li>
		<li>Optional: depending on the transport time from the operation room to laboratory, consider using gas-tight bottle caps to ensure stable pH of aCSF during longer transportation periods.</li>
		<li>Store the final solution at 4&#8211;8&#176;C until further use.</li>
		<li>On the day of operation, chill 1x choline aCSF on ice and carbogenate for at least 10&#8211;15 min using a glass gas disperser connected to carbogen gas (5% CO2, 95% O2). 
		NOTE: Consider keeping a gas bottle accessible to the operation room in the case of longer waiting times, which will require re-carbogenation of the transport solution. However, we have transported hippocampal tissue without re-carbogenation before long and short transportation times (15 min vs. 60 min) and did not observe differences in induction of epileptiform activity.</li>
	</ol>
	</li>
	<li>aCSF for storage and recording
	<ol>
		<li>For a final 2 L solution, add 200 mL of 10x solution 1.2 (aCSF) and 200 mL of 10x solution 2 and glucose (Table 1) to ~1500 mL of ddH2O. 
		NOTE: Volumes of the final solutions depend on applied experiments and the type of chamber used for storing and recording.</li>
		<li>Add ddH2O to a final volume of 2 L and measure osmolarity (300 mOsm &#177;10 mOsm).</li>
		<li>Prewarm the solution to 35 &#176;C and carbogenate for at least 10&#8211;15 min before use.</li>
	</ol>
	</li>
	<li>HighK++4-AP aCSF for induction of burst activity
	<ol>
		<li>For a final 1 L solution, add 100 mL of 10x solution 1.3 (highK++4-AP aCSF) and 100 mL of 10x solution 2 to ~700 mL of ddH2O.</li>
		<li>Add glucose and 4-AP (final concentration = 100 &#181;M) according to Table 1.</li>
		<li>Add ddH2O to final volume of 1 L and measure osmolarity (300 mOsm &#177;10 mOsm).</li>
		<li>Prewarm the solution to 35 &#176;C and carbogenate for at least 10&#8211;15 min before use.</li>
	</ol>
	</li>
	<li>LowMg2++BIC aCSF for induction of seizure-like events (SLEs)
	<ol>
		<li>For a final 1 L solution, add 100 mL of 10x solution 1.4 (lowMg2++BIC aCSF) and 100 mL of 10x solution 2 to ~700 mL of ddH2O.</li>
		<li>Add glucose and BIC (final concentration = 10 &#181;M) according to Table 1.</li>
		<li>Add ddH2O to final volume of 1 L and measure osmolarity (300 mOsm &#177; 10 mOsm).</li>
		<li>Prewarm the solution to 35 &#176;C and carbogenate for at least 10&#8211;15 min before use.</li>
	</ol>
	</li>
</ol>
3. Preparation of interface chamber
<ol>
	<li>In an interface chamber, slices rest on three layers of filter paper to ensure sufficient amount of solution below the slice. To do this, cut two ~4 cm x ~2 cm pieces of filter paper for each slice-holding compartment (the described&#160;interface chamber&#160;consists of two compartments) and place them on top of each other.</li>
	<li>Place thin cotton strings around the 4 cm x 2 cm filter papers inside the compartments to break the tension of the solution. Ensure even flow (here, black nylon tights cut to&#160;thin-cut strings ~10 cm in length are used; for placement, see Figure 1).</li>
	<li>Place small pieces of filter paper on top of the larger filter papers inside the slice-holding compartments. Small filter tissue pieces should be roughly the size of one brain slice (~1.5 cm x ~1.0 cm) and will enable further handling of individual slices. Place three to four small filter paper pieces in each compartment.</li>
	<li>Ensure an aCSF flow rate of 1.8 mL/min with a peristaltic pump.</li>
	<li>Carbogenate and prewarm the interface chamber to ~35 &#176;C (final temperature of the slice should be ~32 &#176;C).</li>
</ol>
4. Set-up of preparation area
NOTE: Preparation can be performed under sterile conditions to avoid contamination and elongate slice survival. However, not all vibratomes fit under a sterile hood, and other measures are required to reduce contamination during preparation. This section describes some of these measures.
<ol>
	<li>Wipe the preparation area with 70% EtOH and place either aluminum foil or sterile covers on top of the area.</li>
	<li>Prepare super glue, two sharp tweezers, a spatula, a scalpel with blades, and a blade for rough cutting of the brain tissue. Tools can be sterilized prior to the procedure to reduce contamination.</li>
