저자는 자신의 지속적인 기술 지원 Ikiel Ptito 감사하고 싶습니다. MP에 NSERC 부여합니다.

1 부 : 조직의 사전 처리 <ol><li> 조직은 잘 paraformaldehyde, 글루 타 알데히드, 또는 포르말린과 perfused해야합니다. 이것은 일반적으로 다른 장기를 수확하는 데 사용되는 표준 transcardial 재관류를 통해 달성될 수있다. 현재 연구에서 주제가 깊이 나트륨 pentobarbital (25 MG / kg, IV)의 과다로 euthanized 케타민을 하이드로 클로라이드 (10 MG / kg, IM)과 진정했고 완전히 exsanguinated.This 미행 때까지 0.1 M PBS로 transcardially perfused 5 분 (~ 1리터)을 PBS에 4 % paraformaldehyde 용액에 의해. </li></ol> 2 부 : Stereotaxic는 차단 <ol><li> 이전 stereotaxic 프레임에 머리를 배치하는 Horsley-Clarke/interaural 비행기 제로의 좌표를 이동해야합니다. 이것은 귀 사이의 이론적 미드입니다. 이 비행기를 측정하려면, 귀 바 모두가 장치에 장착되어해야 다음 stereotaxic 조작하는 사람의 팔에 메스 블레이드를 배치하고 귀 막대 사이의 미드를 측정합니다. 이 연구의 필요에 따라 조직을 차단하는 위치를 결정하는 것이 중요합니다. 이것이 완료되면 두개골은 stereotaxic 장치로 고정을위한 준비를해야합니다. </li><li> stereotaxic 프레임에 머리를 배치하기 위해 아래턱은 뼈 rongeurs와 메스로 제거해야합니다. 또한 피부, 근육 및 결합 조직은 두개골을 노출하기 위해 제거되어야합니다. 결합 조직은 두개골에서 제거되면, calvaria을 (후두 골의 표 부분뿐만 ​​아니라 정수리, 광고 이마 뼈) 날 무시하여 뇌를 노출. calvaria의 현재 실험 부분에 모든 준비가 제거되었습니다. 귀 운하는 stereotaxic 프레임에 머리를 장소에 그대로 있어야 때문에 완전히 관자놀이 뼈를 제거하지 않도록주의하십시오. 나머지 두라의 문제가 노출 두뇌에서 제거되어야합니다. 두개골은 이제 stereotaxic 프레임에 배치 준비가되어 있습니다. </li><li> 한 stereotaxic 수술을하는 것이 동일한 방식으로, 눈, 미각, 그리고 머리가 안전하게 stereotaxic 장치에서 해결 그러한 귀 막대를 조정합니다. 예정된 (A / P) 후부 / 앞쪽에 좌표에 stereotaxic 조작하는 사람을 놓고 두뇌의 측면으로 조작하는 사람을 이동합니다. 천천히, 두뇌에 날개를 낮은 완전히 두뇌의 칼날을 마련, 다음 medially 이동하고 다시 칼날을 낮추십시오. 날개가 반대 북반구의 측면에 도달하기 전까지는이 두 단계를 반복합니다. 이것은 첫 번째 코로나 블록을 완료합니다. 전체 두뇌가 차단되었습니다 때까지 후속 코로나 블록 / P 축과 반복에서 조작하는 사람의 1cm를 이동하십시오. </li></ol> 파트 3 : 두개골에서 뇌를 제거 <ol><li> stereotaxic 프레임에서 머리를 제거하고 손바닥에 노출된 머리를 잡아. 가볍게 손바닥에 coddling 두뇌를 배치하고 이마 엽 (叶), 두개골에 뇌이 최소화 움직임에 걸쳐 새끼 손가락을 배치하여 두뇌를 확보하려고합니다. 두뇌의 pial 표면의 건조를 방지하기 위해, PBS의 일부가 뇌 전체 거즈를 dampened 놓으십시오. 단단히 척추 열의와 함께 두개골과 칩 거리에 남아있는 후두와 시간적 뼈의 머리를 잡아. 이것은 뇌의 바닥과 측면 측면을 제공합니다. 마지막으로 후각 구근에 대한 액세스를 허용하는 모든 나머지 이마 뼈와 코뼈가 골절을 제거합니다. 당신은 두개골의 기초를 제거할으로 두뇌가 약간 이동하고 이마 엽 (叶)이 fontal 뼈의 계단 모서리에 손상이 될 수 있기 때문에 마지막으로 전두엽 뼈를 제거하는 것이 중요합니다. 멀리 잘라 나머지 두라의 문제를 제거합니다. 신중하게, 두뇌의 전면을 리프트 두개골에서 두뇌 무료 두뇌에서 메스를 슬라이드. </li></ol> 4 부 : 측정 <ol><li> 이전 동결로 만들 수있는 유용한 측정은 여러 가지가있을 수 있습니다. 