这项研究是由美国国立卫生研究院授予NS047073和国家科学基金会资助DMS1010434。

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+multiple+identified+motor+neurons+recorded+intracellularly+during+evoked+feedinglike+motor+programs+in+Aplysia.">Activity of multiple identified motor neurons recorded intracellularly during evoked feedinglike motor programs in Aplysia.</a> J. Neurophys. 72 (4), 1794-1809 (1994).</li><li>McManus, J. M., Lu, H., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vitro+Preparation+for+Eliciting+and+Recording+Feeding+Motor+Programs+with+Physiological+Movements+in+Aplysia+californica.">An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica.</a> J. Vis. Exp. (70), e4320 (2012).</li><li>Cullins, M. J., Chiel, H. 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我们什么都没有透露。

典型的做法是在动物与大确定的神经元，如软体动物（例如， 椎实螺 ， 螺旋 ，和海兔 ），分析电机池使用细胞内记录1,2,3,4。在这个协议中，我们描述了一个过程，用于唯一地识别用于电动机的运动神经元池使用一种细胞外的技术。作为一个例子，这个过程中，我们使用的测力。也可以使用肌电图测量肌肉的神经支配。简要地说，这样做的，该协议附加钩电极I1/I3肌...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>氯化钠</td><td> Fisher Scientific则</td><td> S671 </td><td>生物，认证</td></tr><tr><td>氯化钾</td><td> Fisher Scientific则</td><td> P217 </td><td>认证ACS </td></tr><tr><td>氯化镁六水合物</td><td> Acros Organics公司</td><td> 19753 </td><td> 99％ </td></tr><tr><td>七水硫酸镁</td><td> Fisher Scientific则</td><td> M63 </td><td>认证ACS </td></tr><tr><td>二水氯化钙</td><td>费舍尔Scientifc </td><td> C79 </td><td>认证ACS </td></tr><tr><td>葡萄糖（右旋糖） </td><td> Sigma-Aldrich公司</td><td> G7528 </td><td> BioXtra </td></tr><tr><td> MOPS buffeŕ </td><td> Acros Organics公司</td><td> 17263 </td><td> 99％ </td></tr><tr><td>卡巴胆碱</td><td> Acros Organics公司</td><td> 10824 </td><td> 99％ </td></tr><tr><td>氢氧化钠</td><td> Fisher Scientific则</td><td> SS255 </td><td>认证</td></tr><tr><td>盐酸</td><td> Fisher Scientific则</td><td> SA49 </td><td>认证</td></tr><tr><td>单管毛细管玻璃</td><td> AM系统</td><td> 6150 </td><td></td></tr><tr><td>火焰山 - 布朗微量移液器车夫模型P-80/PC </td><td>萨特仪器</td><td></td><td>灯丝用的：FT345B </td></tr><tr><td>搪瓷涂层的不锈钢丝</td><td>加州细线</td><td></td><td> 0.001D，涂料H </td></tr><tr><td>家用硅II胶</td><td>通用电器</td><td></td><td></td></tr><tr><td>杜罗快速凝胶强力胶</td&gt; <td>汉高公司。 </td><td></td><td></td></tr><tr><td>系统模型1700放大器</td><td> AM系统</td><td></td><td>过滤器设置：10-500赫兹的I2神经/肌肉300-500赫兹的所有其他神经</td></tr><tr><td> Pulsemaster多通道刺激</td><td>世界精密仪器</td><td> A300 </td><td></td></tr><tr><td>刺激隔离器</td><td>世界精密仪器</td><td> A360 </td><td></td></tr><tr><td> AxoGraph X </td><td> AxoGraph科学</td><td></td><td>录音软件</td></tr><tr><td>金连接器引脚</td><td> BULGIN </td><td> SA3148 / 1 </td><td></td></tr><tr><td>黄金的连接插座</td><td> BULGIN </td><td> SA3149 / 1 </td><td></td></tr><tr><td> Sylgard的184有机硅弹性体</td><td>道康宁</td><td></td><td></td></tr><tr><td> 100 x 15毫米Crystalizing的菜</td><td>高硼硅</td><td></td><td></td></tr><tr><td>高真空润滑脂</td><td>道康宁</td><td></td><td></td></tr><tr><td>枪头</td><td> Fisher Scientific则</td><td> 21-375D </td><td></td></tr><tr><td> Minutien销</td><td>精细科学工具</td><td> 26002-10 </td><td></td></tr><tr><td>橡皮泥</td><td>萨金特艺术</td><td> 22-4400 </td><td></td></tr><tr><td>护舒宝充气泵</td><td>利</td><td> 77849 </td><td></td></tr><tr><td>水族馆管</td><td> EHEIM </td><td> 7783 </td><td> 12/16毫米</td></tr><tr><td>精英AIRSTONE </td><td>哈根</td><td> A962 </td><td></td></tr><tr><td> Vannas春风似剪刀</td><td>精细科学工具</td><td> 15000-08 </td><td></td></tr><tr><td>杜蒙＃5精细镊子</td><td>精细SciencE工具</td><td> 11254-20 </td><td></td></tr><tr><td> Kimwipes </td><td> Kimberly-Clark公司</td><td> 34155 </td><td></td></tr></tbody></table>

