Name: laser nanosurgery of cerebellar axons in vivo
Uploaded: 2014-07-28T13:00:00
Description: Watch this Scientific Journal Video about Laser Nanosurgery of Cerebellar Axons In Vivo at JoVE.com

We would like to thank Erica Lorenzetti for technical assistance on the injections and Irene Costantini for making figure 1. The research leading to these results has received funding from LASERLABEUROPE (Grant 284464, European Commission&#8217;s Seventh Framework Programme). This research project has also been supported by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX and by Italian Ministry of Health in the framework of the &#8220;Stem Cells Call for Proposals.&#8221; This work is part of the research activities of the European Flagship Human Brain Project and has been carried out in the framework of the International Center of Computational Neurophotonics foundation supported by &#8220;Ente Cassa di Risparmio di Firenze&#8221;.

1. 축삭 라벨링 <ol><li> 클라이밍 섬유는 고 분자량 덱스 트란 또는 형광 단백질 26-29의 발현을 유도하는 플라스미드 /의 바이러스에 접합 된 유기 염료 하나를 주사하여 표시 할 수있다. 이 프로토콜, 유기 염료 알렉사 플 루어 488 덱스 트란은 등반 섬유 레이블 (그림 1) 소뇌 피질을 시각화하기 위해 하부 올리브에 주입된다. 여기에 설명 된 모든 절차는 건강의 이탈리아 교육?...

<ol>
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이 프로토콜은 형광 염료와 하부 올리브의 뉴런 레이블을하는 방법을 보여줍니다. 이어서, 소뇌 피질에 두개 창을 수행하는 방법을 설명한다. 이 기술은 olivocerebellar 신경, 등산 섬유의 단자 부분에 광 액세스를 제공합니다. 불행하게도, 라벨 및 개두 수술 모두의 결과는 (일반적으로 3 마우스 중 1 표시, 1 3 중 두개골 창 1 ~ 2 주 후에 명확 유지됩니다)도 숙련 된 운영자의 손에 아주 낮다. <p class=...

기계적 손상, 독성 모욕 또는 신경 변성 질환으로 인한 절개 축삭은 보통 10-13 전지 본체에서 분리 축삭의 말단 부분의 변성에 의해 이어진다. 몇 가지 예외를 제외하고 2,7,14,15로, 성인 동물의 CNS에서 절단 축삭은 일반적으로 재성장 프로그램 (16)을 활성화 할 수 없습니다. 약간은 세포와 세포 이하의 수준에서 퇴행성 이벤트의 실시간 역학에 대한 알려져있다. 신경 세포의 손상을 제한하고 신경 세포의 재성장을 촉진하기위한 새로운 전략의 개발은 단독으로 부상당한 신경 세포가 퇴화하고 재생하는 메커니즘을 명확히, 첫 번째 단계로,이 필요합니다. 이 연구는 가장 직접적으로 생체 내에서 단일 신경 세포의 역학을 모니터링하여 해결됩니다. 일 광자 형광 이미징 기술은 가시광의 산란 강도에 의해 제한되는 반면, 두 개의 광자 여기가 리 깊은 피질 층에 도달세포 내 해상도 3,4,17와 마우스를했습니다. 형광 단백질을 선택적으로 신경 18-20의 모집단에서 발현되는 형질 전환 마우스의 활용, TPF 현미경은 생체 내 21, 22의 개발 과정에서 시냅스 가소성과 축삭 신장의 탐사에 적용되었습니다. 유일하게 손상된 신경 세포의 T 그 기능에 부상이 특히 관심의 축삭을 대상으로 상해의 모델 두 광자 이미징에 의해 생체 모니터링에 커플 링에 의해 조사 할 수 있습니다 후 다시 자라게. 펨토초 펄스의 다 광자 흡수는 하나의 수상 돌기 또는 한 쪽이 5,23을 방해하는 데 사용되었습니다. 또한,이 부상 패러다임은 연락 덴 드라이트 (dendrite) 6을 방해하지 않고 하나의 축삭 가지를 절단 할 수 있습니다. 한 번 손상, 소뇌 등산 섬유 (CFS) 자신의 축삭을 재생하는 특정 신경 세포의 인구 수 있도록 기능을 해부의 컨텍스트 내에서 유용한 모델 SI는후부 그들은 심지어 성인 동물 (24, 25)의 부상 후 뛰어난 플라스틱의 특성을 유지합니다. 최근 CF 및 장기 이미징은 이러한 축삭 레이저 axotomy 6에 따라 일 재성장 할 수 있다는 것을 보여 주었다. 이 프로토콜은 선행 성 추적을 통해 olivocerebellar 뉴런과 자신의 축삭 신장 레이블을하는 방법에 대해 설명합니다. 관심의 신경 세포가 형광 표지되면, 그들은 두개골 창 아래에 몇 주 또는 몇 달 임의의 시점에서 반복적으로 모니터링 할 수 있습니다. 생체 내에서 레이저 axotomy에 의해 하나의 축삭 가지를 해부하는 절차는 설명한다. 여기에 제시된 기술은 생체 내에서 축삭 리모델링의 메커니즘에 새로운 통찰력을 제공하고 신경 세포의 변성을 제한하고 축삭 재성장을 촉진하는 치료 전략의 개발을하는 데 도움이 될 수 있습니다.

