eoe sub articles

EoE Sub Articles

1. Preparation of Acute Slices
<ol>
	<li>Prepare ACSF, cutting solution, tools (scissors, scalpel, forceps, spatula, Pasteur pipette), and agar blocks. 
	NOTE: 1 L of artificial cerebrospinal fluid (ACSF) is required per experiment. As previously published, ACSF is prepared by dissolving chemicals in double-distilled H2O. The cutting solution is prepared by supplementing 200 ml of ACSF with 0.87 ml of 2 M MgSO4 stock solution.</li>
	<li>Oxygenate ACSF and cutting solution throughout the experiment with 95% O2 and 5% CO2.</li>
	<li>Deeply anesthetize the mouse using a small animal anesthesia machine, using isoflurane (3% oxygen). Check the anesthesia depth using the limb withdrawal reflex before continuing.</li>
	<li>Decapitate the mouse using large scissors and immediately chill the head in an ice-cold cutting solution.</li>
	<li>Open the skull by a single midline incision from caudal to rostral and gently push pieces of the skull to the sides using forceps. Rapidly remove the brain by gently lifting it out of the skull using a small rounded spatula. Cut off cerebellum with scalpel. Place the brain in an ice-cold cutting solution.</li>
	<li>For mPFC injection sites, use a scalpel to cut off the anterior part of the brain (containing mPFC) and put it in an ice-cold cutting solution until slicing.</li>
	<li>Remove the excess cutting solution with filter paper and glue the posterior part of the brain onto the vibratome stage.</li>
	<li>For coronal amygdala slices, MGm/PIN injection sites, and mPFC injection sites, glue the brain directly on stage (Figure 1D, left and right). Place an additional agar block behind the brain for stability while slicing.</li>
	<li>Place the stage in a cutting chamber with an ice-cold oxygenated cutting solution maintained at 4 &#176;C using a cooling unit. Using a sapphire blade, prepare coronal slices of the amygdala. Place the slices in an interface chamber supplied with oxygenated ACSF at RT.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Artificial cerebrospinal fluid (ACSF)</td>
			<td>for composition see references #16 and #23</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Internal patch solutions</td>
			<td>for composition see references #16 and #23</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slicer, Microm HM650V</td>
			<td>Fisher Scientific, Germany</td>
			<td>920120</td>
			<td></td>
		</tr>
		<tr>
			<td>Cooling unit for tissue slicer, CU65</td>
			<td>Fisher Scientific, Germany</td>
			<td>770180</td>
			<td></td>
		</tr>
		<tr>
			<td>Sapphire blade</td>
			<td>Delaware Diamond Knives</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscope, SZX2-RFA16</td>
			<td>Olympus, Japan</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generating a coronal brain slice exposing the amygdala regions from a mouse brain

Source: Bosch, D., et al. <a href="https://app.jove.com/v/53628">Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices.</a> J. Vis. Exp. (2016).
This video demonstrates the preparation of thin coronal brain slices from a murine brain, exposing the amygdala regions rich in synaptic connections from the medial prefrontal cortex. The slices are then recovered in oxygenated artificial cerebrospinal fluid to maintain neuronal viability.

<img alt="Figure 1" src="/files/ftp_upload/22604/22604_Figure1.jpg" /> 
Figure 1. Stereotactic Injections, Preparation of Acute Brain Slices, and Visualization of Presynaptic Fibers. (A, B) Stereotactic virus injection. A) Picture of an anesthetized mouse placed in a stereotactic frame with the skull exposed and the injection pipette. Inset: Zoom in the picture of the injection pipette filled with virus solution mixed with fast green. Scale bar: 3 mm...

Generating a Coronal Brain Slice Exposing the Amygdala Regions from a Mouse Brain

1. Preparation of Acute Slices

	Prepare ACSF, cutting solution, tools (scissors, scalpel, forceps, spatula, Pasteur pipette), and agar blocks.
	NOTE: 1 L ...

Take a mouse brain in an ice-cold buffer and remove its posterior portion containing the cerebellum and the anterior portion with the medial prefrontal cortex, or mPFC.
The middle portion contains amygdala regions that process emotions like fear and anxiety.
Move the brain section onto a filter paper to remove the excess buffer.
Glue it onto a stage and fix an agar block behind the section to prevent its movement during slicing.
Place this stage into a vibratome cutting chamber with the ice-cold buffer.
Cut the section to obtain a thin coronal brain slice.
This exposes the amygdala regions, which contain synaptic connections between the amygdala neurons and the projections of mPFC neurons that regulate the amygdala&#39;s responses.&#160;
Cut the slice into two halves, each containing an amygdala region.
Next, transfer them to an interface chamber containing oxygenated artificial cerebrospinal fluid.
Recover the slices at physiological temperature to maintain neuronal viability.

generating-coronal-brain-slice-exposing-amygdala-regions-from-mouse

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

132 조회수. 출처: Bosch, D., et al. 뇌 절편에서 공포 회로의 생체 내 광유전학적 해부. J. Vis. 특급 (2016). 이 비디오는 쥐의 뇌에서 얇은 관상 뇌 절편을 준비하여 내측 전전두엽 피질에서 시냅스 연결이 풍부한 편도체 영역을 노출시키는 방법을 보여줍니다. 그런 다음 조각은 신경세포의 생존력을 유지하기 위해 산소가 공급된 인공 뇌척수액에서 회수됩니다. 

