肯塔基大学生物系，本科课程和艺术与科学学院办公室支持。

膜电位早在1902年，伯恩斯坦在乌贼轴突的静息电位的问题处理。这是耐人寻味的，要考虑如何将这些早期思想和Berstein（1902年）和能斯特（1888）的意见后在膜生理的影响研究。 （见Malmivuo和Plonsey，1995年的审查;在WWW上也<a href="http://www.bem.fi/book/">可用</a> http://www.bem.fi/book/） 。还有，这一天，正在对离子通道的功能和属性是非常理解这涉及到组织，器官和系统功能的细胞生理学的重要的生物膜突破。 的实验和理论推导的外部效应的比较[K +]静息膜电位表示离子的膜电位的影响。使用此相同的编制的其他实验，仍然要执行，以解决基本的生理问题。有些人早在1968年突出阿特伍德和帕纳斯还没有得到完全解决。在这次演习中获得的技术，可以继续回答其余的许多问题在其他实验的准备工作以及在相关的医学和健康的生理应用。我们已经展示了一个模型的无脊椎动物准备解决的根本问题有关的所有动物的实用性。 就在这上面的练习中的离子的电化学梯度获得的知识，你现在可以提前膜的兴奋性突触传递的研究在小龙虾的神经肌肉准备。 测量突触反应这个实验室的第一部分，以及相关的影片提供的细节，都提供了记录膜电位，调查肌肉结构的关键步骤。在本实验室的第二部分，解剖和阶段性和补品电机单位NMJs记录突触传递的示范提供了一个暴露在生理学的基本概念。一个神经回路，它可以在部分可以用来解释相关的行为，在完整的动物，有潜力，不仅为学生，调查他们的实验室内行使各种开放式问题，也为未来的研究神经回路在一个良好的曝光建立无脊椎动物的准备（Kennedy 等，1969; Antonsen和爱德华兹，2003） 这些准备工作也可以用于研究突触的便利，抑郁症和长期可塑性（而不是在本实验室研究调查）。 ，即使是在小龙虾一些物种，取决于神经可塑性的实验刺激条件（Mercier和阿特伍德，1989年Cooper 等人，1998年）以及他们的自然环境。在何种程度上改变突触效能和肌肉动态服务能力的动物还有待调查。由于小龙虾不改变其季节变化和蜕皮周期中的行为，也有相对长期在他们的神经肌肉系统的活动的分歧。它已被证明阶段性电机爪接近肌肉的神经末梢在冬季表现出经典的阶段性形态，但膨胀，并成为更多静脉曲张在暑假期间（Lnenicka 1993年; Lnenicka，赵，1991）沿码头的长度。 在小龙虾横向巨头（LG）的interneurons腹神经索内进行一些早期的研究表明，存在的差距路口（1924年生，1961年）;渡边Grundfest，。这是人所共知的CO 2有电气通信偶联缝隙连接（阿雷利亚诺等，1990）的影响。这是最近出现的神经线，并在本报告所述，在感觉CNS电机肌肉电路的通信也很敏感，CO 2曝光，表明存在的差距路口（Bierbower 2010年; Bierbower和库珀， 2010） 3 路电机根已自发活动的主题，因为1960年的时，埃克特（1961年）研究如果进补射击静态肌肉受体的器官（MRO）在相同或邻近的段占自发电机驱动。在这些早期的研究中，很明显，这项活动是在腹神经索（VNC）的驱动可能从较高的中心（埃克特，1961年，肯尼迪和武田，1965a，B;斯特朗等， 2000）。由于存在的CO 2停止自发的活动，可以假设驱动器中的运动神经元，可能有差距路口或谷氨酸的兴奋驱动。 NMJs是阻止或表现出的敏感性降低谷氨酸的CO 2的存在，以及他们可能会集团中枢神经系统内以及KED（Bierbower，2010; Bierbower和Cooper，2010;也见Badre 等人，2005年）。 各种神经调的动作也很容易在NMJs（Cooper和Cooper，2009; Griffis等，2000; Southard等，2000;。斯特朗等，2000）的各类研究。此外，施加各种影响中枢神经系统的电路上的神经调。有人曾建议的5 -羟色胺和octopaminergic神经元可能会改变神经回路的输出（ 马等，1992;。施耐德等，1996;霍纳等，1997年为“增益制定”功能;爱德华兹 。 等，2002）。之前，我们完全可以理解的个别靶细胞的神经调的影响，还有许多工作有待完成。由于不同的神经调质可能与另一个演唱会的工作，分析其混合行动是为未来的研究（Djokaj等。2001）领域。此外，研究较少，特别是在脊椎动物，解决整个通路的神经调质，它可以调节的具体行为的影响。在这种感觉CNS电机单位编制，可以检查的感觉输入和上运动神经元（Kennedy等，1969）活动的神经调质的影响。 既然已经推测，5 -羟色胺起着一定的作用，在规范国家行为的小龙虾，龙虾，螃蟹（利文斯通等，1980; Sneddon 的等，2000），作了一些尝试，以确定其浓度VNC的，血淋巴中分离出神经节龙虾（利文斯通等人 ，1980年;哈里斯瓦里克和Kravitz的1984年; Fadool 等 ，1988）。然而，有相当大的变化，在记录的测量，排除特定的剂量 - 反应关系，这可以解释为行为的行动。 一直被认为与中提出的立场和下腹部卷起的尾巴举行的爪小龙虾，表现出主宰的姿态（ 利文斯通等，1980 ）。小龙虾腹部屈曲状态，不会出现的架势，占主导地位的小龙虾，在一对，展览期间的社会互动，或同时保持一个占主导地位的等级地位（ Listerman等，2000）。唯唯诺诺的小龙虾，甚至会掖下自己的肚子，因为他们从对手撤退。这种尾巴每天进食也被看作是一个防御姿态（Listerman 等，2000）。这些行为已经很容易观察到的在野外和实验室设置（Bovbjerg，1953年，1956年; Bruski和邓纳姆，1987年，李等，2000;。Listerman等 ，2000）。有趣的是，行为的姿势，在龙虾（ 利文斯通等 ，1980）指出，扭转了5 -羟色胺和章鱼胺注射在澳洲龙虾 ，Cherax析构函数（麦克雷，1996年）。可能，完全不同的反应，将观察到浅屈肌准备在澳洲小龙虾。此外，因为优势是一般大小之间的小龙虾相关，期望塑料响应系统为迅速改变的社会条件（ 斯特朗等人，2000年） 。在无脊椎动物的神经调质可塑性响应是一个开放的调查面积。 Wyttenbach，Johnson和霍伊（1999年）已经产生了数字媒体实验室手册和涉及相同的肌肉在本报告除了其他小龙虾准备提交的各种小龙虾experimentations。这是一个优秀的资源为学生练习。

