This project was funded by the DFG Excellence Cluster 171, the Center for Nanoscale Microscopy and Molecular Physiology of the Brain, the Bernstein Center for Computational Neuroscience and the ENC-Network, an Erasmus Mundus Joint Doctoral Program. The authors thank Stephan Junek, Mihai Alevra and Guobin Bao for providing MATLAB codes and custom-written programs for image evaluation and data analysis.

<ol>
	<li>Mamasuew, K., Breer, H., Fleischer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grueneberg+ganglion+neurons+respond+to+cool+ambient+temperatures.">Grueneberg ganglion neurons respond to cool ambient temperatures.</a> Eur J Neurosci. 28 (9), 1775-1785 (2008).</li><li>Brechb&#252;hl, J., Moine, F., Broillet, M. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Grueneberg+ganglion+neurons+share+molecular+and+functional+features+with+C.+elegans+amphid+neurons.">Mouse Grueneberg ganglion neurons share molecular and functional features with C. elegans amphid neurons.</a> Front. Behav. Neurosci. 7 (193), (2013).</li><li>Gaudin, A., Gascuel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+atlas+describing+the+ontogenic+evolution+of+the+primary+olfactory+projections+in+the+olfactory+bulb+of+Xenopus+laevis.">3D atlas describing the ontogenic evolution of the primary olfactory projections in the olfactory bulb of Xenopus laevis.</a> J Comp Neurol. 489 (4), 403-424 (2005).</li><li>Stosiek, C., Garaschuk, O., Holthoff, K., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+two-photon+calcium+imaging+of+neuronal+networks.">In vivo two-photon calcium imaging of neuronal networks.</a> Proc Natl Acad Sci U S A. 100 (12), 7319-7324 (2003).</li><li>Kludt, E., Okom, C., Brinkmann, A., Schild, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+temperature+with+odor+processing+in+the+olfactory+bulb.">Integrating temperature with odor processing in the olfactory bulb.</a> J Neurosci. 35 (20), 7892-7902 (2015).</li><li>Junek, S., Chen, T. W., Alevra, M., Schild, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+correlation+imaging:+visualizing+function+and+structure+of+neuronal+populations.">Activity correlation imaging: visualizing function and structure of neuronal populations.</a> Biophys J. 96 (9), 3801-3809 (2009).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis. , (1994).</li><li>Schild, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computer-controlled+device+for+the+application+of+odors+to+aquatic+animals.">A computer-controlled device for the application of odors to aquatic animals.</a> J Electrophysiol Tec. 12 (2), 71-79 (1985).</li><li>Bao, G., Schild, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+Accurate+Fitting+and+Filtering+of+Noisy+Exponentials+in+Legendre+Space.">Fast and Accurate Fitting and Filtering of Noisy Exponentials in Legendre Space.</a> PLoSONE. 9 (3), (2014).</li><li>Terasaki, M., Schmidek, A., Galbraith, J. A., Gallant, P. E., Reese, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+cytoskeletal+elements+in+the+squid+giant+axon.">Transport of cytoskeletal elements in the squid giant axon.</a> Proc Natl Acad Sci U S A. 92 (25), 11500-11503 (1995).</li><li>Haas, K., Sin, W. C., Javaherian, A., Li, Z., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Electroporationfor+Gene+Transfer+In+Vivo.">Single-Cell Electroporationfor Gene Transfer In Vivo.</a> Neuron. 29 (3), 583-591 (2001).</li><li>Haas, K., Jensen, K., Sin, W. C., Foa, L., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+electroporation+in+Xenopus+tadpoles+in+vivo-from+single+cells+to+the+entire+brain.">Targeted electroporation in Xenopus tadpoles in vivo-from single cells to the entire brain.</a> Differentiation. 70 (4-5), 148-154 (2002).</li><li>Paredes, R. M., Etzler, J. C., Watts, L. T., Lechleiter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+Calcium+Indicators.">Chemical Calcium Indicators.</a> Methods. 46 (3), 143-151 (2008).</li><li>Salome, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+random-access+scanning+in+two-photon+microscopy+using+acousto-optic+deflectors.">Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors.</a> J Neurosci Methods. 154 (1), 161-174 (2006).</li><li>Keller, P. J., Ahrens, M. B., Freeman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-sheet+imaging+for+systems+neuroscience.">Light-sheet imaging for systems neuroscience.</a> Nature Methods. 12 (1), 27-29 (2015).</li><li>Hjorth, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+silent+cells,+synchronization+and+modulatory+activity+in+developing+cellular+networks.">Detection of silent cells, synchronization and modulatory activity in developing cellular networks.</a> Dev Neurobiol. , (2015).</li></ol>

