，F30 DC011673（GFH：NINDS，NIH）和得克萨斯大学西南的启动资金（JPM）：这项研究是由R00 DC011780（NINDS，NIH JPM）的支持。

所有实验均按照批准的得克萨斯大学西南机构动物护理和使用委员会的协议，并被选为以尽量减少应激，不适，疼痛经历的实验动物。 1，解剖商会自定义夹层室，小，薄塑料板所需要的最好的结果（ 图1）。提前尝试此协议的建造或取得该室。 2，解剖解决方案<ol><li>准备1升的标准人工脑脊液（ACSF）中的步骤3.22-5.4使用。加氯化钠125毫米，氯化钾2.5毫米，氯化钙2为2mM，MgCl 2的1毫米，的NaHCO 3的25mM，将NaH 2 PO 4 1.25毫米，肌醇3毫米，丙酮酸钠2mM的，抗坏血酸钠0.4毫米，和葡萄糖25 1mM至1升的无热原的水。 </li><li>准备初步解剖学联（200毫升）在步骤3.3-3.22使用。采取200毫升标准学联和提高氯化镁浓度由9毫米加入0.366克的氯化镁六水。 </li><li>制备标准林格氏液通过导管用于进行刺激的VNO中含有氯化钠115毫米，氯化钾5毫米， 氯化钙，2毫米， 氯化镁 ，2毫米， 碳酸氢钠 25毫米，HEPES 10 mM的步骤4.11-5.4，葡萄糖10毫米。 </li><li>泡的0.8 L标准学联和0.5升林格与95％O 2，5％CO 2气体在37℃水浴中至少15分钟，直到使用。 </li><li>气泡的200毫升初始剥离的aCSF入95％O 2，5％CO 2的气体在冰上15-20分钟，直到达到4℃。 </li></ol> 3。主要解剖<ol><li>准备解剖区域。将3张35毫米培养皿，含氧学联，斩首剪刀，弯解剖剪刀，直解剖剪刀，镊子阿德森，一个＃11的头皮埃尔刀片和手柄，在冰刀片。 </li><li>后的含氧脑脊液冷却至4℃，深深地麻醉动物中使用2-5毫升Isothesia（99.9％异氟烷）一干燥室中。执行的尾巴和脚捏测试，以确保动物深度麻醉。 </li><li>一旦深麻醉确认，杀头的动物，并立即将头在培养皿中充满了冷剥离学联。注：浸入冷剥离学联组织要爽保持它在整个过程在不使用时。 </li><li>用弯剪卸下下纵横（腹侧咽，下颌部和舌）。 </li><li>使用阿德森镊子deglove（剥离）的浅表皮肤和颅骨肌肉附着。从剥离尾头皮，直到喙附件的唯一网站是在门牙。使用弯曲的剪刀切割组织的附着点。用抓钳将其取下眼睛和配制而成其拉出。 Ñ耳朵的皮肤脱套过程结束，通过使用阿德森镊子从上颚骨游离肌肉附着避免对精致的鼻软骨过大的力量。注：从直剪头骨的尾部方面拆下头皮。 </li><li>与直剪，剪下来的尾头骨吻端的中线，直到剪技巧是几个毫米到额骨（只是之前的鼻腔鼻窦）。 </li><li>删除使用阿德森镊子头骨的顶骨和额骨。注：额骨饮片往往会保持连接到鼻骨尾椎到鼻腔鼻窦。移除这些作品小心注意不要接触嗅球。 </li><li>使用手术刀通过额叶2毫米尾椎到鼻窦，使冠状切口，然后取出大脑尾的切割。保证手术刀接触腹头骨的骨性轮廓的尖端彻底断绝后路组织。注意：这使除去大部分的脑的不上施加机械应力侧嗅束或嗅球。 </li><li>取出头骨腹直剪刀尾外露方面。离开鼻窦和剩余的神经组织。 </li><li>采用直剪刀去除颧弓和面部肌肉的解剖右侧颅骨</li><li>把鼻子比（腹朝上），暴露上膛。 </li><li>切除多余的口感立即向尾侧门牙。通过插入在平行于口腔顶部硬腭手术刀刀片的尖端做到这一点，然后走向门牙刀片。 </li><li>两手抓，阿德森钳腭的释放（喙）边缘拉尾侧取出。 </li><li>在头骨的解剖左侧，使用手术刀刀片延长腭孔尾端，从靠近磨牙的小孔开始。通过将手术刀BLA的尖端达到这个去到附近的臼齿虚空和旋转手腕。注意：要特别小心，只使用刀片的尖端 - 一个深的伤口可能会损坏室间隔组织。 </li><li>使用手术刀刀片的顶端，切离腭孔穿过门齿，开始延髓的解剖左犁鼻器。使用多个评分削减。通过插入刀片太深不要损坏室间隔组织和犁鼻神经。 </li><li>所以转背头骨朝上的组织，然后定向直刀片垂直（平行于中线）在准备的尾部边缘。 </li><li>在组织的腹缘，触摸刀片立即向中线的左侧的最前沿。轻轻摇动叶片和沿左腭孔（切割步骤3.14引入区域）移动。 </li><li>在组织的背缘，放置在剃刀的刀刃立即向中线的左边（小于1毫米）。 </li><li>保存叶片平行于中线，以朝向组织的前压力摇动刀片缓慢。请注意在筛板阻力。通过这种阻力突破后立即停止。注：在这一点上，右半球是从左脑松动。剩下的唯一连接是沿背吻前方筛板的半球之间。 </li><li>抓住他们靠近尾部两侧用戴手套的手指，轻轻横向和向前旋转它们分开的半球。注：此步骤是实验性的变异的来源，尤其是在小鼠斜井或脆口鼻（ 例如 ，年纪较大的老鼠）。另外，当试图以旋转半球彼此远离，如果强遇到阻力。比分背侧吻（前向筛板），以削弱结缔组织。虽然这样做，采取极端谨慎不深插入的手术刀，它可以破坏的间隔TISSUE和犁鼻神经。 </li><li>适用的组织胶量小（50-100微升），以木板和右半球的横向边缘轻轻地放置到使用阿德森镊子胶水。使用纸巾，以防止过早聚合胶和松散粘附在板准备的侧面除去多余的水分。注：应用数滴于样品冷冻解剖学联的加速胶水聚合后组织被放置到胶水。这确保了组织保持紧密地保持到板条。 </li><li>将在二级清扫室的中间真空润滑脂少量（3-5毫米直径2毫米深），然后将板侧面的组织坚决反对油脂相反。马上补上剥离室中冷藏解剖学联，转移到精细解剖区域，并开始灌注氧合标准学联。定向木板，以便嗅球是直接在新鲜含氧学联标准的流。 </li></ol> 4，二次解剖<ol><li>从用细镊子准备移除任何可见对侧（左）嗅球或皮层组织。捏在一起的细镊子（形成半钝点），然后将其放置在背部和组织的尾侧中线附近。轻轻沿中线运行捏钳，剥离组织远离右半球在缓慢前向运动。删除所有对侧组织，包括对侧嗅球组织。要格外注意不要刺伤通过组织，这可能会导致损坏AOB或犁鼻神经。 </li><li>观察该白色硬膜沿嗅球的背侧/内侧表面。通过抓两对细镊子的硬脑膜最白的部分撕裂这个硬膜沿嗅球的背/尾边和拉他们远离该点。硬脑膜是紧密缠绕在嗅球，并可以很容易地通过本身的组织切片，而它被去除。避免损坏嗅球和AOB本身的内侧表面上。注：如果硬脑膜抵抗在这一点上很容易流泪，引入一小截细春尖解剖剪刀，方便的分​​离。 </li><li>慢慢地，小心地剥开额叶皮层用细镊子不损坏底层的嗅球。观察显示为半透明的净紧接尾于AOB血管的集合。用细镊子取出该网以方便在电生理实验电极渗透。抓住网用钳子，因为它从AOB分离过程中额叶皮层收缩和拉尾侧和内侧取下，小心不要触碰AOB。一旦额叶皮层已经分开嗅球，通过用钳子腹侧和后向的AOB切割移除它。注：取前极端与护理去除净，因为如果它是做了大致的肾小球层可以被损坏。 </li><li>在暴露对侧鼻腔，用细镊子取出所有剩余的对侧间隔组织（包括血管脆弱黄片）。掌握组织在尾/背区（靠近室间隔骨），然后拉/解除对前侧的组织。注意：对侧间隔组织可以在半切步骤（步骤3.20）被无意中删除。在这种情况下，观察到白色的，无血管鼻中隔软骨和略透明的，血管室间隔骨。如果是这样的话，在步骤4.4，可以跳过。 </li><li>小心通过运行一个单点的鼻中隔软骨和犁鼻“翼”（一块薄薄的骨组织沿两个VNOS背边缘之间运行的精细镊子取出对侧VNO。 <ol><li>下面由软骨翼支队，将镊子尖腹到中线附近对侧VNO。在几个地点沿对侧VNO的喙/尾轴松开重复此步骤，使其能够抓住钳和起重远离被删除。 </li></ol></li><li>准备通过使切口在腹部/尾缘（它缩小到一个白色的股骨头缺血性带状运行沿室间隔骨的腹缘）用细镊子取出鼻中隔软骨。 </li><li>按捏细镊子在一起，使一个半钝点取出鼻中隔软骨。识别步骤4.6获得了晋级，后面插入（横向）的镊子夹住该切割，然后慢慢抬起软骨远离室间隔组织和继续吻侧。注意：不要破坏底层的室间隔组织。由于软骨被抬起，观察室间隔组织弹力底层表因粘连松动的软骨。捏细镊子沿松一此站点周期性的，温柔的，且稳定的运动dhesion可以极大地方便了两种结构的成功分离。 </li><li>使用一对＃3镊子与弯曲尖端取出鼻中隔骨。在一个角度接近室间隔骨几乎平行于室间隔骨的平面。插入室间隔组织和中隔骨之间的弧形齿。抓住与骨钳，轻轻移动骨来回，直到筛板附近出现裂缝。注：在某些情况下，隔骨会抵制破坏筛板附近。在这种情况下，用细镊子沿背/腹轴轻轻比分室间隔骨只是前筛板，然后重复步骤4.8。 </li><li>通过与一对细镊子的手捏在入口处，并与另一对细镊子之吻侧拉动取出白色软骨只是喙的VNO。 </li><li>附聚酰亚胺套管以快速灌注装置并启动的林格氏溶液（0.1-0.3毫升/分钟），通过在聚酰亚胺套管的流动。注：测量流速与在线，小体积流量的仪表。利用计算机控制的气动激励开关器件的刺激时可减少流量的变化。比较神经反应与反应的模拟刺激（控制林格氏液）测试激励，以确保响应是刺激特定的。 </li><li>取向平行于VNO入口插管。当套管被令人满意地平行于VNO入口，手表作为出口的压力使所述VNO到“膨胀”，导致血管撤离。立即插入插管进入VNO，保持套管恒定的角度，使之保持平行于VNO开口。注意：它可以帮助保持插管与一对对塑料板材细镊子，并使用另一对细镊子对角套管的末端微微向上。如果无意中置于所述套管的VNO后面，流出也会造成VNO血管撤离，导致印象进行适当的插管。适当的位置可以通过在林格氏溶液掺入染料的确认。当套管被妥善安置，染料应该只通过VNO入口出口。此外，人们可以通过确保套管是容易看到通过所述VNO的半透明的内侧面确认正确放置。 </li><li>将塑料板慢慢地，直到AOB是在标准学联的直接流量，小心不要从VNO打跑套管。解剖完整，生理功能的评估可以立即开始。第5步简要介绍了一种方法作出这样的评估。 </li></ol> 5。评估<ol><li>穿透该AOB的表面与附着于细胞外的放大器和示波器2-4兆欧硼硅酸盐玻璃微电极。 </li><li>慢慢地从推进微电极，以100-200微米的深度面以50微米/分钟的速率。注：在此组织深度，人会遇到由微电极电压尖锐，短暂（约1-2毫秒）挠度例证偶尔自发动作电位波形。 </li><li>当遇到一个动作电位波形，停止前进的微电极。 </li><li>观察和/或记录动作电位的速率，同时改变从控制林格氏刺激交付解决方案，以VSN活动的一个已知的激活剂（ 如含50毫米氯化钾林格氏液）。如果夹层是成功的，在动作电位的速率或局部场电位的刺激依赖的变化可以观察到。注意：并非所有的AOB神经元将每一个刺激响应增加射速（包括含50毫米氯化钾林格氏液）。在射速刺激依赖性下降也预示功能连接和一个成功的清扫。在两个或更多个神经元中遇到的事件是反应迟钝VNO刺激，或者如果没有动作电位的多个电极穿透之后即可观察到，这表明一个不成功的解剖。如果是这样的情况下，一个新的夹层可以在步骤3中被启动。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+anatomy+of+individual+rapidly+adapting+low-threshold+mechanoreceptors+innervating+the+'hairy'+skin+of+newborn+mice:+early+maturation+of+hair+follicle+afferents.">Central anatomy of individual rapidly adapting low-threshold mechanoreceptors innervating the 'hairy' skin of newborn mice: early maturation of hair follicle afferents.</a> J Comp Neurol. 436, 304-323 (2001).