Support by Centre National de la Recherche Scientifique (CNRS, UMR 6612 and 7339) is gratefully acknowledged.

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The authors have nothing to disclose.

NMR 분광법은 매우 정확하고 재현 가능한 방식으로 용액 화학 물질의 농도를 측정하기위한 효율적인 방법이다. 그러나, 고품질의 데이터를 얻기 위해 그 샘플 준비 및 분석에 관한 소정의 규칙에 적용하는 것이 필요하다. 관측 된 신호의 강도가 검출 임계치 (특히 약한 신호)에 접근하지 않는 NMR 분광법에 의한 대사 산물 농도의 결정에있어서, 생성이나 NMR 신호의 수신도는 정량 오류를 지배하고있...

쥐 모델은 뇌 연구 1에서 광범위하게 활용되고있다. 유전자형 표현형 상관 관계는 다른 2-6 한편 RNA 및 / 또는 단백질 수준에서 유전자 발현을 연구함으로써, 마우스 및 래트의 뇌에 조사하고, 형태 학적 기능, 전기 생리 학적 및 / 또는 행동 적 표현형되었다. 그러나, 완전히 유전자형 표현형에 연결 메커니즘을 이해하기 위해서는 하류 단백질 발현, 즉 분자의 사건을 조사하는 것이 필수적이다. 효소는 7 행동을 두는 생화학 기판의 신진 대사. 이 요구 사항은 생물학 8,9의 여러 가지 대사 연구의 르네상스로 지난 10-15년 이상했다. 고전 대사 연구는 종종 특정 경로의 상세에 집중되었지만, 새로운 메타 볼로 접근법 고려 조직의 대사 프로파일의 글로벌 모두 포괄 맞도록 조사된다.이 개념 중 하나 결과는 특정 대사 경로 및 / 또는 화합물의 클래스에 대한 편견을 최소화 분석 도구에 대한 명백한 필요합니다. 그러나, 전통적인 생화학 적 분석은 분석이 수행되기 전에 지정해야 특정 분석 물의 특정 화학 반응에 기초한다. 대조적으로, 핵 자기 공명 (NMR) 분광법 및 질량 분석법 (MS) (I)와 같은 광학적 기술은 생화학 물질, 특정 분자 (물리적) 특성을 기반으로는 각각의 스펙트럼에있어서, 하나 또는 다수의 별개의 신호를 일으킨다 한 실험 과정에서 검출; 및 (ⅱ) 실험마다 상이한 다수의 화합물을 검출한다. 따라서, 각각의 스펙트럼은 대사 산물의 전체 범위의 결합 된 정보가 포함되어 있습니다. 더 이전의 선택은 피 분석의 특성에 관한 8 측정 할 할 필요가 없기 때문에 이러한 이유로, 분 광학적 방법은 대사 체학을위한 적절한 툴, 아르 </sup&gt;. 그들은 매우 예기치 대사 변화의 검출을 용이하게하기 때문에 결과적으로, 이들 기술은 자연 탐색 연구에는 적합. NMR 분광기 및 MS는 많은 대사 분석을 위해 상호 교환 적으로 사용될 수 있지만, 각각의 방법은 특정의 장점과 최근 10를 평가 한 단점을 가지고있다. 간단히, NMR 분광법은 보통 조 추출물에서 수행 할 수 있으며, 분석하기 전에 샘플 화합물을 크로마토 그래피 분리를 필요로하지 않는다. 반면, MS는 질량 분석 이미징 특히 최근 개발을 제외하고, 기체 또는 액체 크로마토 그래피 (GC 또는 LC) 분리와 함께 작동합니다. 다른 당의 공진 라인 (1 H) 양성자 NMR 스펙트럼에서 상당히 중첩 때문에 이러한 당의 분석과 같은 몇몇 특별한 경우에서, LC 분리는 물론 NMR 분광법에 대한 필요성이 될 수있다. CHR없이 그럼에도 1 H NMR 분광법omatographic 분리는 가장 인기있는 거의 보편적으로 적용 메타 볼로 NMR 방법 남아있다. 일반적으로, 샘플 준비는 NMR 분광법에 대해 MS보다 더 많은 시간이 걸리고 복잡한이다. 매트릭스 효과로 인해 심각한 문제가 많은가 상당히 약화 신호로 이어질 수있는 위치 MS보다 NMR 분광법 덜 일반적이다. 대사 정량은 어느 방법으로 달성 될 수있다. 그러나, 여러 표준 화합물 인해 대사 사이의 매트릭스 효과 및 이온화 효율의 변동에 MS 필요하다. 대조적으로, 샘플 당 하나의 표준 때문에 적절한 측정 조건 하에서 NMR 분광 분석에 필요한, ​​후자의 방법은 본질적으로 관측 핵에 의해 엄격하게 선형 응답 NMR로 정량 덕분이다. NMR의 주요 단점은 상대적으로 낮은 민감도이다. MS, 특히 LC-MS에 몇 배에 의해 NMR보다 더 민감하다; 이러한 이유로, MS는 NMR에 대해 선호 될매우 낮은 농도에서 발생하는 화합물의 분석. 한편, NMR 실험의 비파괴 특성은 MS 위에 분명한 장점은; 이러한 방식으로, NMR은 1 H, 인-31 (31 P), 탄소 -13 (13 C) 같은 다른 NMR-활성 핵 위해, 예를 들어 동일한 샘플에 반복적으로 수행 할 수있는 불소 - 19 (19 F) 등., MS 측정 반대로 어떤 물질이 NMR에 의해 소비되지 않은 것처럼. NMR 및 MS 모두는 상이한 모드로 사용될 수 있으며, 각각의 하나는 특정의 화학적 특성을 갖는 화합물의 검출에 최적 인. 거의 모든 대사 인산화도 양자를 포함 할지라도, 예를 들어, 31 P NMR 종종 적당히 농축 인산화 분석 화합물의 1 H NMR보다 더 적합하다. 그러나, 그들의 1 H NMR 신호는 후자 반면, 다른 비 - 인산화 된 화합물 1 H NMR 신호에 의해 가려 질 수도분명히 31 P NMR 스펙트럼에서 배경 신호를 발생하지 않습니다. 13 C NMR의 특별한 경우는 거의 독점적으로 관심있는 동안 아날로그 상황에서, 19 F NMR 분석, 불소 화합물, 예를 들면, 플루오르 화 약물 (내인성 대사에서 배경 없음 신호)에 바람직 할 경우 13 C-의 운명 표시 외인성 대사 전구 물질로 인해 13 C 동위 원소 (약 1 %)의 매우 낮은 자연 풍부 따라야합니다. 