예를 들어, 캘리퍼스로 두뇌의 A / P의 축. 또한 특정 밀도는 다음 볼륨 변위 (표 1) 체중을 나누어 눈금 실린더에 물을 변위에 의해 음량을 측정, 뇌의 무게로 측정할 수 있습니다. </li></ol> 제 5 부 : 마침이 머리를 차단. <ol><li> 일반적으로 메스 블레이드가 두뇌에 넣었을 때 그것은 완전히 두뇌의 전체 지느러미 - 복부 범위를 뚫을 수있을 정도 오래되지 않습니다. 두뇌가 제거되고 총 측정 취득되면 조직 깔끔히 칼날을 가지고 두뇌의 복부 측면 (그림 2)을 통해 뇌를 차단 마칩니다. </li><li> 전에 블록을 냉동하기 위해서는 4 ˚ C까지 두뇌 싱크에서 유지 (10, 20, 그리고 30 %) 등급 PBS 버퍼 자당 솔루션의 조직을 cryoprotect 필요합니다. 그것은 일반적으로 20 % 2-3일 및 컨테이너의 하단에 싱크하는 블록에 대해 30 % 추가 3~5일 10 %에서 하룻밤 배양 걸립니다. 일의 일일 변화E 30 % 자당 솔루션을 권장합니다. </li></ol> 6 부 : 대표 결과 두뇌는 두개골에서 제거되었습니다 일단 만들 수 있습니다 총 형태학의 측정​​은 여러 가지가있을 수 있습니다. 이들은 A / P의 길이, 무게, 그리고 구체적인 밀도 (표 1)을 포함합니다. 우리는 일반적으로 1cm를 (그림 1) 측정 6-7 블록에 두뇌를 차단합니다. 각 조각은 다음 사진이다 (그림 2)와 추가 연구 필요에 따라 해부 또는 등급 자당 솔루션에 냉동 준비하실 수 있습니다. <table border="1"><tbody><tr><td> 제목 </td><td> A / P 길이 (㎜) </td><td> 무게 (g) </td><td> 워터 배기량 (ML) </td><td> 특정 밀도 (G / ML) </td></tr><tr><td> O2303 - 2 - 1 - 1 </td><td> 64.3 </td><td> 28.1 </td><td> 24 </td><td> 1.171 </td></tr><tr><td> O5180 - 1 </td><td> 71.3 </td><td> 38.7 </td><td> 34 </td><td> 1.138 </td></tr><tr><td> O2708 - 3 - 1 </td><td> 62.8 </td><td> 28.7 </td><td> 26 </td><td> 1.104 </td></tr><tr><td> O9184 - 4-2 </td><td> 65.3 </td><td> 29.5 </td><td> 24 </td><td> 1.229 </td></tr><tr><td> N459 - 1 - 14-2 </td><td> 68.2 </td><td> 31.6 </td><td> 26 </td><td> 1.215 </td></tr><tr><td> 평균 </td><td> 66.38 </td><td> 31.32 </td><td> 26.8 </td><td> 1.171 </td></tr><tr><td> STD DEV </td><td> 3.38 </td><td> 4.33 </td><td> 4.17 </td><td> 0.052 </td></tr></tbody></table> 5 월 올드 Vervets의 오른쪽 뇌를 표 1. 그로스 형태학의 측정 <img src="/files/ftp_upload/1259/1259fig1.jpg" alt="그림 1" /> <img src="/files/ftp_upload/1259/1259fig2.jpg" alt="그림 1.5" /> 그림 1. 두뇌를 차단에 사용되는 코로나 비행기에 대한 설계도. 이것은 externalized 성인 vervet 두뇌의 예입니다. 차단 절차의 예. 여기에 수직선은 일반적으로 각 반구에서 7 코로나 블록을 생산, 1cm의 간격입니다. <img src="/files/ftp_upload/1259/1259fig3.jpg" alt="그림 2" /> 그림 2. stereotaxic 공간에서 뇌 조직의 블록. 각 블록은 50μm에서 찍은 약 200 섹션을 얻을 것입니다. 코로나 비행기에 얇게 때 샘플링 방식과 피질을 통해 무려 1200 쪽이 넘는 방대한 부분은 소뇌의 품을 추가로 400-500 것입니다.

