Name: minimizing hypoxia in hippocampal slices from adult and aging mice
Uploaded: 2020-07-02T14:00:00.000+00:00
Duration: 8 min 58 s
Description: Watch this Scientific Journal Video about Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice at JoVE.com

I thank Dr. Carla J. Shatz for advice and support, and Dr. Barbara K. Brott and Michelle K. Drews for critically reading the manuscript. The work is supported by NIH EY02858 and the Mathers Charitable Foundation grants to CJS.

The protocol is carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Stanford University Institutional Animal Care and Use Committee. Methods are also in accordance with the Policies of the Society for Neuroscience on the Use of Animals and Humans in Neuroscience Research.
NOTE: All mice were maintained in a pathogen-free environment. Wild-type mice on mixed C57Bl/ 6 x SV/ 129J genetic background were used here, unless otherwise noted.
1. Setup
<ol>
	<li>Prepare 1 L of 1x aCSF&#160;(in mM): 125 NaCl, 26 NaHCO3, 2.3 KCl, 1.26 KH2PO4, 1.3 Mg2SO4&#183;7H2O, 2.5 CaCl2, 25 glucose (Table 1). Use this solution for the recovery chamber and subsequent recordings. 
	NOTE:&#160;Store aCSF as a 10x stock solution that contains NaCl, NaHCO3, KCl, and KH2PO4. Store Mg2SO4&#183;7H2O and CaCl2 as 1 M stock solutions. Prepare the working solution on the day of experiment from the above stock solutions, and add glucose before adjusting the final volume with 18 M&#937; water. 
	NOTE: Depending on the brain region or mouse strain, chilled aCSF can be also used for transcardial perfusion with success11,15,26.</li>
	<li>Prepare 300 mL of NMDG-aCSF&#160;(in mM): 135 NMDG, 1 KCl, 1.2 KH2PO4, 1.5 MgCl2, 0.5 CaCl2, 20 choline bicarbonate, 10 glucose (Table 1). This volume of NMDG-aCSF is sufficient for both transcardial perfusion and cutting steps. 
	NOTE:&#160;Store NMDG-aCSF as a 3x stock solution at 4 &#176;C. Prepare working solution on the day of the experiment; add choline bicarbonate and glucose before adjusting the final volume with 18 M&#937; water. Bubble the solution with 95%O2/5%CO2 to buffer it, and saturate with oxygen. 
	NOTE: NMDG stock starts as highly alkaline, and requires concentrated hydrochloric acid (HCl) to adjust pH to ~7.4. Addition of hydrochloric acid should be slow once below pH 8, as it is easy to acidify the solution to ~pH 3 with excess HCl. This adjustment should be done prior to adding divalent cations. This NMDG-aCSF recipe is sodium-free. Previously published NMDG recipes use 30 mM NaHCO3 for buffering, which results in 30 mM sodium present in NMDG-aCSF13,20.</li>
	<li>Set the vibrating microtome cutting tray and mounting disk to -20 &#176;C.</li>
	<li>Prepare the recovery chamber.
	<ol>
		<li>Fill the recovery chamber to just above the slice-holding mesh, keep it on the bench at room temperature, and bubble. 
		NOTE: The slice recovery chamber used here is very similar to the classical chambers described&#160;previously by Edwards and Konnerth3. aCSF is kept in a glass beaker (400 mL) that holds a round acrylic frame with black nylon mesh glued to the bottom. The acrylic frame is suspended in the middle of the beaker via an acrylic hook resting over the edge of the beaker. The glass bubbler is inserted all the way to the bottom. The design allows for oxygenation of both sides of slices. Bubbling also provides constant mixing of aCSF in the recovery chamber. The black mesh provides a high contrast against the off-white slices, which are then easier to see.</li>
	</ol>
	</li>
	<li>Prepare the transcardial perfusion and cutting solution.
	<ol>
		<li>Chill the entire 300 mL of NMDG-aCSF in a freezer, until ice crystals start to form on the surface and the walls of the bottle. DO NOT over-freeze!</li>
		<li>Place the bottle with chilled NMDG-aCSF on ice and bubble. The solution should be between 0&#8210;2 &#176;C. 