The authors have nothing to disclose.

本明細書に提示される方法は、 アフリカツメガエルの嗅球に記録温度処理を目指すオタマジャクシをアフリカツメガエル 。第一プロトコル汚れや嗅球における二次ニューロンとは、嗅覚系は、主に無傷のままであるサンプル調製物を提供します。従って、温度感受性γ-糸球体の活性化をモニターし、その化学感受性隣接糸球体と比較することができます。この糸球体のユニークな二国間の神経支配は、スペクトルの異なる色素で細胞エレクトロポレーションにより可視化されます。また、ボーラスローディングは嗅球内の大ボリュームにまたがる僧帽細胞の染色を可能にします。神経回路網の処理温度によって誘起される信号は、反復刺激アプリケーションでカルシウム測定を行うと、その後の活性相関イメージングを使用してデータを分析することによって明らかにされます。 プロトコルは、2つの洗練された染色proceを強調します<html>満足と再現性のある結果を達成するために慎重な操作と実践を必要とどちらもデュレス、。エレクトロポレーションの間に、動物の任意の損傷は、鼻孔に電極を配置する場合は特に、避けなければなりません。最適には、嗅上皮との接触は発生しません。動物は依然としてエレクトロポレーション手順の後に住んでいると、その回復時間が考慮されなければならないことに注意してください。染色は、使用される染料の種類に応じて発生することができるエレクトロポレーションの一巡後弱すぎるままである場合、その強度は、鼻孔内の色素濃度を増加させることによって高めることができます。デキストラン結合分子は（1-2 MM / 10日目の速度で）遅い軸索輸送と受動拡散を含むいくつかのメカニズムを介して輸送されているので、別の代替は、動物を犠牲にする前に、エレクトロポレーション後48時間を待つことです。あるいは、エレクトロポレーションは、回復の1日後に繰り返してもよいです。 jove_content &quot;&gt;ボーラスローディング色素の量は、僧帽細胞に入るので、重要なステップは、制御が困難であり、ピペットチップのサイズとアプリケーションの位置など様々なパラメータに依存する。共焦点蛍光顕微鏡下で手順の監視をするために有用であることがわかります染料適用の持続時間を調整するので、製剤にわたって同様の染色結果を生成する。また、以前にエレクトロオタマジャクシは、（γ-糸球体を含む）小クラスターの位置を特定することにより、色素のアプリケーションに最適な位置を決定するために使用されるべきである。最も測定中の重要なステップは、サンプルのシフト及び退色の両方を回避することである。シフト注意深く顕微鏡下リンガーフローを配置することによって回避することができる。関心領域の漂白を制限するように、測定時間が重要に低減されるべきです。 カルシウム感受性色素を持つボーラスローディング染色は非常に提供します健康な細胞は一般的に低カルシウムレベルを有し、したがって、弱い基底蛍光を示すので、限られたコントラスト。活動の相関イメージングを適用すると、同様のカルシウムシグナルとの活動やハイライトの構造に基づいて、コントラストを生成することにより、この制限を回避します。このポスト取込み分析法は、3Dボリューム内で選択された関心領域（基準トレース）、各個々のピクセルのカルシウム信号との間の相関係数を算出します。したがって、強く得られた結果は、基準トレースとして選択された活動パターンに依存しています。主な焦点は、僧帽細胞の神経支配のパターンを可視化する場合、自発的神経活動に由来する基準信号が好ましく、最も活発な僧帽細胞を選択することが最良の結果を生成します。僧帽細胞の化学療法または感熱ネットワークを明らかにするために、唯一のヒスチジンまたは冷たいリンゲルのいずれかへの応答を含む参照トレースを選択する必要があります。全体糸球体oの選択rを関心領域として僧帽細胞体は、常に2つの異なる刺激に応答する構造が互いの上に横たわっている場合は特に、明確な基準トレースを提供することはできません。このような場合には、関心領域として糸球体または細胞体のより小さな領域を選択することがしばしば有用です。 過去数十年間では、エレクトロポレーションは、単一または複数のセル11,12を染色するための効率的な方法として記載されています。ここでは、特に嗅覚受容体ニューロンを標識するために使用されます。デキストランコンジュゲート分子は、最高の効率を与え、非カルシウム感受性色素のために、選択の範囲が広く、一般的に蛍光顕微鏡13で使用される完全なスペクトルをカバーしています。