preparation of brain sections using a nmdg-based protective recovery method

Preparation of Brain Sections Using a NMDG-Based Protective Recovery Method

Gently scoop out the intact brain into the beaker of prechilled NMDG-HEPES aCSF and allow the brain to uniformly cool for about 1 minute. Now, use a large spatula to transfer the brain from the beaker onto the Petri dish, covered with filter paper.
Trim and mount the brain, according to the preferred angle of slicing and desired brain region of interest. Work quickly to avoid prolonged oxygen deprivation during handling. Afterward, affix the brain block to the specimen holder, using adhesive glue.
Retract the inner piece of the specimen holder to withdraw the brain block fully inside. Then, pour the molten agarose directly into the holder until the brain block is fully covered in agarose. Subsequently, clamp the precooled accessory chilling block around the specimen holder for 10 seconds until the agarose has solidified.
Insert the specimen holder into the receptacle on the slicer machine and verify proper alignment. Following that, fill the reservoir with the remaining prechilled, oxygenated NMDG-HEPES aCSF from the 250-milliliter beaker and place a bubble stone into the reservoir for the duration of slicing to ensure adequate oxygenation.
Next, adjust the micrometer to begin advancing the agarose-embedded brain specimen. Start the slicer and empirically adjust the advanced speed and oscillation frequency to the desired level. Continue advancing and slicing the tissue in 300-micrometer increments until the brain region of interest is fully sectioned. The total time for the slicing procedure should be less than 15 minutes.
For initial NMDG recovery, upon completion of the sectioning procedure, collect all of the slices using a cut-off plastic Pasteur pipette and transfer them into a 34 degrees Celsius recovery chamber, filled with 150 milliliters of NMDG-HEPES aCSF.
The timing of the acute brain slice recovery step is very important to be able to strike a balance between morphological preservation and the functional recovery of the electrophysiological properties of each neuron.