		NOTE: Slushy NMDG-&#160;aCSF implies having most of the solution as liquid, while a small fraction of slushy ice present will keep the solution near 0 &#176;C throughout perfusion and cutting. Care should be taken to keep away ice crystals from the brain during cutting.</li>
	</ol>
	</li>
	<li>Prepare the tissue-mounting disk.
	<ol>
		<li>Take the disk out of the freezer, wipe it dry if needed.</li>
		<li>Cut out a block of 5% agar from the previously prepared agar plate, and glue it in the center of the disk using a thin layer of cyanoacrylate glue. The agar piece should be about the size of a mouse brain. Place the disk with glued agar on ice, and cover with paper towels until it is ready to use.
		<ol>
			<li>For 5% agar plate, dissolve 5 g of agar in 100 mL of 18 M&#937; water by microwaving, and pour into a clean petri dish. Keep at 4 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Take the cutting tray out of the freezer, place it into the microtome, surround it with ice, and load the blade.</li>
</ol>
2. Transcardial perfusion and brain extraction
<ol>
	<li>Overdose mice via an intraperitoneal injection of anesthetic&#160;cocktail. Check for the anesthesia depth by checking the pain reflex (toe pinch); a mouse should not exhibit the reflex once it reaches deep anesthesia. 
	NOTE: Rodent anesthesia cocktail recipe: Ketamine HCl (66 mg/mL), xylazine HCl (6.6 mg/mL), acepromazine maleate (0.1 mg/mL), 18 M&#937; water to final volume. Dose: 0.4 mg/g body weight, 0.04 mg/g body weight, 6 x 10-4 mg/g body weight for ketamine, xylazine, and acepromazine, respectively.</li>
	<li>Set-up the peristaltic pump for transcardial perfusion. Insert one side of the pump tubing into the bottle with iced NMDG-aCSF. Fit the other side of the tubing with the 27 G needle that will be inserted into the left ventricle.</li>
	<li>Set the pump speed at approximately 3.5 mL/min. At this speed, the outflow of NMDG-aCSF is a fast drip, not continuous flow. 
	NOTE: Perfusion by gravity is a good substitute for the peristaltic pump perfusion, as long as the same approximate flow-rate can be achieved. A perfusion at high flow-rate will result in burst blood vessels in the brain. A telltale sign of an overly fast perfusion and increased pressure in blood vessels&#160;is solution coming out of the nose of the animal.</li>
	<li>Perfusion
	<ol>
		<li>Place the properly anesthetized mouse on its back on a diaper. Using paper tape, tape down its front and hind legs so that the chest and abdomen are exposed.</li>
		<li>Cut out a large patch of the skin atop of the chest, going from below the sternum to the throat; this should provide a large working area. Grab the sternum with forceps, lift gently, and start cutting through the rib cage on both sides until the chest cavity is exposed.</li>
		<li>Cut through the diaphragm in order to expose the chest cavity. The flap of the rib cage should be left attached via a thin piece of muscle. It should be possible to set it on a side without having it fall back onto the exposed chest cavity. Check that the heart is still beating. Ensure that most of the liver is visible.</li>
		<li>Insert the 27 G needle into the left ventricle; the mouse&#8217;s left ventricle looks lighter in color than the right. Locate the dark red-colored right atrium. Cut through the right atrium with small scissors; the blood should start flowing out.</li>
		<li>Start the pump that has been preset to the correct flow speed. If everything was done correctly, the liver should start to change color from red to brown soon after the start of perfusion. Monitor the liver color to determine the length of the perfusion; the liver should turn pale brown.</li>