まだ市販されている場合は、成功した嗅覚受容ニューロンにエレクトロポレーションされているカルシウム感受性色素は、カルシウムグリーンデキストランに限定された瞬間にある、とのFluo-4デキストラン。また、録音は主にsuperficiaをターゲット唯一の嗅球の腹面にL層、高速測定技術の侵入深さが限られているからです。二光子イメージングは​​、部分的にこの制限を克服するが、多くの場合、速度を欠き、さらに選択可能なカルシウム感受性色素の量を制限することができます。 ここで嗅球における温度誘導活性を測定するためのプロトコルを記載しています。脳の神経網は、温度の嗅覚処理に関与する複雑な細胞ネットワークを可視化するために、3次元ボリュームとしてスキャンされます。嗅球で温度誘導活性を測定することは非常に最近5を報告し、異なる技術を組み合わせ、特にカスタマイズされた手順を必要とされています。上記の技術の主要な資産は、細胞の数百人が嗅覚系のほとんどが無傷のまま準備に3次元で画像化されていることです。これらの利点は、高い染色技術への要求だけでなく、脳pを置きます償いとイメージング。例えば、セルのエレクトロポレーションおよびボーラスロードは、嗅上皮および球で細胞を大量にヒットし、したがって完全な携帯電話ネットワークの可視化を可能にします。また、代わりに遺伝的にコード化された蛍光体のボーラスロードを介した化学指標の配信は、種の潜在的に大規模なセットでの測定を可能にします。 AM色素とバスのインキュベーションのような他の選択肢は、主に無傷組織のわずか数百マイクロメートルを残し、深刻な嗅球を損傷スライスで働いています。 γ-糸球体の二国間の神経支配は無傷のままと録音は、このように、まだ手術システムに取り込まれることを比較して、我々のプロトコルで使用されるホールマウントの準備は、例えば、保証されます。最後に、撮影自体は、3Dボリュームの取得を可能とするライン照明顕微鏡によって行われます。ライン照明顕微鏡は、可能な限り最高の取得率6を提供する共焦点技術の一つです&lt;/ SUP&gt;嗅球の大部分をカバーするために必要です。遅い取得システムが使用されるが、記録されたボリュームのサイズを小さくしなければならないという欠点を有していてもよいです。近年、高速な画像取得のための他の方法が開発されており、代替14,15として使用することができます。それにもかかわらず、ライン照明顕微鏡は、十分な速度と分解能の両方を獲得するための最も簡単な方法の一つです。ここでは、適切な画像のセットアップを選択するためのガイドラインとして、いくつかの情報を、次の。カルシウムイメージングが厚い脳の調製物からの内で行われているので、セットアップはまともな共焦点を提供すべきであると目的は、1.0以上の開口数を持つ必要があります。基準点のために、ライン照明顕微鏡で撮影した記録は0.5-1軽やかユニットのピンホールサイズの標準的なレーザ走査型顕微鏡で撮影した画像に相当します。高速取得速度が望ましいです。厚さ20μmの体積は、少なくとも5リットルで覆われてエアーズ、100ミクロン×100ミクロンの側方視野と0.5μm以下のピクセルサイズは、スタックあたり1ヘルツの最低速度でスキャンする必要があります。共焦点性を低減することが数えた光子の量を増加させ、必要に応じてこのように高速取得を可能にするが、より焦点外の光を記録するという欠点を有することができます。このようなアプローチは、光学切片の厚さを増加させるので、それは実際にACI 6の適用後に別のz平面を介して樹状突起の追跡を容易にすることができます。 広く嗅球ネットワークに温度処理を研究するために必要なツールは、本明細書で提示されています。温度誘発性の活性は、カルシウム感受性色素との信号到着およびγ-糸球体から逸脱することの両方を介して第1および第2次ニューロンに記録されています。さらに、個々の僧帽細胞は、化学的および温度情報の両方を評価することができるプロセスれる程度。準備LEAので、無傷の嗅球VES、嗅覚処理における二国間の神経支配の役割をさらに研究することができます。手順は、嗅覚ネットワーク5を重ねて符号化されているかどうか、およびどのように熱電とchemoinformation明らかにするのに便利です。最後に、上記の技術は、嗅球における温度応答の研究に限定されないが、大きな三次元ボリュームにおける嗅覚系、特に細胞処理ネットワークのより一般的な評価のために適用することができます。ボーラスロードと活動の相関イメージングは、異なる脳のネットワーク16にそれらを適用すること、ニューロンの数十の活動を観察し、比較するための強力なツールです。

