obtaining horizontal mouse hippocampal brain slices

Obtaining Horizontal Mouse Hippocampal Brain Slices

Place both hemispheres on the freshly-cut dorsal side with the ventral part of the brain facing up, and place a drop of superglue onto a specimen plate. Use a pipette tip to spread the glue into a big area to accommodate both pieces of tissue, and touch a piece of filter paper strap to the ventral side of one hemisphere. Use another filter paper strap to carefully semi-dry the dorsal side of the brain, and position the hemisphere dorsal side down onto the glue on the specimen plate.
Use two additional filter paper straps to position the second hemisphere onto the glue as just demonstrated, and place the specimen plate into the slicing chamber. Then, quickly but carefully, cover the plate with ice-cold high-sucrose slice solution slush. To acquire sections of the brain tissue, position the vibratome blade in front of the medial side of the hemispheres, and lift the vibratome table so that the blade is on the same height as the ventral sides of the hemispheres, which are now facing up.
Use the vibratome control to lower the blade 600 microns further in the dorsal direction, and slice the tissue until the first two slices are completely separated from the two hemispheres. When the first two tissue slices have been obtained, reverse the cutting direction, and lower the blade another 300 microns before slicing again. When the hippocampus becomes visible, use a widened plastic Pasteur pipette to collect and transfer the slices into the recovery chamber in the water bath.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of high-sucrose slice solution and artificial cerebrospinal fluid (ACSF)
<ol>
	<li>Prior to experimental day
	<ol>
		<li>Prepare 1 L of 10x slice pre-solution with laboratory grade Type 1 water containing (in mM): 25 KCl, 20 CaCl2, 10 MgSO4, 12.5 KH2PO4 (Table 1). In order to prevent calcium phosphate precipitation, slowly mix the chemicals in a beaker pre-filled with 800 mL H2O while constantly stirring with a magnetic stirrer. Store the solution at 4 &#176;C or room temperature (RT).</li>
	</ol>
	</li>
	<li>On the experimental day
	<ol>
		<li>Prepare 1 L of 1x ACSF with laboratory grade Type 1 water containing (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, 25 Glucose (Table 2). Use a vapor pressure osmometer to validate the osmolarity between 305&#8211;315 mOsm (pH ~ 7.55&#8211;7.6). 
		NOTE: In order to prevent calcium phosphate precipitation, slowly mix all solid chemicals in a beaker pre-filled with 800 mL of H2O while constantly stirring with a magnetic stirrer. Add MgSO4 and CaCl2 at the very end, slowly dripping in the necessary amount from 1 M stock solutions.</li>
		<li>Continuously bubble 1x ACSF solution at RT with carbogen to set pH between 7.3&#8211;7.4. 
		NOTE: If the pH is slightly too high or too low, small adjustments in the carbogenation strength would be sufficient. If the pH is higher than 7.45 with carbogenation, adjust it by adding few drops of 1 M NaH2PO4 solution.</li>
		<li>Prepare 250 mL (per brain) of 1x high-sucrose slice solution in a beaker containing 25 mL of 10x slice pre-solution and (in mM): 252 Sucrose, 26 NaHCO3, and 10 Glucose (Table 3). Verify that the osmolarity is between 320&#8211;325 mOsm (pH ~ 7.55&#8211;7.6).</li>
		<li>Bubble the high-sucrose slice solution for 10&#8211;15 min with carbogen to control the pH between 7.3&#8211;7.4. 
		NOTE: If the pH is higher than 7.45 with carbogenation, adjust it by adding a few drops of 1 M KH2PO4 solution.</li>
		<li>Store the high-sucrose slice solution for 20&#8211;30 min in the ultra freezer (-80 &#176;C) until it is partially frozen.</li>
	</ol>
	</li>
</ol>
2. Dissection and positioning of the murine brain
<ol>
	<li>Transfer the brain to the chilled 35 mm culture dish and remove all hair or blood particles from the tissue by gently washing the brain with an ACSF-filled Pasteur pipette (blood has cytotoxic effects on brain tissue).</li>
	<li>Transfer the brain with the help of the spatula on the soaked filter paper on top of the chilled 90 mm culture dish (Figure 1A).</li>
	<li>Use a blade to cut the brain in half, separating the two hemispheres, and place both hemispheres on the freshly cut medial side.</li>
	<li>Use a blade to remove the dorsal part (5%&#8211;10%) of the brain from each hemisphere with a parallel cut to the dorsal top (Figure 1C)and place both hemispheres on the freshly cut side with the ventral part of the brain facing upwards.</li>
	<li>Position a drop of super glue on the specimen plate and spread out properly with a pipette tip to accommodate both hemispheres.</li>
	<li>Use a filter paper strap to pick up one hemisphere with capillary forces by touching the ventral side with the filter paper strap, thereby not damaging the tissue.</li>