		<li>Run the pump for few more minutes. If using rectal thermometer, the body temperature should fall down to 28&#8210;29 &#176;C, and animal&#8217;s nose should be cold to the touch.</li>
	</ol>
	</li>
	<li>Brain extraction. 
	NOTE: For this step, have the following dissection tools ready: decapitation scissors, small scissors with straight or angled blade, scalpel and #10 blade, single edge blade, #3 forceps, spatula with one side bent to 90&#176;, a spatula, a &#8220;60&#176;&#8221; tool (Figure 1A,B), and a small soft brush.
	<ol>
		<li>Decapitate the mouse with large decapitation scissors. Using a scalpel with #10 blade, cut open the skin on top of the skull. With small angled scissors, cut the skull at the midline. Next, using the #3 forceps, pry away the right and left halves of the skull, being careful to take the dura away with it. The brain should be exposed now. 
		NOTE: If the dura detaches from the skull, it will stay over the brain and has to be removed separately. The edges of the remaining dura can tighten and slice deep through the brain, potentially damaging the brain region of interest.</li>
		<li>Remove the brain by scooping it out with a small spatula. Drop the brain into the NMDG-aCSF solution placed in a separate small beaker on ice. Leave it there for up to a minute. 
		NOTE: In addition to hypothermia, exposure to the ice-cold saline firms up the brain, which is necessary for even cutting. The entire procedure from decapitation to the brain extraction should be under 30 s.</li>
	</ol>
	</li>
</ol>
3. Slicing
<ol>
	<li>Take the brain out of the NMDG-aCSF and place it on a piece of filter paper.</li>
	<li>Cut and remove a 60&#176; wedge from the rostral end of the forebrain using a &#8220;60&#176;&#8221; tool centered at the midline. Cut surfaces will be used for mounting to achieve the proper angle for transversal hippocampal slices as discussed below&#160;(Figure 1A,B). 
	NOTE: Two single-edged blades held together via a home-made holder make an easy-to-use tool for making the 60&#176; cut (Figure 1A).</li>
	<li>Separate hemispheres down the midline with the scalpel.</li>
	<li>Glue the hemispheres onto the mounting disk as follows. Take the mounting disk that has been on ice until now. If needed, wipe it dry again. Glue each hemisphere in front of the agar block, cut side down.</li>
	<li>Ensure that the ventral side of each hemisphere is touching the agar block. The agar block provides additional support during cutting and is essential for even slices. Dorsal sides of each hemisphere should be facing the blade. When glued on the cut side, each hemisphere is oriented relative to the blade in a way that results in transverse slices of dorsal hippocampus in situ&#160;(Figure 1B).</li>
	<li>Submerge the disk with hemispheres into a cutting chamber containing ice-cold carbogenated NMDG-aCSF.</li>
	<li>Cut 400 &#181;m sections. Cutting should be done in less than 10 min. Different microtomes will require different settings to achieve this time. A total of 8&#8210;10 slices from the dorsal hippocampus region will be obtained.</li>
</ol>
4. Recovery
<ol>
	<li>Transfer slices to a recovery chamber containing carbogenated aCSF at room temperature using disposable transfer pipettes with cut-off tips (Figure 1C). 
	NOTE: It is highly recommended to fortify recovery chamber-aSCF with 5 mM Na-ascorbate and 3 mM Na-pyruvate. Pyruvate is an energy substrate shown to boost the production of ATP in slices28, while Na-ascorbate is a free-radical quencher29.</li>
	<li>Incubate at room temperature (22&#8210;24 &#176;C) for approximately 2 h before recording (up to 4 h). 
	NOTE: A longer incubation at room temperature results in healthier slices for longer periods of time, relative to the standard incubation at 37 &#176;C for 30 min followed by room temperature incubation. Rewarming at 37 &#176;C can introduce cytotoxic edema13.</li>
</ol>

