资金来源为实验室示威是NIGMS授予：GM077119。我们感谢博士约瑟夫·海伍德和彼得·科贝特博士为他们的中西医结合和器官系统药理学支持短的：原因。我们也感谢汉娜·加弗女士的技术支持实验室演示。

在此之前任何动物的过程中，机构的动物护理和使用委员会（IACUC）必须批准该协议。该协议被批准为密歇根州立大学IACUC。 FITC的 - 菊粉解决方法1.实验前的准备<ol><li>暖20毫升盐水至70℃，并慢慢地搅拌在100毫克的FITC-菊粉（5毫克/ ml的FITC-菊粉），直到所有菊粉溶解。 </li><li>凉溶液至室温，并添加了牛血清白蛋白800毫克（40毫克/毫升BSA，冻干粉末，基本上球蛋白自由，低内毒素，≥98％纯度通过琼脂糖凝胶电泳）。 </li><li>过滤用滤纸（1级）的菊粉BSA解决方案。放置在20ml的注射器用注射器尖过滤器（0.2微米）过滤溶液，并盖上铝箔避光。 </li></ol> 2，麻醉和手术<ol><li>将大鼠在感应腔室填充有5％异氟烷诱导麻醉。记录BODŸ重量（250-350克），将老鼠的目的是保持整个实验37℃的体温加热的手术平台。轻轻固定鼠与实验室磁带在爪子的平台。维持麻醉用1-2％异氟醚与医疗级100％O 2在0.8-1.0升/分钟的气流速率。 </li><li>插入一锥形导管（血管内尖端外径，2.7F）插入股动脉血压和心脏速率监控和血液取样。 </li><li>插入一个导管（PE-50）插入股静脉菊糖输注。固定导管周围组织与5-O丝编织手术缝合6。 </li><li>附加的动脉导管以应变仪压力传感器。记录血压，并使用数据采集软件和显示在实时计算机屏幕上心脏速率。该技术表现出在细节上视频6。 </li><li>经耻骨上切口暴露膀胱。切一小孔在膀胱的前端与插入套管（PE-190）用热扩口顶端的膀胱尿液收集内部。固定套管用一个荷包缝合膀胱。 </li></ol> 3，尿液和血液采集<ol><li>放置的FITC-菊粉的注射器在注射器泵的流速设置每100g体重1毫升/小时（3毫升/小时的大鼠称重300克）。附加注射器向股静脉导管。启动菊粉输液，并允许1-2小时平衡期。保持注射器用铝箔纸覆盖避光。 </li><li>确定尿的流速是稳定和足够的试样分析（20微升/分钟）通过收集在预称重的收集瓶尿样，为期10分钟。重力测定尿量用数字刻度。足够的尿量为10分钟收集期为0.2毫升继续收集尿液样本，直到连续两个集合指示20微升/ m的尿流率在以上。 </li><li>预药物样本<ol><li>收集在一个20分钟期间的尿液样本。收集血液样品（0.5毫升），从动脉导管在尿液收集期间的中点。小心生理盐水彻底清除动脉导管含1 U肝素收集瓶采集血样前。使用收集瓶与体积标记，以促进为0.5ml的动脉血收集。 </li><li>冲洗动脉导管与肝素盐水（20单位/毫升）以清除血液（约0.1ml）中的导管。动脉导管的长度应尽可能地短，以限制冲洗所需肝素盐水的体积。 注：稀释血液样本产生GFR和Na和K的排泄分数的计算不准确</li><li>等待10分钟，并重复一第二预药物尿液和血液样品的集合。 </li></ol></li><li>下面的两个预毒​​品样品的收集，管理利尿DRU克，速尿（10毫克/千克），通过动脉导管。冲洗动脉导管与肝素生理盐水以清除药物的导管。照顾，以防止空气注入通过动脉导管。记录速尿注射的时间。 </li><li>用药后的样品：在每个以下3个时间点的，收集在10分钟收集时段尿样和血样（0.5毫升）的尿液收集期间的中点。 <ol><li>对于用药后的样品1 - 收集5分钟后，速尿。 </li><li>对于用药后的样品2 - 收集10分钟后，速尿。 </li><li>对于用药后的样品3 - 收集15分钟后，速尿。 </li></ol></li><li>毕竟样品已收集，安乐死大鼠按照制度程序通过开胸手术和摘取心脏。取出两个肾脏​​。解封装（去掉周围的膜），并吸干肾脏，去除多余血液。权衡肾脏。 </li></ol><ol><li>衡量所有尿样体积与重量分析的数字称重，并记录重量。 </li><li>离心的全血样品与台式离心机（1800×g离心）以分离血浆。转移血浆样本，以小标记的小瓶。 </li><li>与钠/钾分析器分析Na和K浓度的尿和血浆样品中。 </li><li>的FITC-菊粉的血浆和尿的测量<ol><li>稀预药物尿（从1：200至1：400），以及后药物尿（1:10）与HEPES缓冲液（500毫米，pH值7.4）。 </li><li>添加40微升HEPES缓冲标准品或样品和60微升的96孔板（每孔样品）中，并允许混合10分钟，同时用铝箔覆盖。 </li><li>产生的标准曲线的FITC-菊粉的浓度6.25，12.5，25，50，100，200，400微克/毫升（ 图1）。确定在样品和标准的FITC-菊粉荧光用酶标仪与前分别为485和538纳米，引文和发射波长。 </li><li>适合的标准荧光值到4放慢参数逻辑函数的回归分析。回归函数参数来计算血浆和尿液样品（ 见表1）中的FITC-菊粉浓度。 </li></ol></li></ol>结果5后的实验室分析：计算<ol><li>计算尿流量（UV;毫升/分钟）：尿体积收集（毫升）]÷[集合的时间（min）] </li><li>计算肾小球滤过率（GFR毫升/分钟）：尿菊糖浓度（微克/毫升）×紫外线（毫升/分钟）]÷[等离子体菊粉浓（微克/毫升）] </li><li>计算筛选钠负荷（微摩尔/分钟）：血浆钠浓度（微摩尔/毫升）×GFR（毫升/分钟） </li><li>计算钠排泄率（U 娜伏;微摩尔/分钟）：尿钠浓度（微摩尔/毫升）×UV（毫升/分钟） </li><li>计算分数排泄钠（FE娜;％）：[U 娜 V（微摩尔/分钟）]÷[滤过钠负荷（微摩尔/分钟）]×100 </li><li>过滤计算负荷钾（微摩尔/分钟）：血钾浓度（微摩尔/毫升）×GFR（毫升/分钟） </li><li>计算钾排泄率（Uķ伏;微摩尔/分钟）：尿钾浓度（微摩尔/毫升）×UV（毫升/分钟） </li><li>计算分数排泄钾（FE K表;％）：[UķV（微摩尔/分钟）]÷[过滤钾负荷（微摩尔/分钟）]×100 </li></ol>