	<li>Use another filter paper strap to carefully semi-dry the dorsal side of the brain before positioning the hemisphere, dorsal side down, on top of the glue on the specimen plate. Repeat the same procedure with the second hemisphere. 
	NOTE: The hemispheres should be positioned in horizontal alignment in a mirrored fashion on the vibratome plate, with the rostral sides pointing toward the outside and the caudal sides facing (but not touching) each other in the middle. The medial sides of both hemispheres should point toward the vibratome blade and the lateral sides toward the experimenter (Figure 1D).</li>
	<li>Place the specimen plate in the slicing chamber and quickly, but carefully, cover it with ice-cold high-sucrose slice solution slush. As soon as the solution touches the glue, it will solidify and properly glue the hemispheres to the specimen plate.</li>
	<li>Assure that the hemispheres are properly covered with high-sucrose slice solution and confirm that the solution is bubbled with carbogen. 
	NOTE: The entire dissection procedure should be performed as fast as possible. Please ensure that the brain does not stay without the supply of oxygen for a very long time. It should take only around 1&#8211;1.5 min from decapitation to brain submersion in the high-sucrose slice solution slush. This is the most critical requirement for acute brain slice preparations in order to warrant high quality of the slice.</li>
</ol>
3. Horizontal slicing of the brain
<ol>
	<li>Position the vibratome blade in front of the medial side of the hemispheres and lower it to the same height as the ventral sides of the hemispheres that are now facing upwards. Lower the blade with the help of the vibratome control to 600 &#181;m further in the dorsal direction and start slicing. The blade should hit the tissue (if not, reverse the blade and lower it a bit more). Slice until the first two slices are completely separated from the two hemispheres.</li>
	<li>Reverse the blade and lower another 300 &#181;m and slice again.</li>
	<li>When the hippocampus becomes visible (use the mouse brain atlas for help, if necessary) (Figure 1E) collect the slices with the widened plastic Pasteur pipette. Collect the slices until the caudate putamen becomes visible next to the hippocampus. Typically, between 8&#8211;12 slices of 300 &#181;m (4&#8211;6 per hemisphere) can be collected for the mouse brain.</li>
	<li>Use the plastic Pasteur pipette to collect the slices and transfer them to the recovery chamber in the water bath (Figure 1F) (collect slices after each round of slicing to prevent them from floating around in the slice chamber). 
	NOTE: Work as fast as possible and ensure that the high-sucrose slice solution is ice-cold and carbogenated during the entire procedure. If necessary, refill the ice surrounding the slice chamber.</li>
</ol>
4. Recovery of brain slices for electrophysiological recordings
<ol>
	<li>Leave the slices in the ACSF-filled recovery chamber in the 32 &#176;C water bath for 1 h. 
	NOTE: Recovery chambers are also commercially available.</li>
	<li>Take the recovery chamber out of the water bath.</li>
	<li>Place the slices at RT for at least another 30 min for recovery before starting any further application.</li>
</ol>
Buffer and Media Recipes.
Table 1: 10 x slice pre-solution (1 L).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Concentration (mM)</td>
			<td>Molecular weight (g/mol)</td>
			<td>Amount (g)</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>25</td>
			<td>74.55</td>
			<td>1.86</td>
		</tr>
		<tr>
			<td>CaCl2 * 2H2O</td>
			<td>20</td>
			<td>147.01</td>
			<td>2.94</td>
		</tr>
		<tr>
			<td>MgSO4 * 7H2O</td>
			<td>10</td>
			<td>246.48</td>
			<td>2.46</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>12.5</td>
			<td>136.08</td>
			<td>1.7</td>
		</tr>
	</tbody>
</table>
Table 2: 1x ACSF (1 L) (osmolarity between 305&#8211;315 mOsm).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Concentration (mM)</td>
			<td>Molecular weight (g/mol)</td>
			<td>Amount (g)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>125</td>
			<td>58.44</td>
			<td>7.3</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>2.5</td>
			<td>74.55</td>
			<td>0.19</td>
		</tr>
		<tr>
			<td>CaCl2 * 2H2O</td>
			<td>2</td>
			<td>from 1 M CaCl2 solution</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>MgSO4 * 7H2O</td>
			<td>1</td>
			<td>from 1 M MgSO4 solution</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>NaH2PO4 * 2H2O</td>
			<td>1.25</td>
			<td>156.02</td>
			<td>0.2</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>26</td>
			<td>84.01</td>
			<td>2.18</td>
		</tr>
		<tr>
			<td>Glucose * H2O</td>
			<td>25</td>
			<td>198.17</td>
			<td>4.95</td>
		</tr>
	</tbody>
</table>