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The author has nothing to disclose.

The protocol described here demonstrates that hippocampal slices obtained from adult and aging mice can remain healthy and viable for many hours after cutting. The slices prepared using this protocol are&#160;appropriate for patch-clamp recordings, as well as long-lasting field-recordings in the CA1 regions.
There are two critical steps in this protocol. First step is the transcardial perfusion step with an ice-cold solution. Fast clearance of blood is signaled by rapid change of liver color. Extracted brain should be off-white in color. If the brain remains pink, it means that systemic blood was not replaced with the cold NMDG-aCSF, and the drop in body temperature was not achieved. This could be caused by improper placement of the needle into the heart, or because the heart was punctured through. Brains from poorly perfused mice should not be used for slicing. The second critical step is the use of NMDG as a sodium ion substitute. A well-known earlier variation of the protocol that successfully uses sucrose as substitute for Na+ in rat brains10,31 does not produce sufficiently healthy hippocampal slices in mice (also see Ting et al.13). The use of NMDG as a sodium ion substitute is critical for mouse hippocampal slices.
While healthy hippocampal slices are reliably obtained using the described protocol, the hippocampal preparation from adult and aging animals remains challenging. Its difficulty also changes with different mouse lines and genetic backgrounds. Potential modifications to consider are additives to NMDG-aCSF and recovery-aCSF solutions like taurine, D-serine, or N-acetylcysteine (NAC), that could augment neuronal and synaptic function13,29, increase oxygenation29.&#160; Substitution of Cl- ions should also be considered during perfusion and cutting32. Use of an interface recovery chamber is another way to maintain increased oxygenation, which is especially relevant for long-term plasticity field-recordings. The described recovery chamber (Figure 1C) is modifiable for that purpose (e.g., a culture insert dish that is moistened from below by aSCF, could substitute the submerged mesh). Replacing steel blades with sapphire or ceramic blades could decrease tissue compression exacerbated by heavy myelination of white matter around hippocampus; this in turn could further improve the quality of neurons near the slice surface. Using microtomes designed to minimize the vertical vibration (e.g., zero-z in Leica VT1200S), is another modifiable step.&#160;
Other brain regions could be sliced using the protocol described here, with appropriate modifications in cutting angles. In addition, the stringency of the slice preparation can be adjusted, as different brain regions are sensitive to hypoxia to different degrees.. A protocol that uses an NMDG-aSCF has been reported for adult mouse neocortex and striatum slice preparations13,20; it uses an NMDG-aCSF that contains 30 mM NaHCO3 (i.e. it is not Na+-free)20, and in some instances transcardial perfusion is with NMDG-aCSF at room temperature13. However, for a region as susceptible to hypoxia as hippocampus, using Na+-free NMDG-aCSF and ice-cold transcardial perfusion could make a critical difference.&#160;
This protocol is applicable to the vast array of transgenic mouse lines modeling neurodegenerative diseases. Moreover, the protocol could be further modified to brain slices from other mammalian model species, as well. Together, this protocol could serve as a basis for a standardized preparation for acute hippocampal slices from aging animals, and thus facilitate comparisons across studies in the context of disease mechanisms.