<ol>
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J., Zewde, T., Shirley, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+anaesthesia+and+standard+clearance+procedures+on+renal+function+in+the+rat.">The effect of anaesthesia and standard clearance procedures on renal function in the rat.</a> Quarterly Journal of Experimental Physiology. 74, 805-812 (1989).</li><li>Rieg, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+measurement+of+glomerular+filtration+rate+in+conscious+mice.">A high-throughput method for measurement of glomerular filtration rate in conscious mice.</a> Journal of Visualized Experiments. (75), (2013).</li></ol>

作者宣称，他们有没有竞争的财务权益。该意见或此处包含的断言是作者的个人观点，并不能解释为官方或反映了陆军部和国防部的意见。

适当的标记物GFR测量必须符合四个标准：自由过滤在肾小球，可以绑定到血浆蛋白，并没有被吸收，也不分泌的肾单位。菊糖是一种果糖聚合物满足这些标准。其结果是，菊糖的肾清除被认为是金标准测定GFR 7。所表现出的技术代表确定菊粉8,9的恒定输注期间使用定时尿收集菊粉的肾清除率的传统方法。传统菊粉测量已用蒽酮法以产生由分光光度计10,11测得的比色定量测定...

The most important function of the kidney is the homeostatic regulation of extracellular water and electrolyte content. The kidneys closely regulate extracellular water, sodium (Na) and potassium (K) to maintain normal physiological levels. Disturbances in renal function can result in serious metabolic disorders which can be fatal. The basic renal process occurs in the nephron and begins with the filtration of plasma at the glomerulus and ends with the excretion of urine. Other processes that determine the final concentration of water, Na and K in the urine are secretion and reabsorption within the nephron. Measurements of glomerular filtration rate (GFR) and the fractional excretion of Na and K are critical in assessing renal function in health and disease. The reader is referred to previously published review articles and textbooks for a more thorough discussion of kidney function1-4. 
GFR can be measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron5. While this technique requires anesthesia, surgical preparation, and a terminal experiment, it is considered the gold standard of GFR measurement. Using inulin that is tagged with fluorescein-isothiocyanate (FITC), plasma and urine concentration of FITC-inulin can be easily measured in small volumes and used to calculate GFR during multiple time points of an experiment. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. 
The conceptual understanding of how to measure renal function can easily be demonstrated in a short lab designed to allow students to actively participate in some aspects of the experiment. This video depicts the pre-lab preparation, the renal function demonstration, and the post-lab evaluation of results. The surgical techniques necessary for making measurements of GFR are demonstrated in an anesthetized rat. In addition, example calculations for GFR, and the fractional excretion of Na and K are shown before and after administration of a diuretic drug.