Over the last years, temperature sensitivity has no longer been described as a somesthetic sense only, but also as a physiological function relevant for the olfactory system. In rodents, the main olfactory bulb receives input from the Grueneberg ganglion (GG), an organ in the nasal cavity, consisting of thermosensitive neurons. GG neurons respond to cool temperatures1 as well as to chemical stimuli, and their chemosensitivity is modulated by temperature fluctuations2. These observations suggest that the olfactory bulb may integrate chemical and temperature information collected at the nose. In order to explore this hypothesis, we present here a set of experiments enabling the detection of temperature responses in the olfactory bulb of non-transgenic animals, using the Xenopus laevis larva as a model. The organization of the olfactory system in these animals closely resembles that of mammals. The olfactory receptor neurons of premetamorphic X. laevis terminate in tufts, and make synaptic contacts with the dendrites of second-order neurons, the mitral cells. Pre- and postsynaptic fibers intermingle and form skein-like neuropil structures called glomeruli3. The abundant synapses of the glomerular layer represent the first processing center of olfactory information. Mitral cells further integrate the sensory input and convey it to higher olfactory areas.
We have developed a protocol combining electroporation of olfactory receptor neurons (ORNs) with calcium-sensitive and non-sensitive dyes followed by bolus loading of the postsynaptic network of glomeruli and mitral cells. The staining by electroporation of two spectrally distinct dyes loaded in the nasal cavities serves to single out the &#947;-glomerulus3 through its bilateral innervation by ORNs from both olfactory epithelia. Thus, the location of the &#947;-glomerulus is identified prior to further measurements. Subsequently, bolus loading4 with Fluo-8 acetoxymethyl (Fluo-8 AM) is carried out in a volume comprising the &#947;-glomerulus. Imaging calcium changes with fast confocal microscopy allows the visualization of temperature responses in the 3D neuropil surrounding the &#947;-glomerulus, a unique temperature-sensitive glomerulus in this system5. Mitral cells innervating this specific structure can also be identified by their Ca2+ signals responsive to induced temperature drops. Next, activity correlation imaging6 uses the specific Ca2+ traces of these cells to reveal the dendritic morphology of thermosensitive mitral cells. Alternating repeated applications of cold Ringer solution and chemical odorants in one measurement can be used to visualize the mitral cell networks for odor and temperature processing surrounding the &#947;-glomerulus and identify potential overlaps. To unambiguously assign the responses to either the chemical or the temperature stimulus, we constantly monitor temperature at the olfactory epithelium.