extracellularly identifying motor neurons for a muscle motor pool in aplysia californica

在动物与大确定的神经元（ 例如软体动物），分析电机池在使用细胞内的技术1,2,3,4。最近，我们开发了一种技术，细胞外刺激，记录单个神经元在海兔夜蛾 5。现在我们描述了使用这种技术来唯一地识别和表征运动神经元内的电机池的协议。 此外技术方面具有优势。首先，细胞外电极可以刺激并记录神经元通过鞘5，所以，它并不需要被删除。因此，神经元将健康的细胞外实验，在细胞内的。其次，如果神经节是由适当的钉扎的鞘的旋转，外电极可以访问神经元的神经节上的两侧，这使得它更容易和更有效地识别多个神经元中的相同的制备。三，extracellu拉尔电极不需要穿透细胞，从而可以很容易地来回移动，神经元之间，他们造成损伤少。这是非常有用的，当一个人试图记录多个神经元在重复的运动模式，可能只持续几分钟。四，外电极是在肌肉运动更灵活，比细胞内的。细胞内的电极可以拉出来，破坏肌肉收缩的神经元。相反，由于外电极被轻轻地按压到上述鞘神经元，它们通常留上面相同的神经元在肌肉的收缩，因此可以用在更完整制剂。 为了唯一地识别的用于电动机池（尤其是，I1/I3在海兔肌肉）使用细胞外电极的运动神经元，一个可以使用的功能，不需要标准：细胞内测量胞体的大小和位置，轴突投射，和肌肉的神经支配4[6,7]。对于使用特定电机池来说明的技术，我们记录从颊神经测量轴突突起2和3，并测定I1/I3肌肉的收缩力的确定的图案为个别的运动神经元的肌肉的神经支配。 我们证明首先确定运动神经元肌肉神经支配的完整过程，然后描述他们的时间在运动模式，创建一个简单的诊断方法快速鉴定。简化的和更快速的诊断方法是优越的更完整的准备工作， 例如 ，在暂停的的口腔大量制备8 或 9 体内 。这个过程也可以被应用于其它电机池10,11,12在海兔或其他动物系统中2,3,13,14。

图4和图5结果显示了典型的用于识别两个I1/I3运动神经元。期间egestive等和摄食状的图案（ 图4C，4D），图4示出了一个大的运动神经元，B3，苏摩录音。一个一个对应的尖峰上的SOMA通道和同侧的BN2通道（ 图4E）的特异性的B3 SOMA记录的过程中保持模式。 B3火灾过程中的中，后期的收缩阶段的模式。从图4和其他的结果（图中未示出）中，...