	<li>Wipe the buffer tray and specimen plate of the vibratome with 70% EtOH. Once the buffer tray is fully dry, cover it with aluminum foil and place the tray in the ice bath. Fill the ice bath with crushed ice and keep at -20 &#176;C until preparation.</li>
	<li>Wipe the vibratome and razor blade with 70% EtOH and calibrate the vibratome to minimize vertical vibrations and tissue damage during slicing procedure.</li>
</ol>
5. Tissue slicing and storing
<ol>
	<li>Directly after resection, place the tissue immediately in cold, carbogenated choline aCSF and transport quickly to the laboratory. 
	CAUTION: Wear gloves and a face mask at all times during preparation, since human brain tissue can contain potential pathogens. In addition, wearing a face mask when not working under a sterile hood will greatly reduce contamination of solutions and brain tissue.</li>
	<li>Remove tissue from choline aCSF and cut away any burned portions of tissue.</li>
	<li>Cut an even surface to glue tissue piece onto the specimen plate, while considering the cutting angle and tissue layers. Ideally, a hippocampal slice contains DG, CA1-4, and (if possible) subiculum.</li>
	<li>Slice the brain tissue into 400 &#181;m thick slices and adjust the amplitude and speed during cutting. Due to&#160;possible remaining pia mater, human brain tissue shows more resistance and may require slower cutting. 
	NOTE: Slice thickness greatly affects either the available network (more neurons in thicker slices) or viability of the slice (penetration of solution into the slice). We have used 500 &#181;m&#160;slices to increase the potentially available micro-network, and could not observe differences in induction of epileptiform activity. 300 &#181;m slices are commonly used for patch-clamp experiments, though the induction of epileptiform activity in these slices have not yet been tested here. We use 400 &#181;m as a standard slice thickness, though 300&#8211;500 &#181;m slices may be sufficient.</li>
	<li>Before collecting, use a scalpel to reduce the sizes of brain slices to fit into the recording chamber. For use of the membrane chamber (see section 6), slices should be maximally 1.5 cm x 1 cm. While reducing, consider the specific layers and connections needed to be intact for recording (e.g., for recording in the CA1 and DG, cut away the subiculum and surrounding white tissue).</li>
	<li>Using a spatula and small forceps, carefully place slices in the interface chamber on small filter papers and let them rest for ~1 h in aCSF until recording.</li>
	<li>Slices can be recorded for up to 20 h (even longer when under sterile conditions).</li>
</ol>
6. Recording of epileptiform activity
<ol>
	<li>In the membrane chamber (submerged type recording chamber), place the brain slice on a transparent semipermeable membrane, which is glued to a plastic ring24. For this, use super glue to attach the plastic ring to the membrane of a cell culture insert.</li>
	<li>Use a scalpel to remove any membrane on the outside of the plastic ring. Ensure that the membrane is even and fully attached to the ring before placing the membrane in the chamber. 
	NOTE: The membrane can be stored in ddH2O at 4&#8211;8 &#176;C and reused for up to 1 month. Keep the membrane wet at all times.</li>
	<li>Both the inflow and outflow of the membrane chamber are connected to tubes for solution supply. Place the tubes in a peristaltic pump so that the inflow and outflow move in opposite directions.</li>
	<li>Place the inflow and outflow tube in carbogenated, prewarmed aCSF until all tubes and the chamber are filled with solution. Adjust the speed of the peristaltic pump to achieve an even flow rate of 10&#8211;13 mL/min. 
	NOTE: The membrane chamber used here is a high flow rate, submerged type recording chamber enabling a solution flow of up to 14 mL/min24. In the case of using a different submerged type recording chamber, flow rates need to be adjusted. However, for induction of epileptiform activity, it is highly recommended to use the membrane chamber.</li>
	<li>Use a heating element connected to the inflow in close proximity to the membrane chamber to ensure a stable temperature of 32 &#176;C.</li>