The advent of mammalian acute brain slice preparations facilitated experiments at the cellular and synaptic level that were previously possible only in invertebrate preparations like Aplysia1. Development of acute hippocampal slices was of particular significance, as it is a structure responsible for working memory and context formation, and has a specialized tri-synaptic circuitry that is amenable to easy physiological manipulation. However, the vast majority of acute brain slices are still prepared from relatively young mice and rats, as it is easier to preserve healthy neurons and circuits, and the slices remain viable for longer periods of time2,3,4. Here, we introduce modifications to standard slicing protocols that result in increased viability of acute hippocampal slices from adult and aging mice.
The major impediment to the long-term ex vivo viability of mammalian brain parenchyma is the initial hypoxic damage that occurs rapidly once blood flow to the brain stops following decapitation. Loss of oxygen results in fast metabolic consumption of major energy resources in the brain with the loss of phospho-creatine (P-creatine) being the most rapid, followed by glucose, adenosine triphosphate (ATP), and glycogen4. Preservation of ATP is of particular importance for the long-term health of brain slices, as ATP is needed to maintain the membrane potential via the Na-K ATPase, and consequently the neural activity5,6. The ATP level in the adult rodent brain is ~2.5 mM, and it drops precipitously within 20 s of decapitation to reach a basal steady state (~0.5 mM) at around 1 min post-decapitation4,7,8. In young animals, it takes longer to observe the same drop in ATP (~2 min); with phenobarbital anesthesia it is further slowed to 4 min4. These considerations show that preventing loss of ATP and other energy resources is a necessary strategy to prevent hypoxic damage to the brain and in turn to maintain the health of brain slices over longer periods of time, especially in adult animals.
Low temperatures slow down the metabolism. Consequently, it has been demonstrated that modest hypothermia protects brain energy reserves: in young animals, lowering body temperature by six degrees, from 37 &#176;C to 31 &#176;C, preserves ATP levels to around 80% of normal levels over 4 h of controlled hypoxia9. P-creatine levels are similarly preserved, as well as the overall phosphorylation potential9. This suggests that lowering body temperature prior to decapitation could be neuroprotective, as near-normal levels of ATP could be maintained through the slice cutting and slice recovery periods.
To the degree that an ATP drop cannot be completely prevented upon decapitation, a partially impaired function of the Na-K ATPase is expected, followed by depolarization via passive sodium influx. As the passive sodium influx is followed by water entry into cells, it causes cytotoxic edema and eventually pyknosis. In adult rats, replacing Na+ ions with sucrose in slice-cutting solutions has been a successful strategy to alleviate the burden of cytotoxic edema10,11. More recently, methylated organic cations that decrease sodium channel permeability12 have shown to offer more effective protection than sucrose, especially in slices from adult mice, with N-methyl-D-glucamine (NMDG) being most widely applicable across different ages and brain regions13,14,15,16.
Numerous brain-slicing protocols involve using cold temperatures only during the slice-cutting step, sometimes in combination with Na+ ion replacement strategy16,17. In young animals, these protocols appear to offer sufficient neuroprotection since the brains can be extracted quickly after decapitation because the skull is still thin and easy to remove3. However, this strategy does not produce healthy slices from adult animals. Over time, a number of laboratories studying adult rodents have introduced transcardial perfusion with an ice-cold solution to decrease the animal&#8217;s body temperature, and therefore hypoxic damage to the brain, prior to decapitation. This procedure was successfully applied to produce cerebellar slices18, midbrain slices19, neocortical slices11,20, perirhinal cortex21, rat hippocampus10,22,23, olfactory bulb24, ventral striatum25, visual cortex26.
In spite of the advantages offered by transcardial perfusion and Na+ ion replacement in preparing slices from rat and in some brain regions in mice, mouse hippocampus remains one of the most challenging areas to protect from hypoxia13,20. To date, one of the most common approaches to slicing hippocampus from aging mice and mouse models of neurodegeneration involves the classical fast slicing of the isolated hippocampi27. In the protocol described here, we minimize the loss of ATP in the adult brain by introducing hypothermia prior to decapitation by transcardially perfusing the animal with ice-cold Na+- free NMDG-based artificial cerebrospinal fluid (NMDG-aCSF). Slices are then cut in ice-cold Na+-free NMDG-aCSF. With this enhanced protocol we obtain acute hippocampal slices from adult and aging mice that are healthy for up to 10 h after slicing and are appropriate for long-term field-recordings and patch-clamp studies.

