SG는 버팔로의 뉴욕 주립 대학에서와 존 R. Oishei 재단 기금에 의해 지원됩니다. MK는 UB의 CURCA하여 학생 연구 수상에 의해 지원되었다.

1. 시약의 준비 <ol><li> 7.2 및 필터 소독 128 MM NaCl, 1mM EGTA, 4 MM MgCl 2, KCl 2mM, 5mM HEPES, 1000 ML, PH 36 MM의 자당 다음 화합물을 추가하여 해부 버퍼 준비를 준비합니다. </li></ol> 2. 해부를위한 준비 <ol><li> Siligard 큐브 : 겔은 약 인치 와이드, 1 인치 길이 높이 약 1cm로 잘라입니다. 겔 큐브는 절개 중에 유리 슬라이드에 배치됩니다. 큐브는 두 번 이상 사용할 수 있습니다. </li><li> 핀 : 해부에 사용되는 핀 짧게 추가해야합니다. minutien 핀의 날카로운 아래 부분 절개에서 최상의 상태로 작동합니다. </li><li> 한 날카로운 microdissection 스프링 가위가 필요합니다. </li><li> 이 좋은 밀고 포셉 필요합니다. </li></ol> 3. 애벌레 수집 <ol><li> GFP 태그 소포 단백질을 표현 아르 제 3 instar의 애벌레를 방황하는 것은 배양 접시에 저장됩니다. </li><li> 애벌레는 모든 음식을 없애 탈이온수에 씻어 수 있습니다. </li></ol> 4. 애벌레의 라이브 해부 <ol><li> 유충은 유리 슬라이드에 절개의 겔 큐브로 전송됩니다. 유충은 해부 버퍼에 포장되어 있습니다. </li><li> 유충은 앞쪽에와 후부가 등의 측면을 가진 두 개의 좋은 핀을 이용하여 pined 있습니다. </li><li> microdissection 가위를 사용하여, 작은 상처는 사후 끝에 근처에있는 정중선에서 이루어집니다. 이 절개의 상처가 앞쪽이을 통해 이루어집니다. 이 핀을 사용하여 측면 가장자리에 자른 표피를 핀. </li><li> 버퍼를 해부 한 방울을 추가합니다. 조심스럽게 포셉과 창자, 내장 지방의 시체를 제거합니다. 이러한 조직의 대부분은 일반적으로 지느러미 절단하는 동안 밖으로 스며 나오게하다. </li><li> 신선한 해부 버퍼로 버퍼를 교체합니다. 유충은 건조한 않을 것입니다 그리고 지속적으로 버퍼를 해부에 있어야합니다. </li><li> 생체내 마커의 MitoTracker 또는 LysoTracker에서 사용 Incubations는 해부 버퍼의 생체내 마커의를 diluting하여,이 시간에 이루어집니다. 생체내 표지에의 부화 후 해부 유충에게 신선한 해부 버퍼를 사용하여 여러 번 (5 빠른 세척) 씻으십시오. </li><li> 교체 후 마지막 세차 syligard 즉시 coverslip에 핀을 밀어. 애벌레는 이제 관찰을위한 준비가되었습니다. </li></ol> 5. 해부 애벌레의 라이브 영상 <ol><li> 현미경 스테이지와 거꾸로 현미경을 사용하여 이미지에 coverslip와 젤 놓습니다. </li><li> 우리는 애벌레 segmental 신경 이내에 이미지 태그를 vesicles로 100x 렌즈를 사용합니다. segmental 신경이 몇 군데되기 전에 복부 신경절의 세포 기관에서 GFP 발현 먼저 평가됩니다. 강력한 GFP의 얼룩이 복부 신경절에 세포 기관에서 관찰하면, 신경은 다음 몇 군데와 평가를 위해 사용됩니다. </li><li> CFP / YFP 또는 RFP / YFP에 대한 필터와 분할보기 소프트웨어 생체내에서 우리가 할 수있는 동시에 이미지를 두 개의 태그 단백질과 듀얼 뷰 시스템을 사용합니다. </li></ol> 6. 대표 결과 <img src="/files/ftp_upload/2151/2151fig1.jpg" alt="그림 1" /> 그림 1 :. 애벌레 segmental 신경 내에 많은 axonal 트랙에 많은 axonal vesicles을 보여주는 대표 이미지 axonal 쌓일 대표, GFP의 모양을 확인합니다. <img src="/files/ftp_upload/2151/2151fig2.jpg" alt="그림 2" /> 그림 2 : 애벌레 segmental 신경 내에 많은 axonal vesicles로 단일 axonal 트랙을 보여주는 대표 이미지.

