我们要感谢圣灵实验室成员。这项工作得到了国家耳聋与沟通障碍研究所（R01 DC005964）和美国国家科学基金会IGERT 0548890（HAA）。 对动物的实验是在按照美国的动物福利法案和美国国立卫生指引研究院，是由华盛顿大学的动物护理和使用委员会批准。

第1部分。电气记录仪器<ol><li>我们使用一个平面与10微米平面氮化钛，氮化硅绝缘电极的多电极阵列（ALA的科学仪器）。被安排在60个电极组成一个中心，中心间距30微米的5X6格的两个领域。 （其他配置也有可能与记录面直径在10微米的电极比孤立的单个神经元的活动方面有较大的电极。） </li><li>电信号放大（放大1000倍），使用MEA 1060放大器（ALA科学仪器）。信号采集采用数据采集卡（美国国家仪器）和自定义数据采集软件（也可以购买商业软件）在10千赫。 </li><li>该组织举行的“千年生态系统评估”，由尼龙网（53微米），这是连接到一个自定义的插入，紧贴内的“千年生态系统评估”文化管环。 </li></ol>第2部分。液体输送仪器<ol><li>连续灌流，其中我们提供林格解决方案（115毫米氯化钠，5毫米钾氯化物，2 mM的钙氯化物，2 mM的镁氯，25 mM的钠碳酸氢盐，10毫米的HEPES，和10 mM的D -（+保证组织）葡萄糖）从HPLC泵。刺激被执行注射到高效液相色谱阀和流过和循环之间切换。 HPLC泵和注射机器人的注射泵提供与林格氏液。 </li><li>林格液保存在恒温水浴（ISOTEMP 105）在42 ° C，在整个实验过程，并不断冒泡的95％O 2 / 5％的CO 2。准备一个单独的无葡萄糖瓶中林格注射泵为后盾。林格氏液传递到组织，使用一个307 HPLC泵（吉尔森）3毫升/分钟的速度。在一个典型的5-6小时的实验，1 L每个林格氏液就足够了。 </li><li>在整个实验过程中，该组织是连续灌流37℃林格氏液。加热器探头定位直接组织以上。 </li><li>刺激交付使用吉尔森215液体处理程序机械臂。刺激存储在橡胶覆盖的玻璃小瓶，吉尔森软件的控制下，机械臂灌注行提供的刺激，而保持恒定流速和压力。 </li><li> HPLC阀（循环从事/不从事）的状态是由一个电压，这是由收购的计算机作为一个额外的通道，以确保与神经元的活动的同步记录的代表。 </li></ol>第3部分。准备MEA和刺激给药装置<ol><li>进行解剖之前，填写“千年生态系统评估与BSA在dH2O在室温下至少5分钟。 </li><li>使用林格氏液热身冒泡和冲洗“千年生态系统评估”千年生态系统评估“置于冰上。 </li><li>总理的液体处理和HPLC泵根据制造商的指示。 </li></ol>第4部分。 VNO夹层<ol><li>执行剥离之前，填写冒泡林格氏液置于冰上2 60x15毫米的培养皿中。同时填充2 Sylgard涂层与同样大小的林格氏液，并置于冰上的培养皿。冰寒在解剖使用的热身冒泡林格50毫升。 </li><li>鼠标是颈椎脱位， 通过二氧化碳窒息牺牲。 </li><li>从口边双边切口，下颌线平行，到了脑后。加入沿颈后2削减和消除头的背侧部分，留下连接到鼠标的下颚。 </li><li>用细剪刀，切割之间的组织和上颚骨分开的骨组织的两侧。 </li><li>使用＃3钳，剥离腭部组织暴露鼻腔。 </li><li>使用小剪刀或手术刀，上部2门牙两侧之间自由骨犁鼻囊切开。 </li><li>使用＃3镊子，拿起犁鼻囊，扁骨和在培养皿上冰的地方。透过浸泡的步骤应该是在不到3分钟。 </li><li>将菜立体显微镜下，用反射光照亮。在使用＃3钳的时间，从一个VNO移除骨胶囊。一方面要稳定揪心从尾结束，另一方面剥离骨离的VNO，小心不要损坏VNO骨胶囊。 </li><li>当骨被删除，转移到Sylgard涂层的培养皿的VNO。使用其他VNO剩下的菜。切换透射光照明。 </li><li>采用微型剪刀或＃5镊子，血管分开，沿切割边缘的VNO VNO。在这一点上，你会完全从血管分离的神经表。 </li><li>内部（树突）朝上放置的VNO。使用＃3钳，锚VNO Sylgard组织的一个角落。去皮，从基底使用钳位举行聚四氟乙烯涂层剃须刀刀片的一个小片段的椎板神经上皮。在浅的角度（&lt;30 °从盘表面）刀片是最好的。 </li><li>使用的玻璃吸管，从盘的传输神经上皮的一块“千年生态系统评估”。位置与树突状方犁鼻器上的电极领域的顶级。删除多余的林格的解决吸管。放入数组的以及网状组织持有人举行的地方VNO，并填写冷冻林格溶液。 </li></ol>第5部分。记录<ol><li>放置在一个多边环境协定的1060放大器“千年生态系统评估”，并直接组织的上述立场加热器探头。 </li><li>要确定适当的加热探头安置，刺激与林格氏液含有2秒50毫米钾（摩尔钠取代）。调整定位，以尽量减少钾溶液的神经反应延迟。 </li><li>组织Superfuse与林格加热45分钟至1小时前录音，让组织适应环境的解决方案。 </li><li>电信号保存到磁盘使用自定义的录音软件。 </li></ol>第6部分。数据分析<ol><li>离线数据分析，可以使用原始的电极录音。例如，我们在响应特定的配体测量记录细胞的放电率的变化。由此产生的数据集很大，可以用来解决许多不同的问题;熟悉编程或分析语言（C，MATLAB，R，Python中，或类似的）是必需的。 </li></ol>第7部分。代表性的成果本制剂通常是稳定的，足够的记录，从6个小时，产生大量的数据（6小时的录音，由60个电极）。 <img src="/files/ftp_upload/1845/1845fig1tmb.jpg" alt="图1" border="0" /> 图1显示整个数组的一半同时活动的快照。时间窗口跨越400毫秒。请<a href="https://www.jove.com/files/ftp_upload/1845/1845fig1.jpg">点击这里</a>看到图1的放大版本。 <img src="/files/ftp_upload/1845/1845fig2tmb.jpg" alt="图2" border="0" /> 图2：一个典型的实验，我们刺激与感兴趣的特定化合物为10秒。此跟踪是100倍稀释后的雌性小鼠尿液的反应的一个例子。请<a href="https://www.jove.com/files/ftp_upload/1845/1845fig2.jpg">点击这里</a>看大图图2。

