GM077119：ラボのデモンストレーションのための資金源は、NIGMS助成金でした。我々は、統合および臓器システム薬理学におけるショートクーゼの彼らのサポートのために博士ジョセフ·R·ヘイウッドとピーター·コベットに感謝します。また、実験室でのデモの彼女の技術支援のためにさんハンナGarverに感謝します。

任意の動物手順、制度的動物のケアと使用委員会（IACUC）の前には、プロトコルを承認する必要があります。このプロトコルは、ミシガン州立大学IACUCによって承認されました。 FITC-イヌリン溶液の1前のラボの準備<ol><li>全てのイヌリンが溶解するまでゆっくりと加温20〜70℃の生理食塩水のミリリットルとは、FITC-イヌリン（5 mg / mlのFITC-イヌリン）を100mgを加えて混ぜます。 </li><li>室温に冷却液と（基本的に、アガロースゲル電気泳動によりフリー、低エンドトキシン、≥98％純度グロブリン、40 mg / mlのBSA、凍結乾燥粉末）、ウシ血清アルブミン800mgのを追加します。 </li><li>濾紙（グレード1）とイヌリンBSA溶液をフィルタリングします。シリンジチップフィルター（0.2μm）で、20 mlシリンジで濾過した溶液を置き、光から保護するためにホイルでカバーしています。 </li></ol> 2.麻酔と手術<ol><li>麻酔を誘導するために、5％のイソフルランで満たされた導入室にラットを置きます。レコードBODY重量（250〜350グラム）と、実験を通して37℃の体温を維持するように設計された加熱された外科用プラットフォーム上にラットを置きます。ゆっくり足の上に実験用テープでプラットフォームにラットを固定します。 0.8〜1.0リットル/分の空気流量で医療グレード100％のO 2を1〜2％イソフルランで麻酔を維持します。 </li><li>血圧および心拍数のモニタリング、及び血液サンプリングのために大腿動脈にカテーテルテーパー（血管内先端OD、2.7F）を挿入します。 </li><li>イヌリン注入のための大腿静脈にカテーテル（PE-50）を挿入します。 5-O編み絹縫合糸6と周囲の組織にカテーテルを固定します。 </li><li>ひずみゲージ圧力変換器に動脈カテーテルを取り付けます。リアルタイムでコンピュータ画面上のデータ取得ソフトウェア、ディスプレイを使用して記録する血圧と心拍数。この技術は、映像6に詳細に示されています。 </li><li>恥骨上切開により膀胱を公開します。小さなをカット膀胱の先端に穴や熱でカニューレ（PE-190）を挿入するには、尿採取のために、膀胱の内側に先端をフレア。巾着縫合で膀胱にカニューレを固定します。 </li></ol> 3.尿や血液コレクション<ol><li> 1ミリリットル/体重（300グラムの重さのラットについて3ミリリットル/時）の100グラムあたりの時間の流速が設定されたシリンジポンプにFITC-イヌリンの注射器を置きます。大腿静脈カテーテルに注射器を取り付けます。イヌリンの注入を開始し、1〜2時間の平衡期間を可能にします。光から保護するために箔で覆われた注射器を保管してください。 </li><li>尿流量が安定しており、10分の期間、予め秤量した回収バイアル中の尿サンプルを採取することにより、試料分析（20μL/分）のために適切であるかどうかを確認します。デジタルスケールで重量測定により尿量を決定します。 10分の回収期間のための適切な尿量は、0.2 mlです。二つの連続コレクションが20μL/ Mの尿流量を示すまでの尿サンプルを収集し続けますで以上。 </li><li>プレドラッグサンプル<ol><li> 20分の期間中に尿サンプルを採取します。尿収集期間の中間点において動脈カテーテルから血液サンプル（0.5ml）に収集します。 1 Uのヘパリンを含むコレクションバイアルに血液サンプルを収集する前に生理食塩水を完全に透明に動脈カテーテルに注意してください。動脈血の0.5ミリリットルの収集を容易にするために、ボリュームマーキングコレクションバイアルを使用します。 </li><li>血液（約0.1ミリリットル）のカテーテルをクリアするためにヘパリン生理食塩水（20 U / ml）で動脈カテーテルをフラッシュします。動脈カテーテルの長さは、洗い流すために必要なヘパリン - 生理食塩水の量​​を制限することが可能な限り短くすべきです。 注：希釈された血液サンプルはGFRおよびNaとKの分数排泄の不正確な計算を生成します</li><li> 10分待ってから、第2のプリ薬物の尿と血液サンプルの収集を繰り返します。 </li></ol></li><li> 2つの事前薬物サンプルの収集に続いて、利尿DRUの管理G、フロセミド（10mg / kgの）、動脈カテーテルを介し。薬物のカテーテルをクリアするためにヘパリン化生理食塩水で動脈カテーテルをフラッシュします。動脈カテーテルを介して空気を注入しないように注意してください。フロセミド注射の時間を記録します。 </li><li>尿収集期間の中間点で下3時間点の各々で、10分間の収集期間中に尿サンプルを採取し、血液試料（0.5mL）を：ポスト薬剤サンプル。 <ol><li>後薬物サンプル1について - フロセミド後5分を収集します。 </li><li>後薬物のサンプル2について - フロセミド後10分を集めます。 </li><li>後薬物サンプル3について - フロセミド後に15分を集めます。 </li></ol></li><li>全てのサンプルが収集された後、開胸し、心臓を除去することによって機関の手順に従ってラット安楽死させます。両方の腎臓を削除します。逆カプセル化（周囲の膜を除去）し、過剰の血液を除去するために腎臓をブロット。腎臓を計量。 </li></ol><ol><li>デジタルスケールで重量測定により、すべての尿サンプル量を測定し、記録の重み。 </li><li>テーブルトップ遠心機（1,800 XG）で遠心分離し、全血サンプルは、血漿を分離しました。小さなラベル付きバイアルに血漿サンプルを転送します。 </li><li>ナトリウム/カリウムアナライザーで尿および血漿試料中のNa、Kの濃度を分析します。 </li><li>血漿および尿中のFITC-イヌリンの測定<ol><li> HEPES緩衝液（500mMの、pH7.4）で、ポスト薬物尿（1:10）（400：1：200〜1）から予め薬物尿を希釈します。 </li><li> 96ウェルプレート（ウェルごとに1つのサンプル）にHEPES緩衝液の標準またはサンプルを60μlの40μlを添加して、アルミホイルで覆いながら10分間混合することができます。 </li><li>の濃度についてのFITC-イヌリンの標準曲線を生成6.25、12.5、25、50、100、200、400 / mlの（ 図1）。元にマイクロプレートリーダーを使用してサンプルと標準でFITC-イヌリン蛍光を決定それぞれ485および538 nmでの引用及び発光波長。 </li><li> 4-PARAMTERロジスティック関数の回帰分析に標準の蛍光値を取り付けます。回帰関数のパラメータは、血漿および尿サンプル（ 表1）にFITC-イヌリン濃度を計算するために使用されます。 </li></ol></li></ol>結果5.ポスト​​ラボ分析：計算<ol><li> 【尿量収集（ミリリットル）]÷[収集の時間（分）]：;尿流量（ml /分UV）を計算します</li><li>糸球体濾過率を計算（GFR、ml /分）：尿イヌリン濃度（/ mlの）UV（ml /分）をX]÷[血漿イヌリン濃濃度（μg/ ​​ml）を] </li><li>計算フィルタリングさナトリウム負荷（μモル/分）：血漿ナトリウム濃度（モル/ ml）をGFR（ml /分）をxは</li><li>尿中ナトリウム濃度（モル/ ml）をUV（ml /分）をX：;ナトリウム排泄率（μモル/分U のNa V）を計算します</li><li>ナトリウム排泄分画（;％FE NA）を計算します：[U のNa V（マイクロモル/分）]÷[濾過ナトリウム負荷（マイクロモル/分）]×100 </li><li>計算フィルタリングさカリウム負荷（μモル/分）：血漿カリウム濃度（モル/ ml）をGFR（ml /分）をxは</li><li>尿中カリウム濃度（モル/ ml）をUV（ml /分）をX：;カリウム排泄率（μモル/分U K V）を計算します</li><li>計算カリウムの分別排泄（FE K;％）：[U K V（マイクロモル/分）]÷[濾過カリウムロード（マイクロモル/分）]×100 </li></ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+filtration+rate+determinations+in+conscious+type+II+diabetic+mice.">Glomerular filtration rate determinations in conscious type II diabetic mice.</a> Americam Journal of Physiology. 300 (3), F618-F625 (2011).</li><li>Rosen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+anaesthesia+and+surgery+on+renal+hemodynamics.">Effects of anaesthesia and surgery on renal hemodynamics.</a> British Journal of Anesthesiology. 44, 252-258 (1972).</li><li>Cousins, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+the+kidney.">Anesthesia and the kidney.</a> Anaesthesia and intensive care. 11 (4), 292-320 (1983).</li><li>Walter, S. 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>