비디오: Generating a Coronal Brain Slice: Exposed the Amygdala Regions from a Mouse Brain (코로나 뇌 슬라이스 생성하기: 쥐의 뇌에서 편도체 영역 노출) - 개념

Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala &#8211; peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 &#176;C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases.

Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

심근 경색 후 Spectrofluorometry에 의해 측정 된 쥐 편도체에서 카스파 제 3 활동

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still ...

연구

JoVE Journal

신경과학

Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this purpose, an in vivo imaging technique that detects calcium transients in neurons using genetically encoded calcium indicators (GECIs), a miniaturized fluorescence microscope, and a gradient refractive index (GRIN) lens has been developed and successfully applied to many brain structures1,2,3,4,5,6. This imaging technique is particularly powerful because it enables chronic simultaneous imaging of genetically defined cell populations for a long-term period up to several weeks. Although useful, this imaging technique has not been easily applied to brain structures that locate deep within the brain such as amygdala, an essential brain structure for emotional processing and associative fear memory7. There are several factors that make it difficult to apply the imaging technique to the amygdala. For instance, motion artifacts usually occur more frequently during the imaging conducted in the deeper brain regions because a head-mount microscope implanted deep in the brain is relatively unstable. Another problem is that the lateral ventricle is positioned close to the implanted GRIN lens and its movement during respiration may cause highly irregular motion artifacts that cannot be easily corrected, which makes it difficult to form a stable imaging view. Furthermore, because cells in the amygdala are usually quiet at a resting or anesthetized state, it is hard to find and focus the target cells expressing GECI in the amygdala during baseplating procedure for later imaging. This protocol provides a helpful guideline for how to efficiently target cells expressing GECI in the amygdala with head-mount miniaturized microscope for successful in vivo calcium imaging in such a deeper brain region. It is noted that this protocol is based on a particular system (e.g., Inscopix) but not restricted to it.

In vivo microendoscopic calcium imaging is an invaluable tool that enables real-time monitoring of neuronal activities in freely behaving animals. However, applying this technique to the amygdala has been difficult. This protocol aims to provide a useful guideline for successfully targeting amygdala cells with a miniaturized microscope in mice.

자유롭게 행동하는 마우스의 편도체에 있는 머리 산 소형 현미경을 가진 성공적인 생체 내 칼슘 화상 진찰

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this ...

Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few studies have described the experimental protocol in detail. This paper illustrates the whole process of amygdala electrical kindling epilepsy model making, with the introduction of a method of bipolar electrode fabrication. This electrode can both stimulate and record, reducing brain injury caused by implanting separate electrodes for stimulation and recording. For long-term electroencephalogram (EEG) recording purposes, slip rings were used to eliminate the record interruption caused by cable tangles and falling off.
After periodic stimulation (60 Hz, 1 s every 15 min) of the basolateral amygdala (AP: 1.67 mm, L: 2.7 mm, V: 4.9 mm) for 19.83 &plusmn; 5.742 times, full kindling was observed in six mice (defined as induction of three continuous grade V episodes classified by Racine's scale). An intracranial EEG was recorded throughout the entire kindling process, and an epileptic discharge in the amygdala lasting 20-70 s was observed after kindling. Therefore, this is a robust protocol for modeling epilepsy originating from the amygdala, and the method is suitable for revealing the role of the amygdala in temporal lobe epilepsy. This research contributes to future studies on the mechanisms of mesial temporal lobe epilepsy and novel antiepileptogenic drugs.

The amygdala plays a key role in temporal lobe epilepsy, which originates in and propagates from this structure. This article provides a detailed description of the fabrication of deep brain electrodes with both recording and stimulating functions. It introduces a model of medial temporal lobe epilepsy originating from the amygdala.

양극성 전극을 사용하여 편도체의 전기적 점화에 의한 측두엽 간질 마우스 모델 생성

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few ...

Generating a Coronal Brain Slice Exposing the Amygdala Regions from a Mouse Brain

내레이션 대본

Generating a Coronal Brain Slice Exposing the Amygdala Regions from a Mouse Brain