私たちは、聖研究室のメンバーに感謝したいと思います。この作品は、聴覚障害とコミュニケーション障害研究所（R01 DC005964まで）とNSF IGERT 0548890（HAA）によってサポートされていました。 動物実験は米国の動物福祉の行為と健康ガイドラインの国立研究所に準拠して行われたとワシントン大学の動物実験委員会によって承認された。

パート1。電気的記録計<ol><li>我々は、窒化ケイ素で絶縁さ10μmの平坦な窒化チタン電極を有する平面多電極アレイを（ALAサイエンティフィックインスツルメンツ）を使用します。 60電極が30μmの中心間距離と5X6のグリッドで構成される二つのフィールドに配置されています。 （その他の構成も可能です。直径の記録面は10μmで電極が単一ニューロンの活動を分離するという点で大きな電極よりも優れています。） </li><li>電気信号は、MEA 1060アンプ（ALAサイエンティフィックインスツルメンツ）を使用して（1000倍の増幅）増幅されています。信号は、データ集録カード（ナショナルインスツルメンツ）と、カスタムデータ集録ソフトウェア（1つにも商用ソフトウェアを購入できます）を使用して10 kHzで取得されています。 </li><li>組織はMEAの培養チューブのリングの内側にぴったり合うカスタムインサートに接続されているナイロンメッシュ（53ミクロン）、によってMEAを下に開催されます。 </li></ol>パート2。液体供給計装<ol><li>組織は、我々はリンゲル溶液（115 mM塩化ナトリウム、5mMの塩化カリウム、2mMの塩化カルシウム、2mMの塩化マグネシウム、25mMの重炭酸ナトリウム、10mMのHEPES、10 mMのD -（+を供給することにより保証する連続的な灌流を必要とし、 HPLCポンプから） - グルコース）。刺激はHPLCバルブに注射を行うとフロースルーとループとの間で切り替えることにより表示されます。 HPLCポンプとインジェクションのロボットのシリンジポンプの両方は、リンゲル液に付属しています。 </li><li>リンゲル液は、実験全体を通して42℃温水槽（ISOTEMP 105）° Cに保持され、連続して95％O 2 / 5％CO 2でバブリングする。グルコースのないリンゲルの別のフラスコにシリンジポンプをバックアップ用に準備されています。リンゲル溶液を3mL /分の速度で307 HPLCポンプ（ギルソン）を使用して組織に配信されます。典型的な5〜6時間の実験では、各リンゲル溶液1Lで十分です。 </li><li>実験全体を通して、組織は継続的に37℃のリンゲル液で灌流される。ヒータープローブが直接組織上方に位置している。 </li><li>刺激はギルソン215液体ハンドラロボットアームを使って配信されます。刺激はゴムキャップ付きガラスバイアルに保存され、一定の流量と圧力が維持されている間ギルソンソフトウェアの制御下で、ロボットアームは、灌流ラインに刺激を提供しています。 </li><li> HPLCバルブ（ループが従事/従事していない）の状態は、神経活動との同期を確実にするために付加的なチャネルとして、買収のコンピュータによって記録される電気の電圧で表されます。 </li></ol>第3部。 MEAと刺激の配信デバイスの準備<ol><li>解剖を実行する前に、室温で少なくとも5分間dH2OでBSAとMEAの井戸を埋める。 </li><li>ウォームバブルリンガー溶液を用いてMEAをすすぎ、氷上にMEAを置きます。 </li><li>プライム液体ハンドラとHPLCは、製造元の指示に従ってポンプ。 </li></ol>パート4。 VNOの解剖<ol><li>解剖を行う前に、氷の上で泡立てリンゲル液と場所で2 60x15ミリメートルのペトリ皿を埋める。また、氷の上でリンゲル溶液と場所と同じサイズの2 Sylgardコーティングされたシャーレを埋める。解剖時に使用するためのウォームバブリングリンゲルの追加の50mLのを冷やします。 </li><li>マウスは頚椎脱臼に続くCO 2窒息を経由して犠牲にされています。 </li><li>頭の後ろに、顎のラインと平行に走る、口の側面から二国間の切開を加えます。首の後ろに沿って2カットに参加して、マウスに接続されている下顎を残して、頭部の背の部分を削除してください。 </li><li>細かいハサミを使用して、組織や骨から組織を分離するために上顎骨の両側の間にカット。 </li><li>鼻腔を公開するために口蓋の組織を剥がし、第3位鉗子を使用する。 </li><li>小さなはさみや骨から鋤鼻カプセルを解放する間、および上位2前歯の両側にカットメスを、使用する。 </li><li> ＃3鉗子を使用して、氷の上でペトリ皿の平らな骨と場所によって鋤鼻カプセルを拾う。浸漬を通ってステップは3分未満で実行する必要があります。 </li><li>反射光で照らされた、実体顕微鏡下で皿を置きます。 ＃3鉗子を使用して、一度VNOから骨嚢を取り除く。 、離れてVNOから骨剥離する尾方端と他方の把持VNOを傷つけないように配慮して骨のカプセルを安定させるために片手で行ってください。 </li><li>骨が除去されると、SylgardコーティングしたシャーレにVNOを転送する。他のVNOの残りの皿を使用してください。透過光に照明を切り替えます。 </li><li>マイクロシザーまたは＃5ピンセットを使用して、VNOの端に沿って切断することにより血管からVNOを分離。この時点では、なります完全に血管から分離された神経のシートを持っている。 </li><li>内部（樹）面を上にVNOを置きます。 ＃3鉗子を使用して、組織の隅にSylgardにVNOを固定。クランプで開催されたテフロンコーティングされたかみそりの刃の小さな断片を用いて基底膜からの神経上皮ピール。浅い角度（&lt;30 °皿の表面から）でブレードを保持することが最善の方法です。 </li><li>ガラスピペットを用いて、皿からMEAへの神経上皮の一部を譲渡する。上樹側で電極のフィールドの上にVNOを置きます。ピペットと過​​剰リンゲル液を取り外します。場所にVNOを保持し、冷却したリンゲル液で格納する配列のウェルにメッシュティッシュホルダーを置きます。 </li></ol>第5部。録画<ol><li> MEA 1060アンプと位置を直接組織上のヒータープローブでMEAを置きます。 </li><li>適切なヒータープローブの配置を確認するには、リンゲル溶液が2秒間50 mMリン酸カリウムを（等モルナトリウムの代わりに）を含むと刺激する。カリウム溶液への神経応答の待ち時間を最小限に抑えるために位置を調整。 </li><li>組織が順応できるように、記録前の1時間に45分間加熱したリンゲル液で組織を表面かん流する。 </li><li>電気信号は、カスタムレコーディングソフトウェアを使ってディスクに保存されています。 </li></ol>第6部。データ解析<ol><li>オフラインデータ解析は、生の電極の録音を使用して行うことができます。例えば、我々は、特定のリガンドに反応して記録された細胞の発火率の変化を測定する。結果のデータセットが大きく、さまざまな質問に対処するために使用することができます。プログラミングや解析言語（C言語、MATLAB、R、Pythonの、または類似の）の熟知が必要です。 </li></ol>パート7。代表的な結果この準備は、通常、データ（60の電極から記録の6時間）を大量にもたらして、6時間から記録するのに十分安定しています。 <img src="/files/ftp_upload/1845/1845fig1tmb.jpg" alt="図1" border="0" /> 図1：配列の半分にわたって同時活動のスナップショットが表示されます。時間ウィンドウは、400ミリ秒に広がっています。してください<a href="https://www.jove.com/files/ftp_upload/1845/1845fig1.jpg">ここをクリックして</a>図1の拡大バージョンを参照すること。 <img src="/files/ftp_upload/1845/1845fig2tmb.jpg" alt="図2" border="0" /> 図2典型的な実験では、我々は10秒間、関心のある特定の化合物で刺激する。このトレースは、100倍希釈した雌マウスの尿中への応答の例です。してください<a href="https://www.jove.com/files/ftp_upload/1845/1845fig2.jpg">ここをクリックして</a>図2の拡大バージョンを参照すること。