	<li>Prepare 1&#8211;2 M&#8486; glass pipettes using a vertical puller. Fill pipettes with 154 mM NaCl solution and place them in an electrode holder.</li>
	<li>Using tweezers and a spatula, remove a hippocampal slice from the interface chamber by taking the slice with the small filter paper and placing both in a Petri dish filled with carbogenated aCSF. Remove the small filter paper from the hippocampal slice, and (if necessary) apply some force using a pipette to separate the slice from the filter paper. Be careful not to flip the slice.</li>
	<li>Place the slice in the recording chamber and hold it in place using slice mesh. 
	NOTE: Due to Bernoulli&#8217;s principle, in the used submerged type membrane chamber, slices are usually stable without use of an additional slice mesh.</li>
	<li>Place electrodes in the region and layer of interest (here, CA1) and begin recording.</li>
	<li>Record field potential activity in current clamp mode with a sampling rate of 10&#8211;20 kHz and low-pass filtered at 2 kHz.</li>
	<li>Record basal activity in aCSF for up to 5 min.</li>
	<li>Switch the inflow tubes from aCSF to highK++4AP or lowMg2++BIC aCSF and the outflow tube to a waste container to prevent mixing of solutions. After 2 min, place the outflow tube in the same solution as the inflow to conserve solution.</li>
	<li>Burst activity induced by highK++4-AP should be visible 2&#8211;5 min after the wash in. However, induction of SLEs by lowMg2++BIC can take up to 30 min. If necessary, change the positions of electrodes carefully to obtain optimal results.</li>
	<li>Once in the final position, record baseline activity for at least 20 min. If you record SLEs, consider longer baseline recordings due to low frequency of SLEs.</li>
	<li>&#160;In the case that baseline activity is stable (plateau of event frequency), wash in the desired drug. Note that due to the high flow rate wash in of drugs takes only 2&#8211;5 min, allowing for fast solution exchange.</li>
	<li>Record activity during &#160;drug application of at least 20 min, following wash out. Activity should be stable for at least 60&#8211;90 min, allowing for longer recordings.</li>
</ol>
7. Analysis
<ol>
	<li>Analysis of frequency and amplitude can be performed with any available software. So far, we have not been able to establish a reliable automatic analysis of SLEs or burst activity, and instead used a semi-automated analysis with visual confirmation of identified activity.</li>
	<li>Burst activity is characterized by biphasic, positive, and negative deflection and a duration of &#8805;100 ms. All events visually identified as burst activity (e.g., semi-automatically by threshold analysis) should be manually indicated for further analysis of event frequency (inter-event interval, IEI), amplitude, and total number of events during the analyzed time frame. 
	NOTE: Due to the high frequency of burst activity, the last 5 min of each application phase is typically analyzed20.</li>
	<li>SLEs can be analyzed as described in Heuzeroth et al. Identified SLEs can be further analyzed for duration, amplitude, spike frequency, and duration of tonic (high frequency spiking) vs. clonic (low frequency spiking) phase duration. SLEs that are &#60;10 s in duration should be excluded from analysis. 
	NOTE: For preclinical evaluation of possible antiepileptic substances, effects on burst activity (induced by highK++4AP) are being investigated, due to established induction in resected hippocampal tissue. Preliminary results on induction of SLEs using lowMg2++BIC have been reported (Figure 2), though analysis of this data is not included here.</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Approach+of+Modified+Submerged+Patch+Clamp+Recording+Reveals+Interneuronal+Dynamics+during+Epileptiform+Oscillations.">A New Approach of Modified Submerged Patch Clamp Recording Reveals Interneuronal Dynamics during Epileptiform Oscillations.</a> Frontiers in Neuroscience. 10, 519 (2016).</li><li>Valeeva, G., Valiullina, F., Khazipov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitatory+actions+of+GABA+in+the+intact+neonatal+rodent+hippocampus+in+vitro.">Excitatory actions of GABA in the intact neonatal rodent hippocampus in vitro.</a> Frontiers in Cellular Neuroscience. , (2013).</li></ol>