Table 3: 1x high-sucrose slice solution (250 mL) (osmolarity between 320&#8211;325 mOsm).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Concentration (mM)</td>
			<td>Molecular weight (g/mol)</td>
			<td>Amount (g)</td>
		</tr>
		<tr>
			<td>10x slice presolution</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>252</td>
			<td>342.3</td>
			<td>21.57</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>26</td>
			<td>84.01</td>
			<td>0.55</td>
		</tr>
		<tr>
			<td>Glucose * H2O</td>
			<td>10</td>
			<td>198.17</td>
			<td>0.49</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Carbogen tank</td>
			<td>Air Liquide</td>
			<td>Alphagaz mix B50</td>
			<td>Gasmixture CO2/O2: 5/95, purity 5</td>
		</tr>
		<tr>
			<td>Culture dish (35 mm)</td>
			<td>Corning Life Sciences</td>
			<td>353001</td>
			<td>https://ecatalog.corning.com/life-sciences/b2c/US/en/Cell-Culture/Cell-Culture-Vessels/Dishes%2C-Culture/Falcon&#174;-Cell-Culture-Dishes/p/353001</td>
		</tr>
		<tr>
			<td>Culture dish (90 mm)</td>
			<td>Thermo Fisher Scientific</td>
			<td>101VR20</td>
			<td>https://www.thermofisher.com/order/catalog/product/101R20#/101R20</td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Fine Science tools</td>
			<td>11270-20</td>
			<td>https://www.finescience.de/de-DE/Products/Forceps-Hemostats/Dumont-Forceps/Dumont-7b-Forceps/11270-20</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose monohydrate</td>
			<td>Sigma Aldrich</td>
			<td>16301</td>
			<td>https://www.sigmaaldrich.com/catalog/product/sial/16301?lang=en&#38;region=BE</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>VWR</td>
			<td>516-0818</td>
			<td>grade 413</td>
		</tr>
		<tr>
			<td>Fine brush</td>
			<td>Raphael Kaerell</td>
			<td>8204</td>
			<td>Size #1</td>
		</tr>
		<tr>
			<td>Loctite 406</td>
			<td>Henkel Adhesive technologies</td>
			<td>Loctite 406</td>
			<td>Super glue</td>
		</tr>
		<tr>
			<td>Potassium chlorid</td>
			<td>Chem-lab</td>
			<td>CL00.1133</td>
			<td>https://www.chem-lab.be/#/en-gb/prod/1393528</td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Merck</td>
			<td>104873</td>
			<td>https://www.merckmillipore.com/BE/en/product/Potassium-dihydrogen-phosphate,MDA_CHEM-104873?ReferrerURL=https%3A%2F%2Fwww.google.com%2F</td>
		</tr>
		<tr>
			<td>Razor blade to prepare hemispheres</td>
			<td>SPI supplies</td>
			<td>Safety Cartridge Dispenser - Pkg/10</td>
			<td>GEM Scientific Single Edge Razor Blades</td>
		</tr>
		<tr>
			<td>Razor blade for vibratome</td>
			<td>Ted Pella Inc</td>
			<td>121-6</td>
			<td>double edge breakable style razor blades (PTFE-coated stainless steel)</td>
		</tr>
		<tr>
			<td>Recovery chamber</td>
			<td>home made - Generic</td>
			<td>N/A</td>
			<td>to collect and store brain slices in (see details in manuscript)</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Any company</td>
			<td>N/A</td>
			<td>Blade should be well sharpened and at least 15 cm long for easy decapitation</td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate dihydrate</td>
			<td>Merck</td>
			<td>106342</td>
			<td>https://www.merckmillipore.com/BE/en/product/Sodium-dihydrogen-phosphate-dihydrate,MDA_CHEM-106342?ReferrerURL=https%3A%2F%2Fwww.google.com%2F</td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>Alfa Aesar</td>
			<td>14707</td>
			<td>https://www.alfa.com/en/catalog/014707/</td>
		</tr>
		<tr>
			<td>Sodium chlorid</td>
			<td>Fisher Scientific</td>
			<td>S/3160/60</td>
			<td>https://www.fishersci.co.uk/shop/products/sodium-chloride-certified-ar-analysis-meets-analytical-specification-ph-eur/10428420</td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Sigma Aldrich</td>
			<td>S9147-12EA</td>
			<td>https://www.sigmaaldrich.com/catalog/product/sigma/s9147?lang=en&#38;region=BE</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>VWR International Ltd.</td>
			<td>102745C</td>
			<td>https://es.vwr-cmd.com/ex/downloads/magazine/lupc_userguide_uk.pdf</td>
		</tr>
		<tr>
			<td>Tubing for carbogen, perfusion and suction lines 1</td>
			<td>Warner Instruments</td>
			<td>64-0167</td>
			<td>Tygon tubing (TY-50) for standard valve systems</td>
		</tr>
		<tr>
			<td>Tubing for carbogen, perfusion and suction lines 2</td>
			<td>Fisher Scientific</td>
			<td>800/100/200 &#38; 800/100/280</td>
			<td>Smiths Medical Portex Fine Bore LDPE Tubing</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>14912000001</td>
			<td>Semi-automatic vibrating blade microomei VT1200</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Memmert</td>
			<td>WNB 7</td>
			<td>https://www.memmert.be/wp-content/uploads/2019/09/Memmert-Waterbath-WNB-7.en_.pdf</td>
		</tr>
		<tr>
			<td>Water purification system</td>
			<td>Merck</td>
			<td>Synergy millipore</td>
			<td>to obtain highly purified water</td>
		</tr>
	</tbody>
</table>