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1. Brain Dissection and Slicing
<ol>
	<li>Decapitate the animal. Use a scalpel to open the skin on the head and expose the skull cap.</li>
	<li>Use fine super-cut scissors to cut away the skin over the skull cap and make small incisions laterally on either side at the caudal/ventral base of the skull. Make additional shallow cuts starting at the caudal/dorsal aspect of the skull, moving in the rostral direction up the dorsal midline taking care not to damage the underlying brain. Make a final &#39;T&#39; cut perpendicular to the midline at the level of the olfactory bulbs. 
	NOTE: Care must be taken to ensure no damage is done to the brain region(s) of interest. In particular, at no time should there be any compressive force applied to the brain itself.</li>
	<li>Use the round-tip forceps to grasp the skull, starting at the rostral-medial aspect and peel back towards the caudal-lateral direction. Repeat for both sides to crack open and remove the dorsal halves of the skull cap to expose the brain. Gently scoop out the intact brain into the beaker of pre-chilled NMDG-HEPES aCSF. Allow the brain to uniformly cool for ~1 min.</li>
	<li>Use the large spatula to lift the brain out of the beaker and onto the Petri dish covered with filter paper. Trim and block the brain according to the preferred angle of slicing and the desired brain region of interest. Work quickly to avoid prolonged oxygen deprivation during handling. 
	NOTE: Many slicing angles are possible. The exact blocking method and slicing angle will depend on the exact brain region, cell type, and circuit to be studied.</li>
	<li>Affix the brain block to the specimen holder using adhesive glue. Retract the inner piece of the specimen holder enough to withdraw the brain block fully inside. Pour the molten agarose directly into the holder until the brain block is fully covered in agarose. Clamp the pre-cooled accessory chilling block around the specimen holder for ~10 s until the agarose has solidified.</li>
	<li>Insert the specimen holder into the receptacle on the slicer machine and verify proper alignment. Fill the reservoir with remaining pre-chilled, oxygenated NMDG-HEPES aCSF from the 250 mL beaker and move a bubble stone into the reservoir for the duration of slicing to ensure adequate oxygenation.</li>
	<li>Adjust the micrometer to begin advancing the agarose-embedded brain specimen. Start the slicer and empirically adjust the advance speed and oscillation frequency to the desired level. 
	NOTE: Both settings should be in the low range. For best results, a single pass of the blade arm should take approximately 20 s and the oscillation should produce a very smooth and gentle humming noise with no overt buzzing.</li>
	<li>Continue advancing and slicing the tissue in 300 &#181;m increments (or other preferred thickness) until the brain region of interest is fully sectioned; the total time for the slicing procedure should be less than 15 min.</li>
</ol>
2. Optimized NMDG Protective Recovery Procedure
<ol>
	<li>Initial NMDG recovery step (critical step): Upon completion of the sectioning procedure, collect up all of the slices using a cut-off plastic Pasteur pipette and transfer them into a pre-warmed (34 &#176;C) initial recovery chamber filled with 150 mL of NMDG-HEPES aCSF. Transfer all slices in short succession and start a timer as soon as all slices are moved into the recovery chamber.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compresstome VF-200</td>
			<td>Precisionary Instruments</td>
			<td>VF-200</td>
			<td>Vibrating tissue slicer (recommended)</td>
		</tr>
		<tr>
			<td>N-methyl-D-glucamine</td>
			<td>Sigma Aldrich</td>
			<td>M2004</td>
			<td>aCSF constituent</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td>aCSF constituent</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P5405</td>
			<td>aCSF constituent</td>
		</tr>
		<tr>
			<td>Sodium Phosphate monobasic dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>71505</td>
			<td>aCSF constituent</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td>aCSF constituent</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td>aCSF constituent</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G7021</td>
			<td>aCSF constituent</td>
		</tr>
		<tr>
			<td>Sodium Ascorbate</td>
			<td>Sigma Aldrich</td>
			<td>A4034</td>
			<td>aCSF constituent</td>
		</tr>
		<tr>
			<td>Thiourea</td>
			<td>Sigma Aldrich</td>
			<td>T8656</td>
			<td>aCSF constituent</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma Aldrich</td>
			<td>P5280</td>
			<td>aCSF constituent</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C7902</td>
			<td>aCSF constituent</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate heptahydrate</td>
			<td>Sigma Aldrich</td>
			<td>M1880</td>
			<td>aCSF constituent</td>
		</tr>
		<tr>
			<td>Curved blunt forceps</td>
			<td>Fine Science Tools</td>
			<td>11065-07</td>
			<td>Brain dissection tools</td>
		</tr>
		<tr>
			<td>Fine dissecting scissors (supercut)</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td>Brain dissection tools</td>
		</tr>
		<tr>
			<td>Large heavy duty scissors 7&#39;&#39;</td>
			<td>Fine Science Tools</td>
			<td>14000-18</td>
			<td>Brain dissection tools</td>
		</tr>
		<tr>
			<td>Metal spatula</td>
			<td>Sigma Aldrich</td>
			<td>Z511455-1PAK</td>
			<td>Brain dissection tools</td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>VWR</td>
			<td>89031-954</td>
			<td>Brain dissection tools</td>
		</tr>
		<tr>
			<td>Brain Slice Keeper-4</td>
			<td>Automate Scientific</td>
			<td>S-BSK4</td>
			<td>brain slice holding chamber</td>
		</tr>
		<tr>
			<td>nylon netting</td>
			<td>Warner Instruments</td>
			<td>64-0198</td>
			<td>For building small slice recovery chambers</td>
		</tr>
		<tr>
			<td>Pyrex glass beakers (250 mL)</td>
			<td>VWR</td>
			<td>89090-434</td>
			<td>For building small slice recovery chambers</td>
		</tr>
		<tr>
			<td>35 mm plastic dish, round</td>
			<td>VWR</td>
			<td>100488-376</td>
			<td>For building small slice recovery chambers</td>
		</tr>
		<tr>
			<td>Gas diffuser stones (10 &#181;m)</td>
			<td>Sigma Aldrich</td>
			<td>59277</td>
			<td>For constant carbogenation (fine bubbles)</td>
		</tr>
		<tr>
			<td>Agarose Type I-B</td>
			<td>Sigma Aldrich</td>
			<td>A0576</td>
			<td>For embedding brain specimens</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma Aldrich</td>
			<td>H1758-100ML</td>
			<td>For pH adjustment of media</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>221465-25G</td>
			<td>For pH adjustment of media</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>221473</td>
			<td>For pH adjustment of media</td>
		</tr>
		<tr>
			<td>Plastic transfer pipets 3 mL graduated</td>
			<td>VWR</td>
			<td>89497-676</td>
			<td>For slice transfer</td>
		</tr>
		<tr>
			<td>Zirconium ceramic injector blades</td>
			<td>Cadence Specialty Blades</td>
			<td>EF-INZ10</td>
			<td>http://cadenceinc.com/</td>
		</tr>
		<tr>
			<td>Heated water bath (2.5L)</td>
			<td>VWR</td>
			<td>13491-060</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Filter paper rounds</td>
			<td>VWR</td>
			<td>28456-022</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue</td>
			<td>Amazon</td>
			<td>B000BQRBO6</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Glass petri dish</td>
			<td>VWR</td>
			<td>89000-326</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>10X Phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P5493</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Thermomixer (w/1.5 mL tube block)</td>
			<td>VWR</td>
			<td>89232-908</td>
			<td>To keep agarose molten</td>
		</tr>
	</tbody>
</table>

Source: Ting, J. T. et al. <a href="https://app.jove.com/v/53825">Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method.</a> J. Vis. Exp. (2018).
This video describes a protocol to obtain brain slices using an N-Methyl-D-glucamine (NMDG)-based protective recovery method. A dissected brain is incubated in an aCSF with NMDG and HEPES to minimize neuronal cell death and damage during the sectioning. The section is allowed to recover in the aCSF to restore the cellular activities

1. Brain Dissection and Slicing

	Decapitate the animal. Use a scalpel to open the skin on the head and expose the skull cap.
	Use fine super-cut scissors ...

Begin with an isolated brain. Quickly submerge the brain in a pre-chilled artificial cerebrospinal fluid or aCSF to pause the metabolic processes.
The aCSF contains NMDG, a large organic cation that reduces neuronal cell injury and death.
The aCSF also contains HEPES, which maintains pH and salt concentration and prevents cell swelling and damage.
Transfer the brain onto a Petri dish containing a filter paper.
Trim the brain to obtain the desired region.
Fix the brain block in the specimen holder using a suitable glue and embed it in the agarose.
Position the specimen holder in the slicer.
Fill the slicer&#39;s reservoir with the pre-chilled aCSF and aerate the solution to ensure an optimum oxygen supply.
Obtain sections of the brain block.
Transfer the sections into the prewarmed aCSF to restart the metabolic processes, enabling the sections to recover.

33 ビュー. NMDGベースの保護回復法を用いた脳切片の調製

ビデオ: NMDGベースの保護回復法を用いた脳切片の調製 - 実験

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.

脳スライスにおける全細胞パッチクランプ記録

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

JoVE Journal

神経科学

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

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

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

電気生理学的記録のための急性ヒト海馬スライスの調製

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

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

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

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