<ol><li>小龙虾（ 克氏螯虾 ）。阿查法拉亚生物供应公司，Raceland，洛杉矶，美国。 </li><li>小龙虾生理盐水（标准）：范Harreveld的解决方案（1936年）修改。 （205毫米）氯化钠氯化钾5.3; 13.5氯化钙2 · 2H 2 O; 2.45氯化镁2 · 6H 2 O 5 HEPES和调整pH至7.4。羟色胺，谷氨酸和多巴胺是在小龙虾生理盐水。 Bouin氏固定液直接使用和亚甲蓝在小龙虾的盐水。所有化学品都是从Sigma公司（圣路易斯，密苏里州）。 </li><li>解剖工具：精细＃5镊子，精剪，刀刀座，昆虫引脚＃26002-20（所有获得精细科学的工具（美国）公司，373 - G的复古公园径，福斯特市，CA 94404-1139） 。 </li><li> Sylgard平底玻璃皿</li><li>烧杯（持有化学溶液） </li><li>电信号记录在一个PowerLab系统26T接口计算机（ADInstruments，CO，美国科罗拉多州的斯普林斯，）线。我们使用标准软件名为图表或范围ADInstruments。 </li><li> 3000的AC / DC型号为细胞内，以及作为外录音的放大器都可以使用。 </li><li>解剖显微镜与细胞内的录音变焦功能。对于一个正直的目标（4倍和20倍）的复合式显微镜是使用可视终端的联络记录。人们需要一种汞光源。 </li><li>细胞内的录音，我们使用玻璃毛细管（从金控，不伦瑞克，ME，04011，美国的目录＃30-31-0）。细胞内的电极应该有一个20至30毫欧电阻。细胞内的电极填充带3米氯化钾。 </li><li>吸电极是用来记录外信号。 </li><li>法拉第笼。 </li><li>高强度照明灯（光源）。 </li><li>微操作机器人</li></ol>