</li><li>Llinas, R., Muhlethaler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+electrophysiological+study+of+the+in+vitro,+perfused+brain+stem-cerebellum+of+adult+guinea-pig.">An electrophysiological study of the in vitro, perfused brain stem-cerebellum of adult guinea-pig.</a> The Journal of physiology. 404, 215-240 (1988).</li><li>Riviere, S., Challet, L., Fluegge, D., Spehr, M., Rodriguez, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formyl+peptide+receptor-like+proteins+are+a+novel+family+of+vomeronasal+chemosensors.">Formyl peptide receptor-like proteins are a novel family of vomeronasal chemosensors.</a> Nature. 459, 574-577 (2009).</li><li>Meeks, J. P., 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=An+ex+vivo+preparation+of+the+intact+mouse+vomeronasal+organ+and+accessory+olfactory+bulb.">An ex vivo preparation of the intact mouse vomeronasal organ and accessory olfactory bulb.</a> J Neurosci Methods. 177, 440-447 (2009).</li><li>Leinders-Zufall, T., 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=Ultrasensitive+pheromone+detection+by+mammalian+vomeronasal+neurons.">Ultrasensitive pheromone detection by mammalian vomeronasal neurons.</a> Nature. 405, 792-796 (2000).</li><li>Kato, H. K., Chu, M. W., Isaacson, J. S., Komiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+sensory+representations+in+the+olfactory+bulb:+modulation+by+wakefulness+and+experience.">Dynamic sensory representations in the olfactory bulb: modulation by wakefulness and experience.</a> 76, 962-975 (2012).</li><li>Meeks, J. P., Arnson, H. A., 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=Representation+and+transformation+of+sensory+information+in+the+mouse+accessory+olfactory+system.">Representation and transformation of sensory information in the mouse accessory olfactory system.</a> Nature. 13, 723-730 (2010).</li></ol>