The authors declare no conflict of interest.

Living resected human brain tissue is a highly valuable tool in preclinical evaluation of AEDs, as it properly represents an intact human brain micro-network. The presented protocol describes a method for tissue transport and preparation, which ensures high quality hippocampal slices as well as a stable induction method for epileptiform activity critical for AED evaluation.
Investigation of epileptiform activity as well as methods for chemical or electrical induction in human brain slices have been previously shown by other groups17,20,21,22. This protocol describes the induction of stable burst activity in slices from different patients via application of high K++4-AP as well as induction of SLEs in CA1 area via application of low Mg2++BIC. It was found that the induction of burst activity is more consistent (80% of tested slices in 15 patients) than the induction of SLEs (50% of tested slices in one patient). However, thus far, the induction of SLEs has only been testd in one patient. Nevertheless, induction of SLEs by low Mg2++BIC is recommended, as SLEs have not yet been able to be induced using high K++4-AP.
Several studies have introduced methods for transport and preparation of human brain tissue and often highlight three factors critical to neuronal survival: transportation time, used transport solutions, and storing conditions.
For optimal slice viability, some groups suggest that the transport of resected brain tissue be as short as possible. However, operation rooms and laboratories are rarely in close proximity, meaning that slice quality may be compromised due to long transportation. Some groups have overcome this obstacle by applying constant O2 to the solution during transport12. We have transported brain tissue for short (max = 15 min) and long (up to 1 h) periods of time without constant additional O2 supply during transport, similar to other groups18,25. In these cases, differences in tissue quality were not observed during epileptiform recordings. In communication with other groups at our institute, slice quality did not change for patch-clamp experiments, either. In contrast, variance in tissue quality possibly stems from damage during operations, prolonged resection, and slicing procedure.
Concerning transport and cutting solution, all published methods omit NaCl from solutions to reduce cell swelling due to osmotic pressure, similar to the standard procedure for rodent patch-clamp experiments. However, several substitutes have been introduced so far (i.e., sucrose based aCSF13,22, NMDG-based aCSF12,26, and choline-based aCSF27). Ting and colleagues introduced the NMDG-based aCSF for slice preparation in 201426 and later added a recovery protocol, which slowly reintroduces NaCl to the slices28. However, as described by Ting et al., neurons of brain tissue prepared in NMDG-based aCSF show higher membrane resistance, thus affecting whole-cell seal during patch-clamp experiments26. Therefore, we have transitioned from NMDG-based aCSF to the use of choline-based aCSF20, which yields high quality slices for both field potential and patch-clamp recordings.
Concerning storage of slices, it is generally accepted that interface conditions provide optimal oxygenation critical for long slice survival18. However, other groups show slice survival for up to 72 h under submerged conditions12. Contrary to previous hypothesis, human brain slices seem to be more resistant to low oxygenation or oxidative stress compared to rodent slices. Primarily, interface chambers have been previously used for storing of human hippocampal slices, though submerged conditions are recommended for the maintenance of human brain slices in patch-clamp experiments.
As discussed by other groups, an additional critical step for long slice survival (interface for &#60;48 h18, submerged for &#60;72 h12) is the prevention of bacterial contamination. Rodent brain slices are typically used in electrophysiological recordings for up to 8 h, and bacterial contamination is not considered to affect slice viability during this period. High number of slices prepared from one resection and the uncommon availability of human brain tissue highlights the need to prolong viability of human brain slices. This method successfully describes the preparation of living human hippocampal brain slices, which can easily be adapted to sterile conditions. However, for the recordings performed here, slice survival extending 20 h was not a priority.
Recording in interface chambers has also been shown to be essential for induction of epileptiform activity such as&#160;SLEs22. Submerged conditions, due to low oxygenation, are rarely used for recording of SLEs; though, they are necessary for optical high resolution needed for patch-clamp experiments. The use of an optimized submerged type recording chamber enables the recording of epileptiform activity (extracellular field or single neuron) in human brain slices, due to high oxygenation and fast drug application29. Here, methods and results for field potential recordings are described, but it should be emphasized that patch-clamp recordings have been successfully performed in mouse and human brain slices using this modified recording chamber (data not shown).
Resected human brain tissue has a higher translational value compared to rodent models. It represents an adult, diseased neuronal network that cannot be reproduced by iPSCs. However, as in any in vitro system, human brain slices do not represent an intact human brain. Additionally, the recorded neuronal networks of resected brain tissue can undergo substantial molecular and functional changes due to damage during operation or preparation. Slicing procedures have been shown to affect GABAergic function and may affect the induction of epileptiform activity30. These limitations should be considered while formulating a hypothesis. When testing potential antiepileptic drugs, the use of different brain areas should be considered, as drug targets might not be expressed in all human brain regions or all patients. In particular, the hippocampi of TLE patients often show signs of hippocampal sclerosis accompanied by severe neuronal cell loss. It is recommended to obtain patient information on pathological changes and disease history, such as potential refractory towards medications, and consider this during data interpretation.
In conclusion, this method successfully describes the preparation of living human hippocampal brain slices and induction techniques for recording two different types of epileptiform activity. Since the availability of living human brain tissue is rare, optimized transport and recording conditions should be used to ensure maximum output from experiments using human brain slices. It is suggested that resected human brain tissue can be used as a preclinical validation tool in addition to rodent models and cell culture experiments.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61085/preparation-acute-human-hippocampal-slices-for-electrophysiological">Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings</a>. Step 2.1.3 in the Protocol was corrected.
Step 2.1.3 in the Protocol was updated from:
Add final concentrations of glucose and MgCl, then stir until dissolved (Table 1, solution 1.1).
to:
Add final concentrations of glucose and CaCl2, then stir until dissolved (Table 1, solution 1.1).