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<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>5-0 Braided Silk Surgical Suture</td>
			<td>Surgical Specialties Corp</td>
			<td>SP1033</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay Plate, 96-Well</td>
			<td>Costar&#160;</td>
			<td>3922</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Chemical Co</td>
			<td>A2934-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>MicroFuge 18, 357160</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Sample Tubes</td>
			<td>Dot Scientific Inc.&#160;</td>
			<td>#711-FTG</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicators</td>
			<td>Solon Manufacturing Co</td>
			<td>56200</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition Software</td>
			<td>ADInstruments</td>
			<td>LabChart Pro 7.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Scale&#160;</td>
			<td>Denver Instrument</td>
			<td>APX-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-Inulin</td>
			<td>Sigma Chemical Co</td>
			<td>F3272-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze Sponges</td>
			<td>Covidien</td>
			<td>2146</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated Surgical Bed</td>
			<td>EZ-Anesthesia</td>
			<td>EZ-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagnet</td>
			<td>NDC 25021-402-10</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Chemical Co</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott Animal Health</td>
			<td>IsoFlo, 5260-04-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane Vaporizer</td>
			<td>EZ-Anesthesia</td>
			<td>EZ-190F</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Forceps</td>
			<td>Biomedical Research Instruments Inc.</td>
			<td>70-1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader - Fluoroskan</td>
			<td>ThermoScientific</td>
			<td>Ascent FL, 5210460</td>
			<td></td>
		</tr>
		<tr>
			<td>NOVA 5+ Sodium/Potassium Analyzer</td>
			<td>NOVA BioMedical</td>
			<td>14156</td>
			<td></td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holders with Scissors</td>
			<td>Fine Science Tools</td>
			<td>12002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-190 (for bladder catheter)</td>
			<td>BD Medical</td>
			<td>427435</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Transducer&#160;</td>
			<td>ADInstruments</td>
			<td>MLT1199</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex Culture Tubes</td>
			<td>Corning Inc.</td>
			<td>99445-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Femoral Tapered Artery Catheter</td>
			<td>Strategic Applications Inc.</td>
			<td>RFA-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Salix Furosemide 5%</td>
			<td>Intervet</td>
			<td>#34-478</td>
			<td></td>
		</tr>
		<tr>
			<td>Strabismus Scissors</td>
			<td>Fine Science Tools</td>
			<td>14075-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>91402-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Gloves</td>
			<td>Kimberly-Clark</td>
			<td>Sterling Nitrile Gloves</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Razel Scientific</td>
			<td>R99-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Forceps</td>
			<td>Fine Science Tools</td>
			<td>91121-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Scissors</td>
			<td>George Tiemann&#160; Co</td>
			<td>105-420</td>
			<td></td>
		</tr>
	</tbody>
</table>
5-0 Braided Silk Surgical Suture Surgical Specialties Corp SP1033 Assay Plate, 96-Well Costar&#160; 3922 Bovine Serum Albumin Sigma Chemical Co A2934-25G Centrifuge Beckman Coulter MicroFuge 18, 357160 Conical Sample Tubes Dot Scientific Inc.&#160; #711-FTG Cotton Tipped Applicators Solon Manufacturing Co 56200 Data Acquisition Software ADInstruments LabChart Pro 7.0 Digital Scale&#160; Denver Instrument APX-4001 FITC-Inulin Sigma Chemical Co F3272-1G Gauze Sponges Covidien 2146 Heated Surgical Bed EZ-Anesthesia EZ-212 Heparin Sagnet NDC 25021-402-10 HEPES Sigma Chemical Co H3375 Isoflurane Abbott Animal Health IsoFlo, 5260-04-05 Isoflurane Vaporizer EZ-Anesthesia EZ-190F Micro Dissecting Forceps Biomedical Research Instruments Inc. 70-1020 Microplate Reader - Fluoroskan ThermoScientific Ascent FL, 5210460 NOVA 5+ Sodium/Potassium Analyzer NOVA BioMedical 14156 Olsen-Hegar Needle Holders with Scissors Fine Science Tools 12002-12 PE-190 (for bladder catheter) BD Medical 427435 Pressure Transducer&#160; ADInstruments MLT1199 Pyrex Culture Tubes Corning Inc. 99445-12 Rat Femoral Tapered Artery Catheter Strategic Applications Inc. RFA-01 Salix Furosemide 5% Intervet #34-478 Strabismus Scissors Fine Science Tools 14075-11 Student Surgical Scissors Fine Science Tools 91402-12 Surgical Gloves Kimberly-Clark Sterling Nitrile Gloves Syringe pump Razel Scientific R99-E Tissue Forceps Fine Science Tools 91121-12 Tissue Scissors George Tiemann&#160; Co 105-420

physiology lab demonstration: glomerular filtration rate in a rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

在实验室演示中使用的利尿剂是呋塞米其非常迅速地抑制Na和K的重吸收过滤通过药物给药后几分钟内肾脏导致增加的Na，K和水排泄。由它的主要机制，速尿应该对GFR和Na和K的过滤载荷的影响最小，但会增加尿流Na和K，以及排泄分数在表的代表性的结果3可知 ，在麻醉的大鼠，对于GFR预药物值的平均值为3.2毫升/分钟，钠排泄为0.58微摩尔/分钟（经过滤的...

Watch this Scientific Journal Video about Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat at JoVE.com

生理学实验室演示：大鼠肾小球滤过率 |生物学 |视频

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

生理学实验室演示：肾小球滤过率在一个老鼠

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

physiology-lab-demonstration-glomerular-filtration-rate-in-a-rat

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17.2K 次观看.  U.S. Army Institute of Surgical Research. The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function. 

视频: Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Large Volume (20L+) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column. Two 20L samples can be filtered simultaneously using a single pump unit equipped with four rotating heads. Filtration is done in the field on extended trips, or immediately upon return for day trips. It is important to record the amount of water passing through each sterivex filter unit. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use. This procedure will take approximately 5 hours plus an additional hour for clean up.

Large Volume 20L+ Filtration of Coastal Seawater Samples

This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column.

大容量（20L）过滤沿海海水样品

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, ...