作者没有利益冲突显著披露。

该VNO-AOB 体外在本协议中所述的制备是一个有用的替代体内5-7和急性现场片17实验AOB函数来麻醉。不像急性AOB切片实验，这也暴露了电路元件的电和光学记录，该制剂保留所有感觉传入和国内AOB连接。尽管这也可以在体内方法所述的麻醉剂，麻醉剂的存在不一定会改变神经功能，即兴奋性/抑制性的平衡，这是在这个和其他的嗅觉电路18，用于信息处理的...

在哺乳动物大脑感觉处理通常跨越多个往复连接的神经元回路，其中每个抽取特定的特征从感觉输入。在感觉通路，早期信息处理是正常的认知和行为至关重要。在附件嗅觉系统（AOS）的嗅球（AOB）是感官外围连接到该决定的荷尔蒙平衡1,2，3的侵略和觉醒4下游结构的主要神经回路。因此，该电路内的信息处理是密切相关的变化，动物的行为。 在嗅球位于小鼠和大鼠在主嗅球（MOB）下方的致密的背侧/尾/后一方面，血管化鼻腔鼻窦。该AOB接收传入的神经支配来自驻留在犁鼻器（VNO）的犁鼻周围感觉神经元（的VSN）的轴突，一个SMaL在口鼻前正上方的软腭升盲端管。这些轴突穿越隔组织在鼻腔的内侧边界的微妙片。使用麻醉小鼠5-7或可自由探索动物8一些研究探讨AOB的神经反应，以AOS的气味（如小鼠尿液） 在体内的来源。 在涉及（一）气管切开术，以确保麻醉过深，防止液体刺激5-7的心愿，（二）刺激交感型颈椎节6或犁鼻器5,7的直接插管引进非挥发性气味体内研究英勇麻醉以及（c）开颅手术带或不带额叶消融，以允许电极前进到AOB 6。清醒/行为研究微驱动8-10参与手术植入。总之，这些实验范式是强大的，但非常困难，往往需要麻醉。 体外 ）外取得了一些成功11-15。因为VNO和AOB之间的连接保持同侧，且由于中线室间隔组织可以在单个半球暴露于含氧的灌流，我们试图开发这样的单半球体外方法来分离这些结构，同时保持其功能性连接。最近，我们成功地实现了这一目标16。该制剂保持VNO和AOB活着和功能上连接至少4两 - 6小时，因为无论是轴突（沿中线软室间隔组织）和AOB较浅&lt;600μm的特征都可以访问的灌流充氧人工脑脊液（学联）。这VNO-AOB 体外制备允许引入控制刺激到VNO通过薄插管，并直接的视觉进入小AOB进行有针对性的电极放置和/或现场荧光显微镜。这种方法是有利的，如果一个人渴望学习这些电路在没有麻醉剂。因为这种方法切断离心连接，它不能很好地适合于查询到AOB功能的离心调制。该VNO-AOB 体外的准备是很难学，但一旦实现产生一个可靠的平台，探讨电路组织，信息处理和神经可塑性在这个强大的感测电路。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Straight Scissors</td>
			<td>Fine Science Tools</td>
			<td>14002-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors-Straight</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
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			<td>Fine Scissors-Curved</td>
			<td>Fine Science Tools</td>
			<td>14061-10</td>
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			<td>Adson Forceps</td>
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			<td>#3 Scalpel Handle</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
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			<td>#11 Scalpel Blades</td>
			<td>Fisher Scientific</td>
			<td>3120030</td>
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			<td>Straight Carbon Steel Razor Blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
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			<td>35 mm Petri Dish</td>
			<td>Fisher Scientific</td>
			<td>08-772-21</td>
			<td></td>
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		<tr>
			<td>Dissection Chamber</td>
			<td>Custom&#160;</td>
			<td>N/A</td>
			<td>See Fig. 1</td>
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			<td>Delrin plastic plank 0.6 cm x 1.5 cm x 0.1 cm</td>
			<td>Custom&#160;</td>
			<td>N/A</td>
			<td></td>
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			<td>Dow Corning Silicon Vacuum grease</td>
			<td>Fisher Scientific</td>
			<td>146355D</td>
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			<td>#5 Forceps, Student</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
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			<td>#5 Forceps, Biologie Tip</td>
			<td>Fine Science Tools</td>
			<td>11295-10</td>
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			<td>#5 Forceps, Student</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
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			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
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			<td>0.0045&#34; Polyimide Tubing</td>
			<td>A-M Systems</td>
			<td>823400</td>
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			<td>1/16&#34; Male Luer</td>
			<td>Cole-Parmer</td>
			<td>EW-45505-00</td>
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			<td>1/16&#34; Tubing</td>
			<td>Fisher Scientific</td>
			<td>14-171-129</td>
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			<td>Two ton epoxy</td>
			<td>Grainger</td>
			<td>5E157</td>
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			<td>ValveBank Pressurized Perfusion Kit</td>
			<td>AutoMate Scientific</td>
			<td>09-16</td>
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			<td>ValveLink digital/manual controller</td>
			<td>AutoMate Scientific</td>
			<td>01-18</td>
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			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
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			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
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			<td>CaCl2 dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
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			<td>MgCl2 hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
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			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
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			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
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			<td>myo-inositol</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
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			<td>Na-pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
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		<tr>
			<td>Na-ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
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		<tr>
			<td>HEPES buffer</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
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			<td>glucose</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
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	</tbody>
</table>