<ol>
	<li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Hurd, D. D., Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin+mutations+cause+motor+neuron+disease+phenotypes+by+disrupting+fast+axonal+transport+in+Drosophila.">Kinesin mutations cause motor neuron disease phenotypes by disrupting fast axonal transport in Drosophila.</a> Genetics. 144, 1075-1085 (1996).</li><li>Gunawardena, S., Her, L. S., Brusch, R. G., Laymon, R. A., Niesman, I. R., Gordesky-Gold, B., Sintasath, L., Bonini, N. M., Goldstein, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+axonal+transport+by+loss+of+huntingtin+or+expression+of+pathogenic+polyQ+proteins+in+Drosophila.">Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila.</a> Neuron. 40, 25-40 (2003).</li></ol>

관심 없음 충돌 선언하지 않습니다.

Drosophila 애벌레 segmental 신경 사이 시냅스 소포 수송의 생체내 이미징에서 axonal 교통의 메커니즘을 연구를위한 강력한 도구입니다. 우리는 이전 반복의 확장은 소포 수송 3 역학에 미치는 영향 명료하게하다 확대 polyQ 반복을 표현 애벌레 신경 내에 전송 역학을 평가하기 위해이 프로토콜을 사용하고 있습니다. 이 프로토콜을 사용하는 것은 소포 운동의 속도는 kymograph에 비디...

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<td>1 Pair Micro Dissection Scissors</td>
<td>&nbsp;</td>
<td>Fine Scientific </td>
<td>&nbsp;</td>
<td>Spring scissors &#x2013; 6mm blade. 0.125 mm tip diameter</td>
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<td>2 Pair Forceps</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>&nbsp;</td>
<td>Fisherbrand Dissecting Micro-Adson serrated tip forceps</td>
</tr>
<tr>
<td>Box of Dissection Pins</td>
<td>&nbsp;</td>
<td>Fine Scientific </td>
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<td>Minutien Pins 0.1mm diameter</td>
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in vivo visualization of synaptic vesicles within drosophila larval segmental axons

axonal 수송의 메커니즘을 Elucidating하면 장거리 수송의 결함은 다른 신경 질환에 미치는 영향에 대해 결정에서 매우 중요하다고 표시했습니다. 이 필수 과정에서 결함의 연결 기능 및 개발에 해로운 영향을 미칠 수 있습니다. 우리는을 폭로하기 위해 설계되었습니다 해부 프로토콜을 개발했습니다<em&gt; Drosophila</em실시간 axonal 교통을 보려면&gt; 애벌레 segmental 신경. 우리는 허드와 애벌레 segmental 신경의 immunolocalizatin 사용 색스턴 (1996)에 의해 출판 하나에서 라이브 영상이 프로​​토콜을 적응있다. 주의 해부 적절한 버퍼 조건 해부 애벌레의 수명을 최대화를 위해 중요하다. 제대로 완료되면, 해부 유충이 생리 조건 하에서 2~3시간에 대한 강력한 소포 ​​전송을 보여주었다. 우리는 UAS - GAL4 방법을 사용<sup&gt; 1</sup&gt; 표현하는 GFP - 태그의 연결을 다른 GAL4 드라이버를 사용하여 애벌레 segmental 신경에서 하나의 축삭 또는 여러 axons 이내 APP 또는 synaptotagmin의 vesicles 있습니다. 기타 찬란 태그 마커 예를 들어 mitochrondria (MitoTracker) 또는 lysosomes (LysoTracker)는 또한보기 전에 애벌레에 적용할 수 있습니다. GFP - 소포 운동과 입자의 움직임은 별도의 파장을 사용하여 동시에 볼 수 있습니다.

Watch this Scientific Journal Video about In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons at JoVE.com

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

이 프로토콜은의 라이브 절개를 설명<em&gt; Drosophila</em영상의 목적은 미세 소관 트랙에 GFP 태그 axonal vesicles의 움직임에 대해&gt; 애벌레.

Drosophila 애벌레 Segmental Axons 이내 시냅틱 Vesicles의 생체내 시각화에

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

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Neuroscience

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

9.4K 조회수.  SUNY-University at Buffalo. 이 프로토콜은의 라이브 절개를 설명 Drosophila 애벌레.

비디오: In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3 애벌레 Segmental 신경의 시각화 차 Instar Drosophila 애벌레 준비

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

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Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

의 형태학의 분석 Drosophila</em유전 모자이크 사용하기> 애벌레 주변 감각 신경 세포의 Dendrites 및 Axons

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

의 Proprioceptors의 시각화<em&gt; Drosophila</em&gt; 애벌레와 Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

의 애벌레 먹이 회로의 기능적 분석 초파리</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

소뇌 축삭의 레이저 Nanosurgery<em&gt; 생체</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

의 라이브 영상<em&gt; 초파리</em&gt; 애벌레 Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

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.

Vivo에서경작-Murine 이다 빠르고, 간단 하 고 저렴 한 AWESAM 프로토콜을 사용 하 여 같은

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 ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

초파리 애벌레 신경 근육 학 교차로에서 시 냅 스 전류의 초점 Macropatch 녹음

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

연 접 기능 그대로 신경 회로 내에 유전자 최저 효과 특성 인공 microRNAs Optogenetics 결합

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.