<table border="0" cellpadding="0" cellspacing="0" width="969">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Reagents</td>
			<td style="width:191px;"></td>
			<td style="width:305px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sodium chloride</td>
			<td style="width:191px;">Merck Millipore</td>
			<td style="width:305px;">1064040500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Potassium chloride</td>
			<td style="width:191px;">Merck Millipore</td>
			<td style="width:305px;">1049360250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Calcium chloride dihydrate</td>
			<td style="width:191px;">Merck Millipore</td>
			<td style="width:305px;">1023820250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Magnesium chloride hexahydrate</td>
			<td style="width:191px;">Merck Millipore</td>
			<td style="width:305px;">1058330250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">D(+)-Glucose</td>
			<td style="width:191px;">Merck Millipore</td>
			<td style="width:305px;">1083371000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sodium pyruvate</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:305px;">P2256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HEPES</td>
			<td style="width:191px;">Merck Millipore</td>
			<td style="width:305px;">1101100250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Calcium Green 10 kDa Dextran</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:305px;">C-3713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Alexa Fluor 647</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:305px;">D-22914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Alexa Fluor 546</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:305px;">D-22911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fluo-8 AM</td>
			<td style="width:191px;">TEFlabs</td>
			<td>203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MK571</td>
			<td>Alexis Biochemicals</td>
			<td style="width:305px;">340-021-M005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS-222</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:305px;">E10521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pluronic acid F-127</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:305px;">P2443</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">L-Histidine monohydrochloride monohydrate</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td>53370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td style="width:191px;">Merck Millipore</td>
			<td>1029522500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Equipment</td>
			<td style="width:191px;"></td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Electronic pipette</td>
			<td>BrandTech</td>
			<td style="width:305px;">HandyStep Electronic Repeating Pipette</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NiCr-Ni thermocouple</td>
			<td>Greisinger Elektronik</td>
			<td style="width:305px;">GTF 300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette puller</td>
			<td>Narishige</td>
			<td style="width:305px;">Model PC-10</td>
			<td>two-step puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Funnel applicator</td>
			<td>(Custom-made)</td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Line-illumination microscope</td>
			<td>(Custom-made)</td>
			<td style="width:305px;"></td>
			<td>otherwise, a commercially available spinning disk microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Objective W Plan-Apochromat 63x/1.0&#160;</td>
			<td>Zeiss</td>
			<td style="width:305px;">441470-9900-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Objective W Plan-Apochromat 40x/1.0 DIC</td>
			<td>Zeiss</td>
			<td style="width:305px;">441452-9900-000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Software</td>
			<td style="width:191px;"></td>
			<td style="width:305px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">MATLAB</td>
			<td>The MathWorks</td>
			<td style="width:305px;"></td>
			<td>from R2010b upwards</td>
		</tr>
	</tbody>
</table>

recording temperature-induced neuronal activity through monitoring calcium changes in the olfactory bulb of xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