<ol>
	<li>Dulac, C., Torelo, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+detection+of+pheromone+signals+in+mammals:+from+genes+to+behaviour.">Molecular detection of pheromone signals in mammals: from genes to behaviour.</a> Nat Rev Neurosci. 4, 551-562 (2003).</li><li>Holy, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+Vomeronasal+neurons+to+natural+stimuli.">Responses of Vomeronasal neurons to natural stimuli.</a> Science. 289, 1569-1572 (2000).</li><li>Nodari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfated+steroids+as+natural+ligands+of+mouse+pheromone-sensing+neurons.">Sulfated steroids as natural ligands of mouse pheromone-sensing neurons.</a> J Neurosci. 28, 6407-6418 (2008).</li></ol>

多電極アレイの録音は、私たちの神経細胞の大集団の同時活動を監視する能力を許可する。これは特に、VNOの感覚系の特性をプロービングするための便利なツールです。主嗅覚系とは異なり、VNOは、液相中で刺激を検出します。さらに、VNOは異種の組織です。おそらく別の応答プロファイルとマウス1の受容体の約300の異なる種類があります。 MEAの録音は、私たちは確実にニューロン...

multielectrode array recordings of the vomeronasal epithelium

神経回路を理解することの方法は同時に多数のニューロンから記録する必要があります。の<em&gt; in vitroで</em&gt;研究、一方、現在利用可能な技術は、平面多電極アレイ（MEA）の記録である。ここでは受容体の数百種類を表現する感覚ニューロンの多様な人口を介したフェロモンと社会的手がかりの発見に重要な役割を果たしているマウスの鋤鼻器官（VNO）を、勉強するMEAの使用を文書化する。化学的刺激を提供するロボット液体ハンドラとMEAの記録を組み合わせることで、ニューロンの大規模かつ多様な人口の感覚応答を記録することができます。準備は、私たちは、マウスからVNOの無傷の上皮を除去し、数時間のための神経細胞の電気的活動を監視しながら、化学物質または潜在的リガンドのバッテリーを刺激することができます。したがって、この手法は、リガンド活性を評価するだけでなく、受容体の神経細胞の性質を探求するための有用な方法として機能します。私たちは、鋤鼻上皮、MEAの記録、および化学的刺激を準備するために必要なテクニックを紹介。

Watch this Scientific Journal Video about Multielectrode Array Recordings of the Vomeronasal Epithelium at JoVE.com

Multielectrode Array Recordings of the Vomeronasal Epithelium

多電極アレイ（MEA）の録音は、神経細胞の大規模な集団の電気的活動を研究するための方法を提供する。同時に組織を刺激しながらここで、我々はマウス鋤鼻上皮からのレコードにMEAの準備の詳細を示す。

鋤鼻上皮の多電極アレイ録音

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

multielectrode-array-recordings-of-the-vomeronasal-epithelium

Research

JoVE Journal

Neuroscience

12.1K ビュー.  Washington University School of Medicine. 多電極アレイ（MEA）の録音は、神経細胞の大規模な集団の電気的活動を研究するための方法を提供する。同時に組織を刺激しながらここで、我々はマウス鋤鼻上皮からのレコードにMEAの準備の詳細を示す。

ビデオ: Multielectrode Array Recordings of the Vomeronasal Epithelium

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

シングルユニット in vivoで</emラットの視交叉から>録音

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

神経科学

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

の巨大線維経路からの電気生理学的記録 D.キイロ</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

網膜色素上皮の全マウントするためのマウスの眼の解剖

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官培養における神経雪崩のマルチ電極アレイの録音

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

マウス鋤鼻急性組織スライス標本のイメージングフェロモンセンシング

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

遺伝的にコードされたカルシウムセンサーを発現する鋤鼻器のスライス標本のイメージング神経細胞応答

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

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 ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

マウス鋤鼻器官の急性組織スライスで定義された細胞集団の中で、深さ生理学的解析

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

全細胞電圧クランプ記録のための電気生理学的方法<em&gt;ショウジョウバエ</em&gt;感光体

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

ショウジョウバエ幼虫神経筋接合部のシナプス電流の焦点 Macropatch 録音

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...