<table><tbody><tr><td>&amp;ldquo;60 degree&amp;rdquo; tool</td><td>made in-house</td><td></td><td></td></tr><tr><td>#10 scalpel blade</td><td>Bard-Parker (Aspen Surgical)</td><td>371110</td><td></td></tr><tr><td>1M CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fluka Analytical</td><td>21114</td><td></td></tr><tr><td>95%O2/5%CO2</td><td>Praxiar or another local supplier</td><td></td><td></td></tr><tr><td>Acepromazine maleate (AceproJect)</td><td>Henry Schein</td><td>5700850</td><td></td></tr><tr><td>Agar</td><td>Fisher</td><td>BP1423-500</td><td></td></tr><tr><td>Beakers, measuring cylinders, reagent bottles</td><td></td><td></td><td></td></tr><tr><td>Brushes size 00-2</td><td>Ted Pella</td><td></td><td>Crafts stores are another source of soft brushes, with larger selection and better quality than Ted Pella.</td></tr><tr><td>CCD camera</td><td>Olympus</td><td>XM10</td><td></td></tr><tr><td>Choline bicarbonate</td><td>Pfalz &amp;amp; Bauer</td><td>C21240</td><td></td></tr><tr><td>Cyanoacrilate glue</td><td>Krazy glue</td><td></td><td>Singles</td></tr><tr><td>Decapitation scissors</td><td>FST</td><td>14130-17</td><td></td></tr><tr><td>Feather blades</td><td>Feather</td><td>FA-10</td><td></td></tr><tr><td>Filter paper #2</td><td>Whatman</td><td></td><td>Either rounds or pieces cut from a bigger sheet work well.</td></tr><tr><td>Forceps</td><td>A. Dumont &amp;amp; Fils</td><td>Inox 3c</td><td></td></tr><tr><td>Glass bubblers (Robu glass borosillicate microfilter candles) - porosity 3</td><td>Robuglas.com</td><td>18103 or 18113</td><td>Glass bubblers are more expensive than bubbling stones used in aquaria. However, they are easy to clean and sterilize, and can last a long time.</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>HCl</td><td>Fisher</td><td>A144SI-212</td><td></td></tr><tr><td>Ice buckets</td><td></td><td></td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P4504</td><td></td></tr><tr><td>Ketamine HCl (KetaVed)</td><td>VEDCO</td><td>NDC 50989-996-06</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P0662</td><td></td></tr><tr><td>Leica Tissue slicer VT1000S</td><td></td><td></td><td>The cutting settings are 1 mm horizontal blade amplitude, frequency dial at 9, and speed setting at 2</td></tr><tr><td>Magnetic stirrers and stir bars</td><td></td><td></td><td></td></tr><tr><td>Mg&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt; x 7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>230391</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M9272</td><td></td></tr><tr><td>MilliQ water machine</td><td>Millipore</td><td></td><td>Source for 18 Mohm water</td></tr><tr><td>Na-ascorbate</td><td>Sigma-Aldrich</td><td>A4035</td><td></td></tr><tr><td>Na-pyruvate</td><td>Sigma-Aldrich</td><td>P8574</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>EMD</td><td>SX0320-1</td><td></td></tr><tr><td>Needle 27G1/2</td><td></td><td></td><td></td></tr><tr><td>NMDG</td><td>Sigma-Aldrich</td><td>M2004</td><td></td></tr><tr><td>Paper tape</td><td></td><td></td><td></td></tr><tr><td>Peristaltic pump</td><td>Cole-Parmer</td><td>#7553-70</td><td></td></tr><tr><td>Peristaltic pump head</td><td>Cole-Parmer</td><td>Masterflex #7518-00</td><td></td></tr><tr><td>Personna blades</td><td></td><td>Personna double edge</td><td>Amazon</td></tr><tr><td>pH meter</td><td></td><td></td><td></td></tr><tr><td>Recovery chamber</td><td>in-house made</td><td></td><td></td></tr><tr><td>Scalpel blade handle size 3</td><td>Bard-Parker (Aspen Surgical)</td><td>371030</td><td></td></tr><tr><td>Scissors angled blade</td><td>FST</td><td>14081-09</td><td></td></tr><tr><td>Single edge industrial razor blade #9</td><td>VWR</td><td>55411</td><td></td></tr><tr><td>Spatulas</td><td></td><td></td><td></td></tr><tr><td>Transfer pipettes</td><td>Samco Scientific</td><td>225</td><td></td></tr><tr><td>Upright microscope</td><td>Olympus BX51WI</td><td></td><td></td></tr><tr><td>Xylazine HCl (XylaMed)</td><td>VetOne</td><td>510650</td><td></td></tr></tbody></table>