membrane potentials, synaptic responses, neuronal circuitry, neuromodulation and muscle histology using the crayfish: student laboratory exercises

本报告的目的是帮助开发一个跨生物细胞膜的离子梯度造成的影响的认识。影响细胞的膜电位，并在这些实验中，我们处理的两个方面是：（1）膜外的K +离子浓度，（2）对特定离子的膜通透性。小龙虾腹部伸肌一些补品（慢）和其他阶段性在其生化和生理的表型（快），以及在其结构的分组，支配这些肌肉的运动神经元功能特点也相应不同。我们使用这些肌肉，以及浅，补虚的腹部屈肌证明突触传递的属性。此外，我们引进一个感觉中枢神经系统，运动神经元肌肉电路，演示电路的某些方面的表皮感官刺激的效果以及神经调质的影响。在这次演习中获得的技术，就可以开始回答许多问题，在其他实验的准备工作以及在相关的医学和健康的生理应用的剩余。我们已经展示了模型的无脊椎动物制剂的实用性，以解决有关的所有动物的根本问题。

Watch this Scientific Journal Video about Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises at JoVE.com

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

实验表明一个简单的方法，让学生获得经验，研究肌肉结构，突触反应，离子梯度和膜电位的通透性的影响。此外，一个感觉，中枢神经系统，电机肌肉电路提交显示一个方法来测试化合物在神经元电路的影响。

膜电位，突触反应，神经回路，神经调节和肌肉组织学用小龙虾：学生实验室练习

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

membrane-potentials-synaptic-responses-neuronal-circuitry

Research

JoVE Journal

Neuroscience

58.4K 次观看.  University of Kentucky. 实验表明一个简单的方法，让学生获得经验，研究肌肉结构，突触反应，离子梯度和膜电位的通透性的影响。此外，一个感觉，中枢神经系统，电机肌肉电路提交显示一个方法来测试化合物在神经元电路的影响。

视频: Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

小龙虾的反思记录行为反应

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

科研

神经科学

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.

This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.

小龙虾腿伸肌高和低输出NMJs生理录音

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

小龙虾腹部肌肉受体机关：在本体的学生实验室练习

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings with a force transducer to monitor the strength of contraction. In addition, we demonstrate how to visually monitor peristaltic activity, which can be used as a bioassay for various peptides, biogenic amines and neurotransmitters. This preparation is amenable to student laboratories in physiology and for demonstrating pharmacological concepts to students. This preparation has been in use for over 100 years, and it still offers much as a model for investigating the generation and regulation of peristaltic rhythms and for describing the mechanisms underlying their modulation. The pharmacological assays and receptor sub-typing that were started over 50 years ago on the hindgut still contribute to research today. This robust preparation is well suited to training students in physiology and pharmacology.

In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.

生理实验与小龙虾肠：学生实验室的工作

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings ...

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 ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

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

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

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

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

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

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

本体感觉和张力感受器在蟹四肢：学生实验室练习

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;体内</em&gt;麻醉大鼠超导途径局部场电位和多单位活动的电生理记录

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...