많은 질량 분석기는 음이온 모드 또는 양이온 모드에서 작동합니다. 따라서, 앞서 이온이 부정적 또는 긍정적으로 청구됩니다 관찰 할 수 있는지 여부를 분석 아는 것이 중요합니다. 이 방법은 시료 준비에 필요한 (I)의 시간적으로 저비용으로 중요한 대사 산물 농도의 많은 수를 산출하기 때문에 우리는 1 H, 31 P NMR 분광법에 의한 뇌 조직의 대사 체의 분석 프로토콜에 여기에 집중차 (II) 노력은 대사 산물 정량이 필요합니다. 모든 실험은 표준 습식 화학 실험실의 장비 및 고해상도 NMR 분광법 설비를 사용하여 수행 될 수있다. 또한 요구 사항은 아래의 프로토콜 부분에 설명되어 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="921">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Isoflurane</td>
			<td style="width:144px;">Virbac</td>
			<td style="width:180px;">Vetflurane</td>
			<td style="width:316px;">Anesthetic for animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane vaporizer</td>
			<td>Ohmeda</td>
			<td>Isotec 3</td>
			<td>Newer model available: Isotec 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel, scissors, forceps, clamps</td>
			<td>Harvard Apparatus 
			Fisher Scientific</td>
			<td>various 
			various</td>
			<td>Surgical equipment for animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freeze-clamp tool</td>
			<td>homebuilt</td>
			<td>n/a</td>
			<td>Tong with aluminium plates, to be inserted 
			in liquid nitrogen for cooling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dewar</td>
			<td>Nalgene</td>
			<td>4150-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid nitrogen</td>
			<td>Air Liquide</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrogen gas</td>
			<td>Air Liquide</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrogen evaporator</td>
			<td>Organomation Associates</td>
			<td>N-EVAP 111</td>
			<td>Can be replaced by homebuilt device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mortar</td>
			<td>Sigma-Aldrich</td>
			<td>Z247472</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pestle</td>
			<td>Sigma-Aldrich</td>
			<td>Z247510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue homogenizer</td>
			<td>Kinematica</td>
			<td>Polytron</td>
			<td>With test tubes fitting homogenizer shaft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electronic scale</td>
			<td>Sartorius</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>366910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass centrifuge tubes</td>
			<td>Kimble</td>
			<td>45500-15, 45500-30</td>
			<td>Kimax 15-mL, 30-mL tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td>Kimble</td>
			<td>45150-2</td>
			<td>Kimax 2-mL tube; should replace &#34;Eppen-dorf&#34; tube if compatible with centrifuge rotor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polystyrene pipettes</td>
			<td>Costar Corning</td>
			<td>Stripettes</td>