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61085/preparation-acute-human-hippocampal-slices-for-electrophysiological">Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings</a>. Step 2.1.3 in the&#160;Protocol was corrected.

Erratum: Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy is one of the most common neurological disorders, affecting 1% of the world population, and is associated with increased morbidity and mortality1,2. Unfortunately, one-third of patients suffering from epilepsy are drug-resistant, despite an abundance of available treatment options including more than 20 approved antiepileptic drugs (AEDs)3. Failure to translate results from preclinical animal research to clinical trials is one reason why promising treatment strategies are not effective in many patients4. Recently, neuropeptide Y (NPY) and galanin have been shown to have antiepileptic effects in animal models; though, when tested in resected human brain tissue, only NPY was effective5.
Most of the existing knowledge concerning basic neurological mechanisms and disease therapy approaches stem from animal models and cell culture experiments. Although informative, these models only represent single aspects of complex human diseases and the adult human brain network. Alternatively, human brain tissue has the potential to bridge the translational gap but is rarely available for functional studies. For instance, post mortem brain tissue has been a valuable tool in investigating protein expression, brain morphology, or anatomical connections, though neuronal activity is often compromised is this tissue6,7,8,9,10,11.
In contrast, living resected human brain tissue has been investigated concerning preclinical drug evaluation, basic neuronal functions and gene expression patterns12,13,14,15,16,17. A great advantage of human brain slices compared to rodent slices is the long viability of neuronal tissue after resection and preparation. Compared to rodent brain slices, which can typically be recorded for up to 8 h after preparation, human brain slices show stable neuronal activity for up to 72 h, enabling thorough investigation of these rare and valuable samples12,18.
Several studies have investigated properties of epileptiform activity in various areas of resected cortical and hippocampal human tissue and used different&#160;methods for induction of epileptiform activity. In rodent slices, epileptiform activity can be induced by several methods: electrical stimulation of DG hilar cells, increase of extracellular K+ (8&#8211;12 mM KCl), blocking of GABAA receptors by bicuculline (BIC), blocking of potassium channels by 4-aminopyridine (4-AP), and removing or reducing Mg2+ in extracellular solution19. However, induction of epileptiform activity in human tissue requires the combination of at least two of the abovementioned methods20,21,22.
Presented here is a method for the preparation of human hippocampal brain slices, which are viable for up to 20 h and show induction of epileptiform activity upon application of high K+ (8 mM) and 4-AP or low Mg2+ and BIC.