<ol>
	<li>Gallagher, M., Rapp, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+animal+models+to+study+the+effects+of+aging+on+cognition.+Annu+Rev+Psychol+48.">The use of animal models to study the effects of aging on cognition. Annu Rev Psychol 48.</a> , 339-370 (1997).</li><li>Clancy, B., Darlington, R., Finlay, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+developmental+time+across+mammalian+species.">Translating developmental time across mammalian species.</a> Neuroscience. 105, 7-17 (2001).</li><li>Nowakowski, R. 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The authors have nothing to disclose.

세인트 키츠 vervet (Chlorocebus aethiops sabeus)은 인간의 대뇌 피질과 subcortical하는 두뇌 발달의 패턴과 유사한 속도로 올드 월드 영장류이다. 이 종 불안 행동, 고혈압 8 hemispherectomy 9, 파킨슨병 10 Alzhemier 병 11과 알코올 남용 12에게 같은 복잡한 인간의 행동 장애 증상을 모델로 사용되고 있습니다. 최근,이 수종은 자연 태아 에탄올 노출의 neuroanatomical 효과...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Scalpel</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10003-12</td>
<td>&nbsp;</td>
<tr>
<td>Scalpel blades</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10011-00</td>
<td>&nbsp;</td>
<tr>
<td>Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14090-11</td>
<td>Any surgical scissors are sufficient</td>
</tr>
<tr>
<td>Rongeurs</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>16121-14</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11027-12</td>
<td>&nbsp;</td>
<tr>
<td>Filter paper</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-924-150</td>
<td>&nbsp;</td>
<tr>
<td>Stereotaxic Frame</td>
<td>&nbsp;</td>
<td>Kopf Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue slicing blade</td>
<td>&nbsp;</td>
<td>Thomas</td>
<td>&nbsp;</td>
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dissecting the non-human primate brain in stereotaxic space

비 인간 영장류의 사용은 인간의 발달 및 노화 과정에 대한 우리의 이해를위한 훌륭한 translational 모델을 제공합니다<sup&gt; 1-6</sup&gt;. 또한, 인간이 아닌 영장류의 사용은 최근 자연과 같은 알코올 남용과 같은 복잡한 정신 장애를 모델 수있는 기회를 여유있다<sup&gt; 7</sup&gt;. 여기서 우리는 vervet 원숭이의 코로나 비행기의 뇌는을 차단하는 기술을 (설명<em&gt; Chlorocebus aethiops sabeus</em&gt;) stereotaxic 공간에 그대로 두개골 인치 이 메서드는 현재 블록과 블록 사이의 과목과 최소화 부분 ​​부분 사이의 부분의 표준 평면을 제공합니다 설명했다. 코로나 비행기에서 조직의 블록을 Sectioning하면 관심있는 지역의 묘사를 용이하게합니다. 50μm에서 깔끔히 때 vervet 원숭이의 단일 반구 이상 1,200 섹션을 산출 때문에이 방법은 관리 규모의 블록을 제공합니다. 또한 1cm 단위로 머리를 차단하여, 그것은 cyroprotection에 대한 자당의 침투를 촉진하고 블록이 표준 그라 이오 스탯에 얇게 수 있습니다.

Watch this Scientific Journal Video about Dissecting the Non-human Primate Brain in Stereotaxic Space at JoVE.com

Dissecting the Non-human Primate Brain in Stereotaxic Space

아닌 인간 영장류의 두뇌의 정상적인 처리에 대한 우리의 이해를위한 중요한 translational 수종입니다. 영장류 두뇌의 해부 학적 조직은 인간의 정상 및 병리 학적 조건에 중요한 통찰력을 제공할 수 있습니다.

Stereotaxic 공간에 비 인간 메이트 브레인 해부

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

dissecting-the-non-human-primate-brain-in-stereotaxic-space

Research

JoVE Journal

Biology

10.0K 조회수.  University of Montreal. 아닌 인간 영장류의 두뇌의 정상적인 처리에 대한 우리의 이해를위한 중요한 translational 수종입니다. 영장류 두뇌의 해부 학적 조직은 인간의 정상 및 병리 학적 조건에 중요한 통찰력을 제공할 수 있습니다.