Source: Van Hoeymissen, E., et al. <a href="https://app.jove.com/v/61753">Horizontal Hippocampal Slices of the Mouse Brain.</a> J. Vis. Exp. (2020)
This video demonstrates a detailed protocol for preparing horizontal hippocampal brain slices from mice, focusing on preserving the integrity of hippocampal fiber pathways. The process involves carefully bisecting and securing the brain, using a vibratome for slicing, and maintaining optimal tissue conditions with a carbogenated fluid, resulting in hippocampal brain slices ready for further experimentation.

<img alt="Figure 1" src="/files/ftp_upload/22602/22602_figure1.jpg" /> 
Figure 1: Detailed information on the preparation of horizontal hippocampal brain slices. (A) Image of tools required for dissection and slicing of the rodent brain: (a) &#177;2 cm long and &#177;0.5 cm wide straps of filter paper (e.g., grade 413); (b) blade; (c) super glue; (d) pipette tip; (e) specimen plate (comes with vibratome); (f) 35 mm culture dish; (g) fine brush; (h) spatula; (i) curve...

 All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of ...

Take the mouse brain hemispheres in a high-sucrose solution. Secure the hemispheres with their ventral side facing up on a vibratome specimen plate using an adhesive.
Transfer the specimen plate into the slicing chamber.
Add a semi-frozen high-sucrose solution to maintain the tissue&#39;s physiological conditions and solidify the adhesive.
Supply a carbon dioxide-oxygen mixture to maintain tissue oxygenation.
Using set parameters, obtain the initial slices and remove them. Further, obtain horizontal hippocampal slices.
Carefully transfer these brain slices into a chamber containing ACSF for tissue recovery.
The horizontal hippocampal slices are ready for further experimentation.

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

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

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

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

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Nörobilim

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

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Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

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It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

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It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Obtaining Horizontal Mouse Hippocampal Brain Slices

TRANSKRIPT

Obtaining Horizontal Mouse Hippocampal Brain Slices