<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>

多电极阵列记录，让我们一个人口众多的神经元的同步活动进行监控的能力。这是一个有用的工具，用于探测感官系统的性能，特别是VNO。与主要的嗅觉系统，VNO检测液相的刺激。此外，VNO是一种异质性的组织。有大约300个不同类型的鼠标 1受体可能与不同的反应概况， 。结合机械臂的多边环境协定的录音，让我们能够可靠地提供液体刺激的神经元和身份回应electrophysiologically庞大和多样?...

multielectrode array recordings of the vomeronasal epithelium

了解神经回路需要方法，同时从许多神经元记录。对于<em&gt;在体外</em&gt;研究之一，现有技术是平面的多电极阵列（MEA）记录。在这里，我们记录多边环境协定的使用鼠标犁鼻器（VNO）检测信息素和社会线索，通过感觉神经元表达数百种类型的受体的多样化的人口，它起着至关重要的作用的研究。结合MEA记录机器人的液体处理程序，以提供化学刺激的感官反应，大量的神经元和不同的人口可以被记录下来。准备允许我们从鼠标中删除的犁鼻器与电池的化学品或潜在配体的完整的神经上皮和刺激，同时监控几个小时的神经元的电活动。因此，这项技术作为一个有用的方法评估配体的活动，以及探索受体神经元的属性。我们目前需要准备的犁鼻器上皮，多边环境协定的记录，和化学刺激的技术。

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

Multielectrode Array Recordings of the Vomeronasal Epithelium

多电极阵列（MEA）的录音提供了一个人口众多的神经元电活动的研究方法。在这里，我们目前的一个多边环境协定的准备的细节，从小鼠犁鼻上皮的记录，同时刺激组织。

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 ...

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

Research

JoVE Journal

Neuroscience

12.1K 次观看.  Washington University School of Medicine. 多电极阵列（MEA）的录音提供了一个人口众多的神经元电活动的研究方法。在这里，我们目前的一个多边环境协定的准备的细节，从小鼠犁鼻上皮的记录，同时刺激组织。

视频: 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.

单机在体内</em从大鼠视交叉>录音

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 ...

科研

神经科学

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.

从巨纤维通路的电生理记录 D。果蝇</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.

解剖鼠标眼的视网膜色素上皮的全山

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.

器官型培养的神经元雪崩多电极阵列记录

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.

成像遥感信息素在小鼠犁鼻急性组织切片的制备

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.

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 ...

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.

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 ...

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 ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

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.