이 연구는 NIH 보조금 NS047073 및 NSF 보조금 DMS1010434에 의해 지원되었다.

<ol>
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우리는 공개 할 아무 것도 없습니다.

예 mollusks (예를 들어, Lymnaea, 나선형, 그리고 Aplysia), 모터 풀의 분석과 같은 대형 식별 뉴런과 동물에서 일반적으로 세포 내 기록 1,2,3,4를 사용하여 수행됩니다. 이 프로토콜에서는, 우리는 고유 세포 기술을 사용하여 모터 풀의 모터 뉴런을 식별하기위한 프로세스에 대해 설명합니다. 우리는이 과정을 그림으로 힘 측정을 사용했습니다. 하나는 또한 근육 innervations를 측정하?...

<table border="1"><tbody><tr><td> 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 나트륨 염화물 </td><td> 피셔 과학 </td><td> S671 </td><td> 생물학적, 공인 </td></tr><tr><td> 염화칼륨 </td><td> 피셔 과학 </td><td> P217 </td><td> 인증 ACS </td></tr><tr><td> 마그네슘 염화물 hexahydrate </td><td> Acros의 유기물 </td><td> 19,753 </td><td> 99% </td></tr><tr><td> 황산 마그네슘의 heptahydrate </td><td> 피셔 과학 </td><td> M63 </td><td> 인증 ACS </td></tr><tr><td> 칼슘 염화물 이수화 </td><td> 피셔 Scientifc </td><td> C79 </td><td> 인증 ACS </td></tr><tr><td> 포도당 (덱스 트로 오스) </td><td> 시그마 - 알드리치 </td><td> G7528 </td><td> BioXtra </td></tr><tr><td> 걸레는 buffe연구 </td><td> Acros의 유기물 </td><td> 17,263 </td><td> 99% </td></tr><tr><td> Carbachol </td><td> Acros의 유기물 </td><td> 10,824 </td><td> 99% </td></tr><tr><td> 수산화 나트륨 </td><td> 피셔 과학 </td><td> SS255 </td><td> 인증 </td></tr><tr><td> 염산 </td><td> 피셔 과학 </td><td> SA49 </td><td> 인증 </td></tr><tr><td> 단일 질주 모세관 유리 </td><td> AM 시스템 </td><td> 6150 </td><td></td></tr><tr><td> 불타는 브라운 micropipette 풀러 모델 P-80/PC </td><td> 셔터 악기 </td><td></td><td> 필라멘트 사용되는 : FT345B </td></tr><tr><td> 에나멜 코팅 된 스테인레스 스틸 와이어 </td><td> 캘리포니아 정밀 와이어 </td><td></td><td> 0.001D, 코팅 H </td></tr><tr><td> 가정용 실리콘 II 접착제 </td><td> GE </td><td></td><td></td></tr><tr><td> 겠구나 빠른 젤 superglue </td&gt; <td> 헨켈 주식회사. </td><td></td><td></td></tr><tr><td> 시스템 모델 (1700) 앰프에게 AM </td><td> AM 시스템 </td><td></td><td> 필터 설정 : I2 신경 / 근육에 10-500 Hz에서, 모든 다른 신경에 300-500 Hz에서 </td></tr><tr><td> Pulsemaster 다 채널 Stimulator </td><td> 세계 정밀 계측기 </td><td> A300 </td><td></td></tr><tr><td> 자극 아이솔레이터 </td><td> 세계 정밀 계측기 </td><td> A360 </td><td></td></tr><tr><td> AxoGraph X </td><td> AxoGraph 과학 </td><td></td><td> 레코딩을위한 소프트웨어 </td></tr><tr><td> 골드 커넥터 핀 </td><td> Bulgin </td><td> SA3148 / 1 </td><td></td></tr><tr><td> 골드 커넥터 소켓 </td><td> Bulgin </td><td> SA3149 / 1 </td><td></td></tr><tr><td> Sylgard 184 실리콘 엘라스토머 </td><td> 다우 코닝 </td><td></td><td></td></tr><tr><td> 100 X 15mm Crystalizing 요리 </td><td> Pyrex </td><td></td><td></td></tr><tr><td> 높은 진공 그리스 </td><td> 다우 코닝 </td><td></td><td></td></tr><tr><td> Pipet 팁 </td><td> 피셔 과학 </td><td> 21-375D </td><td></td></tr><tr><td> Minutien 핀 </td><td> 정밀 과학 도구 </td><td> 26002-10 </td><td></td></tr><tr><td> 모델링 점토 </td><td> 사전 트 아트 </td><td> 22-4400 </td><td></td></tr><tr><td> 에어 펌프를 속삭 </td><td> 테트라 </td><td> 77,849 </td><td></td></tr><tr><td> 수족관 배관 </td><td> Eheim </td><td> 7783 </td><td> 16분의 12 mm </td></tr><tr><td> 엘리트 Airstone </td><td> 하겐 </td><td> A962 </td><td></td></tr><tr><td> Vannas 봄 가위 </td><td> 정밀 과학 도구 </td><td> 15000-08 </td><td></td></tr><tr><td> 뒤몽 # 5 좋은 포셉 </td><td> 좋은 Scienc전자 도구 </td><td> 11254-20 </td><td></td></tr><tr><td> Kimwipes </td><td> 킴벌리 - 클락 </td><td> 34,155 </td><td></td></tr></tbody></table>