minimizing hypoxia in hippocampal slices from adult and aging mice

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

We applied the above protocol to generate hippocampus slices from CamKIIa-Cre+;WT&#160;mice on a mixed genetic background C57Bl/ 6 x SV/ 129J, at P &#62; 120. Large numbers of pyramidal cells in the CA1 field (Figure 2A) and subiculum (Figure 2B) appear in low contrast when observed under infrared differential contrast microscopy (IR-DIC), a hallmark of healthy cells in a slice preparation. With this preparation, a high rate of giga-ohm seals (&#62;90%) is routinely achieved when targeting the healthiest cells approximately 20&#8210;50 microns beneath the surface. For this success rate, it is important to use a high NA objective for IR-DIC, such as a 60x water-immersion objective, in order to achieve adequate visualization of pyramidal cells at this depth (Figure 2A,B).
Patch-clamp recordings from single neurons are easily achievable using this hippocampal slice preparation even in mice over six months of age. Figure 2C shows an example experiment using miniature excitatory postsynaptic currents (mEPSCs) recordings from CA1 neurons. Induction of chemical NMDA-LTD with bath application of 20 &#956;M NMDA for 3 min lowers mEPSC frequency in CA1 cells when assessed 60 min post-NMDA treatment. This finding suggests that NMDA-LTD causes activity-dependent pruning of synapses in CA1 in older mice (results adapted from Djurisic et al.15). Change in mEPSC amplitude was not detected. During mEPSC recordings, CA1 cells were also filled with biocytin. Figure 2D reveals an intact dendritic arbor and healthy cell habitus of biocytin-filled CA1 pyramidal neurons. A robust distribution of the fluorescent dye throughout the cell allows for routine evaluation of dendritic spine properties under different experimental conditions.
Using field-recordings, long-term potentiation (LTP) of ~170% of&#160;CA3-CA1 synapses in slices from adult mice was readily observed, suggesting maintenance of signaling cascades needed for LTP (Figure 2E,F). Network connectivity needed for a robust field excitatory postsynaptic potential (fEPSP) signal is also preserved (Figure 2E). The ability to assess synaptic plasticity in hippocampal slices from adult or aging mice is especially relevant for mouse models of neurodegenerative diseases as their hallmark synaptic dysfunction develops later in life.
Together, our results demonstrate that an acute hippocampal preparation from adult and aging mice allows assessment of synaptic function at the level of both single cells and population of cells routinely, as long as transcardial perfusion and ice-cold NMDG-based solutions are used to minimize hypoxic damage.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61377/61377fig1v2.jpg" /> 
Figure 1: Cutting method and recovery of transversal hippocampal slices from adult and aging mice.&#160;(A) A &#8220;60&#176;&#8221; tool used for removing the 60&#176; wedge from the rostral end of the brain, centered on the midline. (B) Positioning the hemispheres for cutting of transversal slices. An illustration of a 60&#176; cut is shown on the left and the upper right panels; positioning of the two hemispheres on the surface with glue in manner shown in the lower right panel ensures a perpendicular orientation of dorsal hippocampus relative to the blade. This orientation results in transversal slices of hippocampus. Yellow structures are a 3D rendering of hippocampi within the rodent brain (gray). This illustration is adapted from SynapseWeb30. All the views are of the dorsal side of the brain. (C) Transversal hippocampal slices cut from a 4-month-old mouse in a recovery chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61377/61377fig1v2large.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/61377/61377fig2v2.jpg" /> 
Figure 2: Patch-clamp and synaptic plasticity measurements in hippocampal slices from mice at P120&#8210;180.&#160;(A) An example IR-DIC micrograph&#160;of CA1 region. (B) Example IR-DIC micrographs of subiculum. In both A and B, images are taken 3 h after cutting with a 60x water-immersion objective. Calibration bar is 10 &#181;m. (C) The effect of chemical NMDA-LTD on mEPSCs in CA1 neurons from P120&#8210;180 mice. Upper panel are example traces of mEPSCs recorded at baseline and after NMDA-LTD pulse. Lower left panel: NMDA-LTD resulted in lower mEPSC frequency. Lower right panel: mEPSC amplitude change after NMDA-LTD is not detected. N = 17 cells at baseline and n = 15 cells for NMDA-LTD, 6 mice. P = 0.004, t-test. This figure has been modified from Djurisic et al.15. (D) Example of biocytin-filled CA1 pyramidal cell. (E) Example of fEPSP signal from CA1 stratum radiatum after Schaffer collateral stimulation. The gray trace is obtained during baseline recording, and the black trace is observed 20 min after tetanic stimulation. Each trace is an average of 30 consecutive traces. (F) Cumulative average of long-term potentiation of CA3-CA1 synapses after 4 trains of 100 Hz induction; n = 23 slices from 8 WT mice at approximately P90. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61377/61377fig2v2large.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="5">Artificial cerebrospinal fluid (aCSF)</td>