<table><tbody><tr><td>(+)-Na L-ascorbate</td><td>Sigma Aldrich</td><td>A4034</td><td></td></tr><tr><td>4-AP</td><td>Sigma Aldrich</td><td>275875-5G</td><td></td></tr><tr><td>Blades</td><td>eliteSERVE GmbH</td><td>HW3</td><td>used for the vibratome</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck</td><td>102382</td><td></td></tr><tr><td>Choline Cl</td><td>Sigma Aldrich</td><td>C1879</td><td></td></tr><tr><td>Filter paper</td><td>Tiffen</td><td>EK1546027T</td><td></td></tr><tr><td>Gas-tight bottle caps</td><td>Carl Roth GmbH+Co.KG</td><td>E694.1</td><td></td></tr><tr><td>Glass filaments</td><td>Science Products</td><td>GB150F-8P</td><td>for recording electrodes</td></tr><tr><td>Glass gas disperser</td><td>DWK Life Sciences GmbH</td><td>258573309</td><td></td></tr><tr><td>Glucose</td><td>Sigma Aldrich</td><td>G7528</td><td></td></tr><tr><td>Interface Chamber</td><td>inhouse made</td><td>-</td><td>see Haas et al., 1979</td></tr><tr><td>KCl</td><td>AppliChem</td><td>131494.1210</td><td></td></tr><tr><td>Membrane (Cell culture inserts)</td><td>Merck</td><td>PICM030050</td><td></td></tr><tr><td>Membrane chamber</td><td>inhouse made</td><td>-</td><td>see Hill and Greenfield, 2011</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;?6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>HNO3.2</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M7506</td><td></td></tr><tr><td>Na pyruvate</td><td>Sigma Aldrich</td><td>P8574</td><td></td></tr><tr><td>NaCl</td><td>Carl Roth</td><td>3957.1</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>106346</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Carl Roth</td><td>HNO1.2</td><td></td></tr><tr><td>Peristaltic pump</td><td>Gilson</td><td>Minipuls 3</td><td></td></tr><tr><td>Slice holder</td><td>Warner instruments</td><td>SHD-41/15</td><td></td></tr><tr><td>Vertical puller</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1200S</td><td></td></tr></tbody></table>

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.