嗅覚受容ニューロン（ORN類）のエレクトロポレーションは、アクティブな軸索輸送を介して、順行性標識のためのデキストランの分子と結合したアレクサフルオロ色素やカルシウム指標で達成されました。前者染料は電球の糸球体層で分岐感覚ニューロンおよびその軸索末端の明るい染色を提供するが、後者は、これらの細胞における神経活動の測定を可能にする（ 図1及び2）。まず、感熱γ-糸球体とその神経支配パターンの位置が左右の嗅覚上皮、（ 図1A）にアレクサフルオロ647デキストランおよびアレクサフルオロ546デキストランをエレクトロポレーションにより可視化しました。手続き後の二十四時間、鼻孔内のORN類は、2嗅神経と両半球における糸球体は、蛍光顕微鏡下で見ることができました。異なる糸球体クラスタはidentifiaましたそれぞれの立場によってBLE、γ-糸球体（ 図1B）を含む、特に小規模なクラスタ。反対側の嗅覚繊維の小さな数は前交連を渡り、同側γ-糸球体（ 図2A）に終了し、反対側の嗅球を駆け抜けました。 γ-糸球体のシナプス前繊維のカルシウム応答を記録するために、カルシウムグリーンデキストランは同じ手順に従って、ORN類に電気穿孔しました。負の温度ジャンプを氷冷リンゲル液の制御放出を介して鼻孔に誘導された（0-1°C）。 γ-糸球体を含む3Dボリュームは、高速のライン照明の共焦点顕微鏡で画像化しました。 -1°CΔTsがはΔF/ FのCa 2+トレースで可逆的なピークとして、γ-糸球体とその求心性神経で冷たい応答を誘発するのに十分に認識した（ 図2B &lt;/ strong&gt;の、C）。 さらに、実験の手順は、そのramifying樹状終末を経由してγ-糸球体に接続僧帽細胞に低温で誘導される活性を測定するために行きました。これらのシナプス後の繊維とその周辺の神経網を効果的に、カルシウム感受性色素のFluo-8 AMのボーラス負荷によって染色されたアレクサ647デキストランは、ORN類（ 図3A、B）にエレクトロポレーションされていた数日後に行われました。二回以下のパラダイムに従って僧帽細胞は、フルオ8 AMを充填した、と嗅上皮を刺激した：冷たいリンガー、ヒスチジン（10μM）と室温リンガーは、続いて適用されます。二つの基準トレースは、記録されたボリューム内の関心領域からの1つだけヒスチジン温度低下、他の1つに排他的に応答を採取しました。アクティビティ相関イメージング（ACI）6は、選択した基準に基づいて計算しました。高コントラストで温度やヒスチジン応答性のCa 2+シグナル（ 図3C、D）のいずれかに対応するシナプス後のネットワークの樹状形態を可視化するためのトレース。最後に、感熱性及び化学感受性マップを色分けし、温度および化学的情報は嗅覚二次ニューロン（ 図3E）に個々の糸球体から搬送される様子を示し、互いの上に重ね。共有嗅覚ネットワークの情報の両方のタイプの統合及び処理の説明については、Kludt ら5参照<img alt="図1" src="/files/ftp_upload/54108/54108fig1.jpg" /> 図1：ORNのエレクトロポレーションおよびボーラスロードの概要 （A）は、Xの両方の鼻腔内の嗅覚受容ニューロン。 ツメガエル幼生は、Alexa色素またはcalciuでエレクトロポレーションしましたデキストラン分子に結合されたメートル感受性色素。蛍光指示薬は、端末軸索樹枝状分岐まで順行性輸送されました。 24時間エレクトロポレーション後、両半球における糸球体層は、蛍光染色を示しました。 （B）1半球における嗅球の細胞組織の模式図。 、内側の小、中、横クラスター：糸球体層は、クラスタ内の電球にまたがります。嗅覚情報は、糸球体における興奮性シナプスを介して、僧帽細胞に受容ニューロンから転送されます。糸球体周辺細胞と顆粒細胞は、嗅覚処理と符号化を調節する抑制性ニューロンです。 γ-糸球体（シアン）を容易に同側（赤）と反対側（オレンジ）嗅覚繊維が合併小さな神経網として同定されました。 （C）ボーラスの負荷は、主に僧帽細胞とそのdendriからなるシナプス後神経網を染色するためにγ-糸球体付近で達成されました糸球体層にかなりの分岐チック木。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54108/54108fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/54108/54108fig2.jpg" /> 図2：ORN類のエレクトロポレーションは、構造的および機能的接続性を明らかにし 、同側（緑）によるγ-糸球体の（A）二国間の神経支配と反対側（赤）嗅覚受容ニューロン（ORN類）。 =50μmのスケールバー。 （B）カルシウム感受性色素カルシウムグリーンデキストランとのエレクトロポレーション後の嗅球。 intermedial（IMC）、内側（MC）と小さなクラスタ（SC）が表示されます。画像は、厚さ100μmの測定体積の最大投影です。 =50μmのスケールバー。 （C）小さなクラスタのクローズアップビュー。記録容量（12μm）があります最大投影で表現。画像は、オーバーレイとしてグレーと色分けされたΔF/ Fマップ内の基底蛍光レベルを示しています。冷リンゲル液での刺激に対する最大応答がプロットされています。隣接する二つの糸球体は黙秘しながら、γ-糸球体は強く反応しました。挿入図は、関心の示された領域に対応するγ-糸球体のためのΔF/ Fトレースを示しています。青いバーは、刺激のアプリケーションを表します。スケールバー=20μmである。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54108/54108fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/54108/54108fig3.jpg" /> 図3：小規模なクラスタで終端ORN類のボーラスロードとACIは、感熱および化学感受性のネットワークを解離 （A）軸索は、非カルチでエレクトロポレーションにより染色しましたええと感受性色素アレクサ647デキストラン。点線は、γ-糸球体の概要を説明します。カルシウム感受性色素のFluo-8 AMとボーラスロード後の第2の測定チャネルにおける（B）（A）と同じ領域の画像。一部の僧帽細胞の細胞体が見られたが、これに対しては限定的でした。矢印以下、2つの応答トレースが活性相関画像（ACI）のために使用された、プロットしました。ブルー、トレース以下の赤と黒のバーは、それぞれの冷リンガー、ヒスチジン（10μM）と室温リンガーなどのネガティブコントロールのアプリケーションの開始を示しています。 2のCa 2+トレースが測定した体積の関心の異なる領域から採取しました。 （C）（B）中の微量のACI結果冷たいリンガーに主に応答する領域を強調表示します。 （D）ヒスチジンに主に応答する（B）ハイライト領域における微量のACI結果。 2 ACIマップの（E）オーバーレイ。 hに応答する僧帽細胞istidineと神経支配糸球体（赤）は感熱僧帽細胞およびγ-糸球体（シアン）から容易に識別可能でした。この図のすべての画像は28ミクロン厚のボリュームの最大強度の投影です。スケールバー=20μmである。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54108/54108fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis at JoVE.com