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

대형 식별 뉴런 (예 : mollusks), 모터 풀의 분석과 동물에서 세포 기술에게 1,2,3,4를 사용하여 수행됩니다. 최근, 우리는 extracellularly 자극하고 Aplysia californica 5 개별 뉴런을 기록 할 수있는 기술을 개발했습니다. 이제 우리는 고유 식별하고 모터 수영장에서 모터 뉴런을 특성화하기 위해이 기술을 사용하기위한 프로토콜을 설명합니다. 이 세포 기술은 장점이 있습니다. 첫째, 세포 전극은 자극하고 기록 뉴런을 피복 1-5하므로 제거 할 필요가 없습니다 수 있습니다. 따라서, 뉴런은 세포들보다 세포 실험에서 건강하게 될 것입니다. 둘째, 신경절 경우하면 집에 걸어주는 적절한하여 회전은, 세포 전극은 쉽고 효율적으로 같은 준비에서 여러 뉴런을 식별 할 수 신경절의 양쪽 뉴런에 액세스 할 수 있습니다. 셋째, extracellu고맙다 전극은 세포를 통과 할 필요가 없습니다, 그래서 쉽게 그들에게 더 적은 손상을 초래 뉴런 사이에 앞뒤로 이동할 수 있습니다. 하나가 단 몇 분 거리에 지속될 수 있습니다 모터 패턴을 반복하는 동안 여러 뉴런을 기록하려고 할 때 특히 유용합니다. 넷째, 세포 전극은 근육 운동 기간 동안 세포들보다 더 유연합니다. 세포 내 전극 꺼내 근육 수축 동안 뉴런가 손상 될 수 있습니다. 세포 전극이 부드럽게 뉴런 위의 집에 눌러되므로 대조적으로, 그들은 일반적으로 근육의 수축시 동일한 뉴런 위 유지, 따라서 더 많은 손상 준비에 사용할 수 있습니다. 소마 크기와 위치, axonal 프로젝션, 그리고 근육 innervation 4 : 고유 세포 전극을 사용하여 모터 풀 (특히, Aplysia의 I1/I3 근육)에 모터 뉴런을 식별하려면, 하나는 기준으로 세포 측정을 필요로하지 않는 기능을 사용할 수 있습니다, 6,7. 기술을 설명하는 데 사용되는 특정 모터 풀의 경우, 우리는 axonal 예측을 측정하기 위해 구강 신경에서 2, 3를 기록하고, 각각의 모터 뉴런에 근육 innervation의 패턴을 결정하기 위해 I1/I3 근육의 수축 힘을 측정. 우리는 빠른 식별을위한 단순화 된 진단 방법을 만들어, 모터 패턴 중에 타이밍을 특성화, 근육 innervation을 사용하여 첫번째 식별 모터 뉴런의 전체 과정을 보여줍니다. 간체 및 더 빠른 진단 방법은 정지 구강 대량 준비 8 또는 생체 9 예를 들어, 더 많은 손상 준비를위한 우수합니다. 이 과정은 2,3,13,14 Aplysia 또는 다른 동물 시스템에서 10,11,12 다른 모터 수영장에서 적용 할 수 있습니다.

그림 4와 5는 두 I1/I3 모터 뉴런을 식별하는 데 사용되는 일반적인 결과를 보여줍니다. 그림 4 egestive 같은과 ingestive 같은 패턴 (인물 4C, 4D) 동안 대형 모터 뉴런, B3의 소마 녹음을 보여줍니다. 일대일의 소마 채널 및 B3 소마 기록의 특이성이 패턴 동안 유지되었다는 ipsilateral BN2 채널 (그림 4E) 쇼에 해당하는 스파이크. B3는 패턴의 중간 - 투 - 늦은 ?...

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

대형 식별 뉴런 (과 동물<em&gt; 예</em&gt; mollusks), 모터 풀의 분석은 세포 기술을 사용하여 수행됩니다<sup&gt; 1,2,3,4</sup&gt;. 최근, 우리는 extracellularly에서 개별 뉴런을 자극하고 기록 할 수있는 기술을 개발<em&gt; Aplysia californica</em<sup&gt; 5</sup&gt;. 이제 우리는 고유 식별하고 모터 수영장에서 모터 뉴런을 특성화하기 위해이 기술을 사용하기위한 프로토콜을 설명합니다.

Extracellularly의 근육 모터 풀에 모터 뉴런을 확인<em&gt; Aplysia californica</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

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JoVE Journal

Neuroscience

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

11.7K 조회수.  Case Western Reserve University. 대형 식별 뉴런 (과 동물 예 mollusks), 모터 풀의 분석은 세포 기술을 사용하여 수행됩니다 1,2,3,4. 최근, 우리는 extracellularly에서 개별 뉴런을 자극하고 기록 할 수있는 기술을 개발 Aplysia californica

비디오: 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.

모터 신경 세포의 이점 패치 클램프 레코딩과 Zebrafish의 대상 골격근

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

연구

신경과학

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

마우스에 모터 조정을 측정

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

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.

FM 염료를 사용하여 양식 소뇌 과립 뉴런에서 시냅틱 소포 풀 보충의 정량 분석

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.

<em&gt; 체외에서</em의 생리적 운동과 수유 모터 프로그램을 도출하도록 의도하고 녹음&gt; 준비<em&gt; Aplysia californica</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.

의 감각 뉴런의 분리<em&gt; 군소 californica</em&gt; 글루타메이트 전류의 패치 클램프 녹음을위한

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.

횡격막 모터 뉴런에 의해 다이어프램 신경 분포의 기능 및 형태 학적 평가

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.

모터 엔드 플레이트 함께 근육 주사 : 직접 모터 뉴런에 셔틀 트레이서에 대한 최소 침습 접근

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.

상기 표현형의 특성을 : 정량이 중요한 이유<em&gt; 초파리</em&gt; 애벌레 신경 근육 학 분기점

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.

마우스에 인할지 모터 신경의 퇴행 성 신경 해부학 추적

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.

Aplysia 칼리포르니카의 오돈토포어에 내재된 근육에 대한 최소 침습 병변 기술

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 ...