		</tr>
		<tr>
			<td>Ingredients</td>
			<td>Mw</td>
			<td>Final conc. (mM)</td>
			<td>g/2 Liters (10X)</td>
			<td>g/Liter (1X)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.44</td>
			<td>125</td>
			<td>146.1</td>
			<td>-</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.01</td>
			<td>26</td>
			<td>43.68</td>
			<td>-</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.55</td>
			<td>2.3</td>
			<td>3.43</td>
			<td>-</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>136.1</td>
			<td>1.26</td>
			<td>3.44</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Mg2SO4*7H2O</td>
			<td>203.3</td>
			<td>1.3</td>
			<td>-</td>
			<td>1.3 ml of 1M stock</td>
		</tr>
		<tr>
			<td>CaCl2*2H2O</td>
			<td>147.02</td>
			<td>2.5</td>
			<td>-</td>
			<td>2.5 ml of 1M stock</td>
		</tr>
		<tr>
			<td>Glucose*H2O</td>
			<td>180.2</td>
			<td>25</td>
			<td>-</td>
			<td>4.5 g</td>
		</tr>
		<tr>
			<td colspan="5">a) Add Mg2SO4*7H2O and CaCl2*2H2O from 1M stocks</td>
		</tr>
		<tr>
			<td colspan="5">b) aCSF 10X keep @RT</td>
		</tr>
		<tr>
			<td colspan="5">c) aCSF 1X @4&#176;C for 24h</td>
		</tr>
		<tr>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td colspan="5">NMDG-aCSF</td>
		</tr>
		<tr>
			<td>Ingredients</td>
			<td>Mw</td>
			<td>Final conc. (mM)</td>
			<td>g/2 Liters (3X)</td>
			<td>g/300 ml (1X)</td>
		</tr>
		<tr>
			<td>NMDG</td>
			<td>195.22</td>
			<td>135</td>
			<td>&#160;158.13</td>
			<td>-</td>
		</tr>
		<tr>
			<td>pH=7.4 with HCl</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.55</td>
			<td>1</td>
			<td>0.4473</td>
			<td>-</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>136.1</td>
			<td>1.2</td>
			<td>0.9799</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MgCl2*6H2O</td>
			<td>203.3</td>
			<td>1.5</td>
			<td>1.8297</td>
			<td>-</td>
		</tr>
		<tr>
			<td>CaCl2*2H2O</td>
			<td>147.02</td>
			<td>0.5</td>
			<td>0.4411</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Choline Bicarbonate</td>
			<td>80%</td>
			<td>20</td>
			<td>-</td>
			<td>1238 &#181;l</td>
		</tr>
		<tr>
			<td>Glucose*H2O</td>
			<td>180.2</td>
			<td>10</td>
			<td>-</td>
			<td>0.54</td>
		</tr>
		<tr>
			<td colspan="5">a) Filter and store 3X stock solution at 4&#176;C</td>
		</tr>
	</tbody>
</table>
Table 1: Media formulations.

Watch this Scientific Journal Video about Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice at JoVE.com

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

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

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

minimizing-hypoxia-in-hippocampal-slices-from-adult-and-aging-mice

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.4K Views.  Stanford University. Het hier gepresenteerde protocol vermindert overmatige hypoxische schade tijdens de voorbereiding van volwassen en verouderende muis hippocampal plakjes, waardoor een groot obstakel voor het bestuderen van de functie van volwassen en veroudering neurale circuits. Introductie van onderkoeling in natriumvrije oplossingen resulteert in hippocampale plakjes die tot 10 uur na het snijden gezond zijn en geschikt zijn voor zowel de lange termijn veldopnames als patchklemstudies. Deze methode voor de...

Video: Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Developmental Biology

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 ...

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 ...

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

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

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

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

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

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

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

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

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

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

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

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

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

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

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

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

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

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

Horizontal Hippocampal Slices of the Mouse Brain

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

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

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

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.

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

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