			<td>5 and 10-mL volumes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deuterochloroform</td>
			<td>Sigma-Aldrich</td>
			<td>431915</td>
			<td>99.96 % deuterated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deuterium oxide</td>
			<td>Sigma-Aldrich</td>
			<td>423459</td>
			<td>99.96 % deuterated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deuterium chloride</td>
			<td>Alpha Aesar</td>
			<td>42406</td>
			<td>20 % in deuterium oxide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium deuteroxide</td>
			<td>Sigma-Aldrich</td>
			<td>164488</td>
			<td>30 % in deuterium oxide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lyophilizer</td>
			<td>Christ</td>
			<td>Alpha 1-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cold centrifuge</td>
			<td>Heraeus</td>
			<td>Megafuge 16R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter</td>
			<td>Eutech Cybernetics</td>
			<td>Cyberscan</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CDTA</td>
			<td>Sigma-Aldrich</td>
			<td>D0922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cesium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>516988</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMR tubes</td>
			<td>Wilmad</td>
			<td>528-PP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMR stem coaxial insert</td>
			<td>Sigma-Aldrich</td>
			<td>Z278513</td>
			<td>By Wilmad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMR pipettes</td>
			<td>Sigma-Aldrich</td>
			<td>Z255688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td>Research</td>
			<td>With tips for volumes from 0.5 to 1000 &#956;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipet-Aid</td>
			<td>Drummond</td>
			<td>XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMR spectrometer</td>
			<td>Bruker</td>
			<td>AVANCE 400</td>
			<td>including probe and other accessories</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMR software</td>
			<td>Bruker</td>
			<td>TopSpin 1.3</td>
			<td>newer version available: Topspin 3.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water-soluble standard compounds</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phospholipid standard compounds</td>
			<td>Avanti Polar Lipids 
			Doosan Serdary 
			Sigma-Aldrich</td>
			<td>various 
			various 
			various</td>
			<td>&#160; 
			Source for plasmalogens, but may be 
			&#60; 70 - 80 % purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylenediphosphonate</td>
			<td>Sigma-Aldrich</td>
			<td>M9508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TSP-d4</td>
			<td>Sigma-Aldrich</td>
			<td>269913</td>
			<td></td>
		</tr>
	</tbody>
</table>