ex vivo preparations of the intact vomeronasal organ and accessory olfactory bulb

鼠标附件嗅觉系统（AOS）是一个专门感觉传导通路检测非易失性社会异味，费洛蒙，和利它素。第一个神经回路在AOS途径，称为嗅球（AOB），它在建立性的典型行为，如领土的侵略和交配的重要作用。这个小（&lt;1 立方毫米）电路具有区分独特的行为状态，如性别，品系，并在同种的分泌物和排泄物化学感受线索压力的能力。虽然这个系统的紧凑型组织呈现为同时从电路的大部分记录了独特的机会，在AOB感觉处理的调查仍然充满挑战，主要是由于其大脑中的实验不利的位置。在这里，我们证明了多阶段夹层，消除了完好的AOB的前小鼠头盖骨的单个半球内，留下连接离子两者的周犁鼻感觉神经元（的VSN）和局部神经回路完好。该过程公开的AOB表面直接目视检查，便于在没有麻醉剂的电生理和从AOB电路元件的光学记录。在插入细导管进入犁鼻器（VNO），这家的VSN，可以直接在外围暴露于社会的气味和信息素，同时记录在AOB下游活动。此过程使控制查询到AOS信息处理，它可以揭示信息素连接变动风险行为机制的光。

取得成功，这种准备需要大量的实践，有几个步骤，在它可能会失败。每个人都应该想到需要很多的尝试取得成功之前。自定义剥离室需要成功完成该协议，并应事先在开始解剖的后期阶段获得。腔室的设计在图1中给出的是足以达到此目的，并且可以由相对廉价的塑料以最小的加工要求。如果一个人没有产生这样的腔容量，本地或在线加工企业可以进行协商，并可以构造室收费。 </p...

Watch this Scientific Journal Video about Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb at JoVE.com

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

鼠标嗅球（AOB）已难以在感官编码的上下文中学习。在这里，我们表明，产生体外制剂，其中AOB神经元保持功能连接到他们的外设输入，便于研究的小鼠费洛蒙和利它素信息处理解剖。

完整的犁鼻器和副嗅球的体外制剂

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

ex-vivo-preparations-intact-vomeronasal-organ-accessory-olfactory

Research

JoVE Journal

Neuroscience

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

10.7K 次观看.  UT Southwestern Medical Center. 鼠标嗅球（AOB）已难以在感官编码的上下文中学习。在这里，我们表明，产生体外制剂，其中AOB神经元保持功能连接到他们的外设输入，便于研究的小鼠费洛蒙和利它素信息处理解剖。 

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

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

多电极阵列的犁鼻上皮录音

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

科研

神经科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

针对嗅球神经元采用组合在体内电穿孔和GAL4基于增强陷阱斑马鱼线

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

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.

在小鼠嗅球成像气味诱发的活动，利用光学反射和自体荧光信号

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

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.

在犁鼻器官的切片准备工作表示一个基因编码的钙传感器成像神经元的反应

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

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

脉络丛上皮细胞的纤毛运动的观察<em&gt;离体</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

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.

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.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

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

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.

在小鼠犁鼻急性组织切片定义细胞群的深入生理学分析

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

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.

嗅球的四次免疫染色用于嗅觉感觉轴突分子识别码的可视化

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