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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Acute Slices
<ol>
	<li>Prepare artificial cerebrospinal fluid (ACSF), cutting solution, tools (scissors, scalpel, forceps, spatula, Pasteur pipette), and agar blocks. 
	Note: 1 L&#160;of artificial cerebrospinal fluid (ACSF) is required per experiment, and is prepared by dissolving chemicals in double-distilled H2O as previously published. The cutting solution is prepared by supplementing 200 ml of ACSF with 0.87 ml of 2 M MgSO4&#160;stock solution.</li>
	<li>Oxygenate ACSF and cutting solution throughout the experiment with 95% O2&#160;and 5% CO2.</li>
	<li>Deeply anesthetize mouse using a small animal anesthesia machine using isoflurane (3% in oxygen). Check anesthesia depth using limb withdrawal reflex before continuing.</li>
	<li>Decapitate mouse using large scissors and immediately chill head in ice-cold cutting solution.</li>
	<li>Open skull by a single midline incision from caudal to rostral and gently push pieces of skull to the sides using forceps. Rapidly remove brain by gently lifting it out of the skull using a small, rounded spatula. Cut off cerebellum with scalpel. Place brain in ice-cold cutting solution.</li>
	<li>For medial prefrontal cortex or mPFC injection sites: cut off anterior part of the brain (containing mPFC) using a scalpel and put in ice-cold cutting solution until slicing.</li>
	<li>Remove excess cutting solution with filter paper and glue the posterior part of the brain onto the vibratome stage.</li>
	<li>For tilted amygdala slices, glue the brain on an agar block (4%) cut at a 35&#176; angle (Figure 1D, middle). For coronal amygdala slices, medial geniculate nucleus/ posterior intralaminar thalamic nucleus (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 brain for stability while slicing.</li>
	<li>Place the stage in cutting chamber with ice-cold oxygenated cutting solution that is maintained at 4 &#176;C using a cooling unit. Prepare acute slices of the amygdala (320 &#181;m) using a sapphire blade. Place slices in an interface chamber supplied with oxygenated ACSF at room temperature (RT).</li>
	<li>After preparation of acute amygdala slices, place the interface chamber in a water bath at 36 &#176;C to recover slices for 35 - 45 min. Subsequently, return the interface chamber to RT.</li>
	<li>During commencement of step 1.10, cut slices of the injection sites as described before for acute amygdala slices (steps 1.8 - 1.9). Recovery of slices in the water bath is not required here. Optional: To quickly estimate injection site location, observe slices on a stereoscope equipped with a fluorescent lamp and appropriate filter sets.</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></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>internal patch solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate</td>
			<td>Roth, Germany</td>
			<td>P027.1</td>
			<td>prepare 2M stock solution in purified water</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>custom order, inquire with company</td>
		</tr>
		<tr>
			<td>Stereoscope, SZX2-RFA16</td>
			<td>Olympus, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Patch microscope, BX51WI</td>
			<td>Olympus, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bath temperature controler, TC05</td>
			<td>Luigs &#38; Neumann, Germany</td>
			<td>200-100 500 0145</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose nitrate filterpaper for interface chamber</td>
			<td>Satorius Stedim Biotech, Germany</td>
			<td>13006--50&#160;&#160;&#160;&#160; ACN</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of tilted amygdala slices 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)
The video demonstrates the generation of tilted amygdala slices from a mouse brain. The brain is positioned at an angle on the vibratome specimen stage to obtain slices containing the amygdala and axons from the medial prefrontal cortex that form synaptic connections with the amygdala. The brain slices are maintained at the physiological temperature to preserve the tissue for further experiments.

<img alt="Figure 1" src="/files/ftp_upload/22603/22603_Figure1.jpg" /> 
Figure 1.&#160;Stereotactic Injections, Preparation of Acute Brain Slices, and Visualization of Presynaptic Fibers.&#160;(A, B) Stereotactic virus injection. A) Picture of anesthetized mouse placed in a stereotactic frame with skull exposed and the injection pipette. Inset: Zoom in picture of injection pipette filled with virus solution mixed with fast green. Scale bar: 3 mm. (<s...

Generation of Tilted Amygdala Slices from a Mouse Brain

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Acute Slices

	...

Take a mouse brain immersed in an ice-cold buffer.
Remove the cerebellum and trim the anterior section containing the medial prefrontal cortex, or mPFC.
Attach the tissue to an angled agar block, which is glued onto a vibratome specimen stage.
Attach additional agar blocks to provide stability during slicing. Place the stage inside the vibratome cutting chamber filled with oxygenated ice-cold buffer.
Generate tilted slices containing the amygdala that respond to fear stimuli and neuronal projections from the mPFC, which form synaptic connections in the amygdala and regulate its activity.
The tilted slices expose the projections, allowing the study of the functional connectivity between the mPFC and the amygdala.
Cut the slices in half and place them in a chamber containing oxygenated artificial cerebrospinal fluid at room temperature to stabilize the tissue.
Incubate the slices at the physiological temperature to recover the tissue for downstream assays.

generation-of-tilted-amygdala-slices-from-a-mouse-brain

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

60 조회수. 출처: Bosch, D., et al. 뇌 절편에서 공포 회로의 생체 외 광유전학적 해부. J. Vis. 특급 (2016년) 이 비디오는 쥐의 뇌에서 기울어진 편도체 슬라이스를 생성하는 방법을 보여줍니다. 뇌는 편도체와 시냅스 연결을 형성하는 내측 전전두엽 피질에서 편도체와 축삭을 포함하는 조각을 얻기 위해 진동 표본 단계에서 비스듬히 배치됩니다. 뇌 절편은 추가 실험을 위해 조직을 보존하기 ?...

비디오: 쥐의 뇌에서 기울어진 편도체 조각의 생성 - 개념

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

자유롭게 움직이는 마우스에 있는 행동을 조절하기 위하여 신경 활동의 광유전학 조작

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

연구

JoVE Journal

행동분석학

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 ...

신경과학

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 ...

Generation of Tilted Amygdala Slices from a Mouse Brain

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