비디오: Dissecting the Non-human Primate Brain in Stereotaxic Space

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

설치류에 살아서 Stereotaxic 외과

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

연구

생물학

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

해부 및 C. 엘레간스 신경근육학 융티온에서 레코딩

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

두뇌에 게이트웨이 : 프리 메이트 &#39;눈&#39;을 분석 해 봅시다

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased stereology, the method accepted as state-of-the-art for quantification of biological objects in tissue sections2, generates reliable structural data for biological features in the mammalian brain3. The key components of the approach are unbiased (systematic-random) sampling of anatomically defined structures (reference spaces), combined with quantification of cell numbers and size, fiber and capillary lengths, surface areas, regional volumes and spatial distributions of biological objects within the reference space4. Among the advantages of these stereological approaches over previous methods is the avoidance of all known sources of systematic (non-random) error arising from faulty assumptions and non-verifiable models. This study documents a biological application of computerized stereology to estimate the total neuronal population in the frontal cortex of the vervet monkey brain (Chlorocebus aethiops sabeus), with assistance from two commercially available stereology programs, BioQuant Life Sciences and Stereologer (Figure 1). In addition to contrast and comparison of results from both the BioQuant and Stereologer systems, this study provides a detailed protocol for the Stereologer system.

The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given reference space1.

비 - 인간 메이트 두뇌의 편견 Stereology를 : 중요 한거지 아는

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased ...

Trajectory Data Analyses for Pedestrian Space-time Activity Study

It is well recognized that human movement in the spatial and temporal dimensions has direct influence on disease transmission1-3. An infectious disease typically spreads via contact between infected and susceptible individuals in their overlapped activity spaces. Therefore, daily mobility-activity information can be used as an indicator to measure exposures to risk factors of infection. However, a major difficulty and thus the reason for paucity of studies of infectious disease transmission at the micro scale arise from the lack of detailed individual mobility data. Previously in transportation and tourism research detailed space-time activity data often relied on the time-space diary technique, which requires subjects to actively record their activities in time and space. This is highly demanding for the participants and collaboration from the participants greatly affects the quality of data4.
Modern technologies such as GPS and mobile communications have made possible the automatic collection of trajectory data. The data collected, however, is not ideal for modeling human space-time activities, limited by the accuracies of existing devices. There is also no readily available tool for efficient processing of the data for human behavior study. We present here a suite of methods and an integrated ArcGIS desktop-based visual interface for the pre-processing and spatiotemporal analyses of trajectory data. We provide examples of how such processing may be used to model human space-time activities, especially with error-rich pedestrian trajectory data, that could be useful in public health studies such as infectious disease transmission modeling.
The procedure presented includes pre-processing, trajectory segmentation, activity space characterization, density estimation and visualization, and a few other exploratory analysis methods. Pre-processing is the cleaning of noisy raw trajectory data. We introduce an interactive visual pre-processing interface as well as an automatic module. Trajectory segmentation5 involves the identification of indoor and outdoor parts from pre-processed space-time tracks. Again, both interactive visual segmentation and automatic segmentation are supported. Segmented space-time tracks are then analyzed to derive characteristics of one's activity space such as activity radius etc. Density estimation and visualization are used to examine large amount of trajectory data to model hot spots and interactions. We demonstrate both density surface mapping6 and density volume rendering7. We also include a couple of other exploratory data analyses (EDA) and visualizations tools, such as Google Earth animation support and connection analysis. The suite of analytical as well as visual methods presented in this paper may be applied to any trajectory data for space-time activity studies.

A suite of spatiotemporal processing methods are presented to analyze human trajectory data, such as that collected using a GPS device, for the purpose of modeling pedestrian space-time activities.

보행자 공간 시간 활동 학습을위한 궤적 데이터 분석

It is well recognized that human movement in the  spatial and temporal dimensions has direct influence on disease transmission1-3.  An infectious disease ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

쥐의 측면 꼬리 정맥에서 혈액을 샘플링

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

Micropuncture of Bowman&#039;s Space in Mice Facilitated by 2 Photon Microscopy

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

보 먼의 공간 2 광자 현미경에 의해 촉진 생쥐에서의 Micropuncture

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

해 부와 영장류의 진화에 DiceCT 공개 그렇지 않으면 숨겨진 데이터

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

시투 혼성화에서 2색 단일 분자 형광을 사용하여 에샤리치아 대장균의 공간과 시간에서 mRNA 운동학을 프로빙

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force.
By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

초고속 포스 클램프 분광법을 사용한 공정 미오신의 기계적 효소 특성 해부

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...

Methods Article

Stereotaxic 공간에 비 인간 메이트 브레인 해부