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Small Volume (1-3L) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents small volume (~1 L) filtration of microbial biomass from the water column. The protocol is an extension of the large volume sampling protocol described earlier, with one major difference: here, there is no pre-filtration step, so all size classes of biomass are collected down to the 0.22 &mu;m filter cut-off. Samples collected this way are ideal for nucleic acid analysis. The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use.

Small Volume 1-3L Filtration of Coastal Seawater Samples

This video documents small volume (~1 L) filtration of microbial biomass from the water column.

沿海海水样品的过滤体积小（1 - 3L）

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s ...

A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; O&#x2019;Keefe and Nadel, 1978; Mittelstaedt and Mittelstaedt, 1980; Landeau et al., 1984; Etienne, 1987; Gallistel, 1990;
Maurer and S&#233;guinot, 1995). Piloting requires the use of the relationships between relatively stable external (visual, olfactory, auditory) cues, whereas dead reckoning requires the integration of cues generated by self-movement. Animals obtain self-movement information from vestibular receptors, and possibly muscle and joint receptors, and efference copy of commands that generate movement. An animal may also use the flows of visual, auditory, and olfactory stimuli caused by its movements. Using a piloting strategy an animal can use geometrical calculations to determine directions and distances to places in its environment, whereas using an dead reckoning strategy it can integrate cues generated by its previous movements to return to a just left location. Dead reckoning is colloquially called "sense of direction" and "sense of distance."

Although there is considerable evidence that the hippocampus is involved in piloting (O&#x2019;Keefe and Nadel, 1978; O&#x2019;Keefe and Speakman, 1987), there is also evidence from behavioral (Whishaw et al., 1997; Whishaw and Maaswinkel, 1998; Maaswinkel and Whishaw, 1999), modeling (Samsonovich and McNaughton, 1997), and electrophysiological (O&#x2019;Mare et al., 1994; Sharp et al., 1995; Taube and Burton, 1995; Blair and Sharp, 1996; McNaughton et al., 1996; Wiener, 1996; Golob and Taube, 1997) studies that the hippocampal formation is involved in dead reckoning. The relative contribution of the hippocampus to the two forms of navigation is still uncertain, however. Ordinarily, it is difficult to be certain that an animal is using a piloting versus a dead reckoning strategy because animals are very flexible in their use of strategies and cues (Etienne et al., 1996; Dudchenko et al., 1997; Martin et al., 1997; Maaswinkel and Whishaw, 1999). The objective of the present video demonstrations was to solve the problem of cue specification in order to examine the relative contribution of the hippocampus in the use of these strategies. The rats were trained in a new task in which they followed linear or polygon scented trails to obtain a large food pellet hidden on an open field. Because rats have a proclivity to carry the food back to the refuge, accuracy and the cues used to return to the home base were dependent variables (Whishaw and Tomie, 1997). To force an animal to use a a dead reckoning strategy to reach its refuge with the food, the rats were tested when blindfolded or under infrared light, a spectral wavelength in which they cannot see, and in some experiments the scent trail was additionally removed once an animal reached the food. To examine the relative contribution of the hippocampus, fimbria&#x2013;fornix (FF) lesions, which disrupt information flow in the hippocampal formation (Bland, 1986), impair memory (Gaffan and Gaffan, 1991), and produce spatial deficits (Whishaw and Jarrard, 1995), were used.

In a piloting scent tracking task, the ability of the rats to return to a refuge with food using visual an odor trail or using dead reckoning in infrared light, the integrated record of previous movements, demonstrates that the hippocampus is necessary for dead reckoning.

录像示范保存鼠气味追踪，但经过伞 - 穹窿病变受损的惯性导航试验

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; ...

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

小龙虾开瓶器肌肉NMJ的历史观和生理学示范

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and 17&amp;#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

在大鼠和小鼠的卵巢切除和17β-雌二醇更换：一个可视化示范

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

高通量清醒小鼠肾小球滤过率的测定方法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

快速分离纯化线粒体移植用组织分离和鉴别过滤

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，<em&gt;白纹伊蚊</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

用小鼠乳腺肿瘤细胞教生物学的核心概念：一个简单的实验模块

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...