著者は、彼らが競合する金融利害関係がないことを宣言します。ここに含まれる意見やアサーションは、作者の個人ビューであり、公式として、または陸軍省や国防総省の見解を反映するものとして解釈されるべきではありません。

血漿タンパク質に結合していないこと、自由に糸球体で濾過され、吸収されたことも、ネフロンに分泌されることもない：GFR測定のための適切なマーカーは、4つの基準を満たす必要があります。イヌリンはこれらの基準を満たすフルクトースポリマーです。その結果、イヌリンの腎クリアランスはGFR 7を測定するためのゴールドスタンダードと考えられています。実証技術はイヌリ?...

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.

ラボのデモに使用される利尿剤は非常に迅速に薬物投与の数分以内に増加したのNa、K、その結果、腎臓、および水の排泄によってフィルタリングNaおよびKの再吸収を阻害するフロセミドました。その主要なメカニズムによって、フロセミドは、GFRおよびNa、Kのフィルタ処理負荷の最小の効果を持っている必要がありますが、尿の流れを増加し、NaおよびKの分数排泄麻酔し...

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

生理学実験室のデモンストレーション: ラットの糸球体濾過率 |生物学 |ビデオ

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.

生理学ラボデモンストレーション：ラットにおける糸球体濾過率

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 ビュー.  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. 

ビデオ: 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.

沿岸海水サンプルの大容量（20L +）ろ過

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

生物学

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.

沿岸海水試料の少量（1 - 3L）のろ過

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.

プリザーブドラットの采 - 脳弓の病変後に香りのトラッキングが障害推測航法による操縦のビデオデモ

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.

ザリガニ開閉筋肉のNMJにおいて歴史的なビューと生理学のデモンストレーション

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.

ラットとマウスにおける卵巣摘出および17β-エストラジオールの交換：ビジュアルデモ

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.

意識マウスにおける糸球体濾過率の測定のためのハイスループット法

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.

組織解離と差動濾過により移植のためにミトコンドリアの迅速な単離および精製

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.

アジアのタイガーモスキートにおける日長休眠のRNA-seqの分析のための実験とバイオインフォマティクス·プロトコル、<em&gt;ヒトスジシマカ</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.

コア生物学の概念を教えるために、マウス乳腺腫瘍細胞を用いた：簡単なラボ·モジュール

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.

ラット腸間膜に微小血管透過性の調節を調査するために、微小灌流法

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