Watch this Scientific Journal Video about Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica at JoVE.com

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

在大确定的神经元（动物<em&gt;例如，</em&gt;软体动物），分析细胞内的技术是通过使用电机池<sup&gt; 1,2,3,4</sup&gt;。最近，我们开发出一种技术，细胞外刺激和记录单个神经元<em&gt;海兔夜蛾</em<sup&gt;</sup&gt;。现在我们描述了使用这种技术来唯一地识别和表征运动神经元内的电机池的协议。

运动神经元细胞外识别的肌肉运动游泳池<em&gt;海兔夜蛾</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

extracellularly-identifying-motor-neurons-for-muscle-motor-pool

Research

JoVE Journal

Neuroscience

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

11.7K 次观看.  Case Western Reserve University. 在大确定的神经元（动物例如，软体动物），分析细胞内的技术是通过使用电机池 1,2,3,4。最近，我们开发出一种技术，细胞外刺激和记录单个神经元海兔夜蛾

视频: Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

科研

神经科学

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors1, allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica provides a tractable system for the study of multifunctionality. During feeding, Aplysia generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper2. However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo, nerve and muscle recordings have been obtained corresponding to feeding programs2,3,4, but it is very difficult to directly record from individual neurons5. Additionally, in vivo, ingestive programs can be further divided into bites and swallows1,2, a distinction that is difficult to make in most previously described in vitro preparations.
The suspended buccal mass preparation (Figure 1) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors - including both biting and swallowing - and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes6. The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo than are motor programs elicited in isolated ganglia. Thus, the motor programs in this preparation can be more directly related to in vivo behavior; at the same time, individual neurons are more accessible to recording and stimulation than in intact animals. Additionally, as an intermediate step between isolated ganglia and intact animals, findings from the suspended buccal mass can aid in interpretation of data obtained in both more reduced and more intact settings. The suspended buccal mass preparation is a useful tool for characterizing the neural control of multifunctionality in Aplysia.

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.

一个<em在体外</em制备与生理变动，为激发和记录进给电机程序<em&gt;海兔夜蛾</em

Multifunctionality, the ability of one peripheral  structure to generate multiple, distinct behaviors1, allows animals  to rapidly adapt their behaviors ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

隔离的感觉神经元中<em&gt;海兔丫</em谷氨酸能电流膜片钳记录

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

隔膜的神经支配膈运动神经元的功能和形态评估

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

肌肉注射沿运动终板：微创方法来接送示踪剂直接为运动神经元

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

为什么量化事项：表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

小鼠膈运动神经元逆行神经解剖学追踪的研究

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

A Minimally Invasive Lesion Technique for Muscles Intrinsic to the Odontophore of Aplysia californica

Aplysia californica is a model system for studying the neural control of learning and behavior. This animal has a semi-open circulatory system, making it possible to access many of its internal structures without causing any significant damage. Many manipulations can be easily performed both in vivo and in vitro, making it a highly tractable model for the analysis of behavior and neural circuitry. To better understand the functions of muscles within the feeding grasper, we have developed a technique for lesioning them without opening the main body cavity of the animal or damaging the outer layers of the feeding organ (i.e., the buccal mass). In this technique, the grasper is partially everted, allowing direct access to the musculature. This procedure allows animals to recover quickly and reliably. This has made it possible to lesion the I7 muscles and sub-radular fibers, allowing us to show that both muscles significantly contribute to the opening in vivo.

A Minimally Invasive Lesion Technique for Muscles Intrinsic to the Odontophore of Aplysia californica

Here we present a protocol for minimally invasive surgical lesioning of muscles intrinsic to the feeding apparatus of the marine mollusk Aplysia californica to understand the roles of these muscles during feeding behavior.

阿普利西亚的奥多托福雷内在肌肉的微创病变技术

Aplysia californica is a model system for studying the neural control of learning and behavior. This animal has a semi-open circulatory system, making it ...