Epileptiform activity has been successfully recorded in resected human hippocampal tissue originating from up to 15 patients. Establishing stable transport and preparation procedures is critical for successful induction of epileptiform activity in human brain tissue. Recently published results have shown 1) stable induction of epileptiform activity in resected tissue of different patients as well as 2) the use of resected human brain tissue as a preclinical tool for evaluation of novel antiepileptic mechanisms14,20.
Application of highK++4-AP induced epileptiform activity in form of burst activity within a few minutes (Figure 2A,B,C,D). Due to low neuronal distribution in human hippocampal tissue or high neuronal cell loss due to temporal lobe epilepsy (TLE), placement of electrodes can be adjusted in the beginning of recording. In cases where burst activity of slices is not visible in the CA1 area after 10 min (independent of electrode placement), slice viability may be compromised, and the slice will need to be replaced.
SLEs, with a duration of &#62;10 s, can be induced with application of lowMg2++BIC (Figure 2E,F). Figure 2E shows stable induction of SLEs after a few minutes and stable frequency throughout the recording. Here, SLE activity was successfully induced in two of four slices from the investigated patient. One slice showed only burst activity after 15 min of SLE activity, whereas the other slice did not show SLEs even after 40 min.
For preclinical evaluation of substance effects, a potential antiepileptic effect on burst activity induced by highK++4-AP was investigated. Known and potential antiepileptic substances (lacosamide, DMEA, dynorphine14) were tested, and examples are shown here for the conventional AED lacosamide (a sodium channel blocker) as well as DMEA (a novel potential antiepileptic substance)20. The number of events and inter-event interval (IEI) of burst events decreased both during application of lacosamide and DMEA (Figure 3C), though amplitudes were mostly unaffected (Figure 3D). In a subset of slices, even though induction of burst events was achieved in the first few minutes, the frequency of activity did not recover during wash out of applied AEDs (data not shown here, see Kraus et al.20). Here, the applied drugs were considered to induce effects; however, decreases in burst activity may have been affected by the gradual decay in activity during long recordings. Thus, results must be interpreted carefully.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61085/61085fig1v2.jpg" /> 
Figure 1: Interface chamber.&#160;For storing of human hippocampal brain slices, an interface chamber with two brain slice holding compartments is used (A); specifically, a Haas-type interface chamber23. Here, hippocampal brain slices rest on (d) three layers of filter paper, (e) smaller pieces to enable handling of individual brain slices, and (f) bigger filter paper pieces to ensure a sufficient layer of solution below the slice. (c) A cotton string surrounding the brain slices, on top of the filter papers, ensures even solution flow from the inlets at the (a) top of the compartment. (b) A cover lid directs oxygen from below the compartment onto the slice. (B) Top view of one slice-holding compartment. (C) Side view to illustrate the layers of filter papers. (g) Bottom of the chamber. (h) Tube for solution inflow, which is connected to a peristaltic pump (blue arrows mark the direction of the solution flow). <a href="https://www.jove.com/files/ftp_upload/61085/61085fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61085/61085fig2v2.jpg" /> 
Figure 2: Epileptiform activity in human hippocampal slices induced by highK++4-AP and lowMg2++BIC.&#160;CA1 example recordings and excerpts of application of highK+ (8 mM)+4-AP (100 &#181;M) (A,B,C,D) and lowMg2++BIC (10 &#181;M) (E,F). (A) Bath application of highK++4-AP induces epileptiform activity within a few minutes, and activity is stable for at least 60 min. Details of (A) can be seen in (B). Two different types of activity are induced in the CA1 area of human hippocampal slices: interictal-like spikes (C, details of [B]) and burst activity (D, details of [B]). Burst activity was shown to be sensitive to antiepileptic drugs and therefore analyzed for the effect of potential antiepileptic substances (Figure 3). (E,F) Application of lowMg2++BIC induces SLEs at a duration of &#62;10 s (F) in CA1 within a few minutes. However, induction of SLEs can take up to 30 min in other slices. Scale bars = 0.2 mV, 2 min (A,E), 5 s (B), 500 ms (C,D), 5 min (E), and 2 s (F). This figure has been adapted from Kraus et al.20. <a href="https://www.jove.com/files/ftp_upload/61085/61085fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61085/61085fig3v2.jpg" /> 