metabolomic analysis of rat brain by high resolution nuclear magnetic resonance spectroscopy of tissue extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

추출 솔루션의 현재 : 두뇌와 다른 조직 추출물의 대사 NMR 스펙트럼에서 최상의 해상도를 얻으려면, 그것은 긴 제거하거나 (상자성 이온 가장 중요한) 마스크 금속 이온을하는 것이 일반적이었다. 이것은, 또는 Chelex-100 (20)로 이온 교환 수지를 통해 추출물을 전달하여 추출물 19 EDTA 또는 CDTA 같은 킬레이트 제를 첨가함으로써 하나를 달성되었다. 도 1에 제시된 결과는 ?...

Watch this Scientific Journal Video about Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts at JoVE.com

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

조직 추출물의 고해상도 핵 자기 공명 분광법에 의한 쥐 뇌의 대사 체 분석

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

metabolomic-analysis-rat-brain-high-resolution-nuclear-magnetic

Research

JoVE Journal

Neuroscience

14.7K 조회수.  Aix-Marseille Université. The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

비디오: Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral line-widths and allows high-resolution spectra to be obtained from extracts, intact cells, cell cultures, and more importantly intact tissue to investigate relationships between metabolites and cellular processes. In vivo HRMAS 1H-MRS studies have yet to be reported in the live fruit fly Drosophila melanogaster. Drosophila, as a simpler genetic organism, allows the multiple biological functions and various evolutionarily conserved signaling pathways to be examined at the whole organism level and it is a useful model for investigating genetics and physiology. To this end, we developed and implemented an in vivo HRMAS 1H-MRS method to investigate live Drosophila at 14.1 T. Here, we outline an HRMAS 1H-MRS protocol for the molecular characterization of Drosophila with a conventional MR spectrometer equipped with an HRMAS probe. This technique is a novel, in vivo, non-destructive Drosophila metabolite measurement approach, which enables the identification of disease biomarkers and thus may contribute to novel therapeutic development.

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

This technique enables the use of high-resolution magic angle spinning proton MR spectroscopy (HRMAS 1H-MRS) for molecular characterization of live Drosophila melanogaster with a conventional 14.1 tesla spectrometer equipped with an HRMAS probe.

라이브의 자기 공명 분광학 Drosophila melanogaster 매직 앵글 스피닝를 사용하여

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral ...

연구

신경과학

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

인간 Midbrain위한 고해상도 기능성 자기 공명 영상 방법

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

쥐 중추 신경계에서 면역 조직 화학 분석 및 주변 림프절 조직 섹션

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

공 초점 빛 시트 현미경 전체 마우스 두뇌의 마이크론 규모의 해상도 광학 단층 촬영

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

일차 운동 피질 대사에 양성 반구 경 두개 전기 자극 효과의 측정을위한 도구로 자기 공명 분광학의 사용

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

높은 해상도<em&gt; 생체</em&gt; 3T 자기 공명 영상을 이용하여 인간의 해마 서브 필드에 대한 수동 분할 프로토콜

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

인간 Subcortex의 고해상도 구조 자기 공명 영상<I&gt; 생체</I&gt; 및 사후

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

망막 리본 시냅스에서 막 수용체의 고해상도 양이 Immunogold 분석

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to technological developments over the last decade and the increase in magnetic field strength, it is now possible to obtain good resolution spectra in vivo in the rat brain. Neuroenergetics (i.e., the study of brain metabolism) and, especially, metabolic interactions between the different cell types have attracted more and more interest in recent years. Among these metabolic interactions, the existence of a lactate shuttle between neurons and astrocytes is still debated. It is, thus, of great interest to perform functional proton magnetic resonance spectroscopy (1H-MRS) in a rat model of brain activation and monitor lactate. However, the methyl lactate peak overlaps lipid resonance peaks and is difficult to quantify. The protocol described below allows metabolic and lactate fluctuations to be monitored in an activated brain area. Cerebral activation is obtained by whisker stimulation and 1H-MRS is performed in the corresponding activated barrel cortex, whose area is detected using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI). All steps are fully described: the choice of anesthetics, coils, and sequences, achieving efficient whisker stimulation directly in the magnet, and data processing.

After checking by blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) that the corresponding somatosensory barrel field cortex area (called S1BF) is correctly activated, the main goal of this study is to quantify lactate content fluctuations in the activated rat brains by localized proton magnetic resonance spectroscopy (1H-MRS) at 7 T.

7 기능 자기 공명 분광학 수염 활성화 중 쥐 신 피 질에서 T

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

용매 클리어 뇌 조직에서 광견병 바이러스 감염의 고해상도 3D 이미징

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...