generation of medial thalamus-anterior cingulate cortex slices from mouse brains

Generation of Medial Thalamus-Anterior Cingulate Cortex Slices from Mouse Brains

Expose the skull of the animal, and trim off the remaining muscle. Next, peel away the dorsal surface of the skull using rongeurs and trim away the sides. Subsequently, using a spatula, cut the olfactory bulbs and nerve connections along the ventral surface of the brain before removing it.
Quickly transfer the brain to a beaker filled with ice-cold oxygenated ACSF. To prepare slices containing the MT-ACC pathway, make a sagittal cut 2 millimeters lateral to the midline in each hemisphere to display the subcortical anatomy.
Then, make an angled crosscut parallel to the visible fiber tract in the striatum. Make the second angled cross-cut from the connection between the cerebellum and visual cortex, to the midpoint between the anterior commissure and the optic tract that are ventral and parallel to the thalamus cingulate pathway. After that, attach the brain block to an angular plate with a cyanoacrylate adhesive.
Make a cut just above the turning point of the pathway. After that, unfold the plate, flatten it, and glue it onto the chamber stage of a vibratome. Subsequently, section the brain slices at 500 micrometers thickness and keep them in ice-cold oxygenated ACSF. Transfer a slice to the recording chamber at 32 degrees Celsius with continuous perfusion of oxygenated ACSF for one hour.

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Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Brain Slice Preparation
<ol>
	<li>Use male C57BL/6J mice, 4-8 weeks old. House the animals in an air-conditioned room (21-23 &#176;C; 50% humidity; 12 hr/12 hr light /dark cycle, lights on at 8:00 AM) with free access to food and water.</li>
	<li>Take a 250 ml&#160;aliquot of the artificial cerebrospinal fluid (aCSF) and place it in a beaker that contains ice. At the same time, supply continuous gas that is composed of 95% O2&#160;and 5% CO2.</li>
	<li>Surgery
	<ol>
		<li>Anesthetize the animal with 4% isoflurane in a glass box for approximately 3 min. Once the animal reaches a surgical depth of anesthesia (indicated by the lack of a response to toe pinch), place it on a shallow tray that is filled with crushed ice, and remove the head using scissors.</li>
		<li>Expose the skull, and trim off the remaining muscle. Next, using rongeurs, peel away the dorsal surface of the skull from the brain. Trim away the sides of the skull using rongeurs. Sterilize all of the surgical instruments with a 75% ethanol solution.</li>
		<li>Using a spatula, cut the olfactory bulbs and nerve connections along the ventral surface of the brain, and remove the brain. After decapitation, quickly transfer the brain to a beaker filled with ice-cold oxygenated aCSF.</li>
	</ol>
	</li>
	<li>Preparation of Medial Thalamus (MT)-Anterior Cingulate Cortex (ACC) Brain Slice 
	Note: Prepare slices that contain the pathway from the MT to ACC.
	<ol>
		<li>Hand-cut the brain block with two sagittal cuts 2.0 mm lateral to the midline in each hemisphere to display the subcortical anatomy. Then make two angled cuts. Make the first cross-cut parallel to the visible fiber tract in the striatum.</li>
		<li>Make the second cross-cut from the connection between the cerebellum and visual cortex to the midpoint between the anterior commissure and optic tract that are ventral and parallel to the thalamocingulate pathway.</li>
		<li>Attach the brain block to an angular plate (~120&#176;) with cyanoacrylate adhesive and make a cut just above the turning point of the pathway. Unfold the plate, flatten it, and glue it onto the chamber stage of a vibratome.</li>
		<li>Make medial thalamus-ACC brain slices (500 &#181;m thick) and then immerse them in ice-cold oxygenated aCSF. Transfer slices to the recording chamber, and keep at 32 &#176;C under continuous perfusion (12 ml/min) with oxygenated aCSF for 1 hr.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthetic:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Halocarbon Products Corporation&#160;</td>
			<td>NDC 12164-002-25</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>aCSF (total:1L):</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>MERCK</td>
			<td>1.08337.1000</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>MERCK</td>
			<td>1.06329.0500</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>MERCK</td>
			<td>1.06404.1000</td>
			<td>124 mM</td>
		</tr>
		<tr>
			<td>(+)-Sodium L-ascorbate, &#62;=98%</td>
			<td>SIGMA</td>
			<td>A4034-100G</td>
			<td>0.15 g / 2 c.c</td>
		</tr>
		<tr>
			<td>Magnesium sulfate, anhydrous, ReagentPlus</td>
			<td>SIGMA</td>
			<td>M7506-500G</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>MERCK</td>
			<td>1.02382.1000</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate monohydrate</td>
			<td>MERCK</td>
			<td>1.06346.1000</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>May &#38; Baker LTD Dagenham England</td>
			<td>MS 7616</td>
			<td>4.4 mM</td>
		</tr>
	</tbody>
</table>

Source: Lu, H., et al. <a href="https://app.jove.com/v/53709">Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation.</a> J. Vis. Exp. (2016)
The video demonstrates the preparation of medial thalamus-anterior cingulate cortex (MT-ACC) slices from mouse brains. A mouse&#39;s brain is dissected, cuts are made to expose the MT-ACC pathway, and slices are generated using a vibratome. The MT-ACC slices are then maintained in oxygenated artificial cerebrospinal fluid.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Brain ...

Begin with a severed mouse head. Remove the skin to expose the skull, and open the skull to access the brain.
Remove the olfactory bulbs and sever any nerve connections to extract the brain into ice-cold oxygenated artificial cerebrospinal fluid, or aCSF.
Take the brain, and make two vertical cuts on each half to expose the internal anatomy.
Make two angled cuts to isolate the region containing the medial thalamus-anterior cingulate cortex or MT-ACC pathway. The MT, a part of the thalamus, acts as a relay station via neuronal projections extending to the ACC in the frontal cortex.
Fix the brain on an angled plate using an adhesive, make a cut to unfold the brain and expose the pathway, and then attach it to a vibratome specimen stage.
Place the stage in the vibratome, add a buffer, and generate slices containing the MT-ACC pathway.
Keep the brain slices in a continuous flow of oxygenated aCSF to maintain their viability.

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Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

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Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...

科研

JoVE Journal

神经科学

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Generation of Medial Thalamus-Anterior Cingulate Cortex Slices from Mouse Brains

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Generation of Medial Thalamus-Anterior Cingulate Cortex Slices from Mouse Brains