Figure 3: Decrease in epileptic burst activity of human slices during application of lacosamide or DMEA.&#160;Burst activity decreased during application of (A) lacosamide and (B) DMEA, a potential new antiepileptic molecule. (A) and (B) show exemplary recordings of the CA1 area with excerpts of regions used for analysis in (C) and (D). Burst activity decreased during lacosamide (100 &#181;M) and DMEA (10 mM) application, as seen by middle excerpts and increases again&#160;during wash out. (C,D) Number and amplitude of burst activity were analyzed for the last 5 min of each application phase (baseline, lacosamide/DMEA, wash out) and shown as summarized results for all patients (number of events, C; amplitude, D) as mean &#177; SD. Each dot indicates one patient. Asterisks mark significant differences as assessed by either Friedman test and post-hoc with Dunnett&#39;s multiple comparison of groups for analysis of lacosamide application (*p &#60; 0.05, n = 4) or by repeated measurement ANOVA and post-hoc with Tukey&#8217;s comparison for analysis of DMEA application (**p &#60; 0.01, n = 10). Scale bars = 0.2 mV, 2 min (full recording, A), 5 s (excerpts, A), 3 min (full recording, B), and 1 s (excerpts, B). This figure has been adapted from Kraus et al.20. <a href="https://www.jove.com/files/ftp_upload/61085/61085fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Solution 1.1 choline aCSF</td>
		</tr>
		<tr>
			<td>Substance</td>
			<td>10x concentration (mM)</td>
			<td>1x concentration (mM)</td>
			<td>Note</td>
		</tr>
		<tr>
			<td>choline Cl</td>
			<td>1100</td>
			<td>110</td>
			<td></td>
		</tr>
		<tr>
			<td>(+)-Na L-ascorbate</td>
			<td>116</td>
			<td>11.6</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2x6H2O</td>
			<td>70</td>
			<td>7</td>
			<td></td>
		</tr>
		<tr>
			<td>Na pyruvate</td>
			<td>31</td>
			<td>3.1</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>25</td>
			<td>2.5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>12.5</td>
			<td>1.25</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>260</td>
			<td>26</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>-</td>
			<td>0.5</td>
			<td>add to final solution</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>-</td>
			<td>10</td>
			<td>add to final solution</td>
		</tr>
		<tr>
			<td colspan="4">Solution 1.2 aCSF</td>
		</tr>
		<tr>
			<td>Substance</td>
			<td>10x concentration (mM)</td>
			<td>1x concentration (mM)</td>
			<td>Note</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>1290</td>
			<td>129</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>12.5</td>
			<td>1.25</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>16</td>
			<td>1.6</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>30</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>18</td>
			<td>1.8</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>-</td>
			<td>10</td>
			<td>add to final solution</td>
		</tr>
		<tr>
			<td colspan="4">Solution 1.3 highK++4-AP aCSF</td>
		</tr>
		<tr>
			<td>Substance</td>
			<td>10x concentration (mM)</td>
			<td>1x concentration (mM)</td>
			<td>Note</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>1240</td>
			<td>124</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>12.5</td>
			<td>1.25</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>16</td>
			<td>1.6</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>80</td>
			<td>8</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>18</td>
			<td>1.8</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>-</td>
			<td>10</td>
			<td>add to final solution</td>
		</tr>
		<tr>
			<td>4-AP</td>
			<td>-</td>
			<td>0.1</td>
			<td>add to final solution</td>
		</tr>
		<tr>
			<td colspan="4">Solution 1.4 lowMg2++BIC aCSF</td>
		</tr>
		<tr>
			<td>Substance</td>
			<td>10x concentration (mM)</td>
			<td>1x concentration (mM)</td>
			<td>Note</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>1300</td>
			<td>130</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>12.5</td>
			<td>1.25</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>16</td>
			<td>1.6</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>30</td>
			<td>3</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>-</td>
			<td>10</td>
			<td>add to final solution</td>
		</tr>
		<tr>
			<td>BIC</td>
			<td>-</td>
			<td>0.01</td>
			<td>add to final solution</td>
		</tr>
		<tr>
			<td colspan="4">Solution 2</td>
		</tr>
		<tr>
			<td>Substance</td>
			<td>10x concentration (mM)</td>
			<td>1x concentration (mM)</td>
			<td>Note</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>210</td>
			<td>21</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Preparation of 10x and final 1x solutions for transport, preparation, and recording.

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Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

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 ...

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JoVE Journal

Neuroscience

7.2K Views.  Charité-Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health. Met behulp van onze techniek kunnen we potentiële anti-epileptische stoffen in menselijke hersenmonsters testen om te helpen bij de ontwikkeling van geneesmiddelen voor temporale kwab epilepsie. Menselijk hersenweefsel heeft het voordeel dat het de translationele dop tussen basisonderzoek en klinische toepassingen wordt geschonden. Op de dag van de procedure of zo laat mogelijk op de dag ervoor, dooi 50 milliliter van een 10x choline aCSF oplossing in een 37 graden Celsius water bad en voeg ...

Video: Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

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 ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

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 ...

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 ...

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 ...

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 ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

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