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

の嗅球にカルシウム変化をモニタリングを通じて温度によって誘発される神経活動を記録<em&gt;アフリカツメガエル</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

recording-temperature-induced-neuronal-activity-through-monitoring

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

7.2K ビュー.  Georg-August-Universität Göttingen. Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose. 

ビデオ: Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

光反射率と自家蛍光信号を用いたマウス嗅球におけるイメージングの臭い誘発活動

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

神経科学

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

軟体​​動物細胞内カルシウム濃度とシナプス伝達効率での変更のモニタ<em&gt;アメフラシ</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

からの神経外植文化<em&gt;アフリカツメガエル</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

の解離、文化、および分析<em&gt;アフリカツメガエル</em&gt;胚性網膜組織

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

ローカスの神経活動を特徴づけるためにマルチユニット記録方法（<em&gt; Schistocercaアメリカーナ</em&gt;）嗅覚回路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

グルタミン酸とGABAの高速マイクロイオン導入：シナプス統合を調査するために便利なツール

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

テスト視機能のための簡単​​な行動アッセイ<em&gt;アフリカツメガエル</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

無傷の鋤鼻器官と副嗅球のex vivoでの準備

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

覚醒マウスにおける神経活性物質のMicroiontophoreticアプリケーションと組み合わせることで神経活動の細胞外記録

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

嗅覚神経軸索分子識別コードの視覚化のための嗅球の四重免疫染色

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...