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	<tbody>
		<tr>
			<td>Lab standard stereotaxic, rat and mouse</td>
			<td>Stoelting&#160;</td>
			<td>51670</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass with filament</td>
			<td>Sutter Instrument Inc</td>
			<td>BF100-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator 500 (Glass bead sterilizer)</td>
			<td>Roboz</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection dispense system</td>
			<td>Picospritzer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small diameter round cover glass, #1 thickness, 3 mm, 100 pack (CS-3R)</td>
			<td>Warner Instruments&#160;</td>
			<td>64-0720</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Sapphire laser, 120 fs width pulses,&#160; 90 MHz repetition rate</td>
			<td>Coherent</td>
			<td>Chameleon&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spongostan, haemostatic sponge&#160;</td>
			<td>Ferrosan</td>
			<td>MS0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Galvanometric mirrors&#160;</td>
			<td>GSI Lumonics</td>
			<td>VM500+</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective&#160;</td>
			<td>&#160;Olympus</td>
			<td>XLUMPLFLN 20XW</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezoelectric stage&#160;</td>
			<td>Physik Instrumente</td>
			<td>P-721</td>
			<td></td>
		</tr>
		<tr>
			<td>Photomultiplier modules&#160;</td>
			<td>Hamamatsu Photonics</td>
			<td>H7710-13</td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW System Design Software</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Voren, 1 mg/ml (dexamethasone-21- isonicotinate)</td>
			<td>Boheringer Ingelheim</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rymadil (carprofen)&#160;</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine clorohydrate 2%</td>
			<td>ATI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 dextran</td>
			<td>Life Technologies</td>
			<td>D22910</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

이 프로토콜은 하나의 뉴런의 축삭 라벨, 생체 내 이미징 및 레이저 axotomy을 수행하는 방법을 설명했다. 실험의 타임 라인은도 1에 도시된다. 알렉사 형석 488 덱스 트란으로 표시하고 생체 이광자 현미경으로 두개 창 아래 시각의 CF의 예는도 2에보고된다. 이전 6,27보고 된 바와 같이, 상승하는 분기 수일의 관찰 기간에 걸쳐 높은...

Watch this Scientific Journal Video about Laser Nanosurgery of Cerebellar Axons In Vivo at JoVE.com

Laser Nanosurgery of Cerebellar Axons In Vivo

레이저 nanodissection 결합 이광자 이미징은, 세포 내 해상도 중추 신경계 퇴행성 및 재생 공정을 연구하는 유용한 도구이다. 이 프로토콜은, 라벨 이미지 및 생체 내에서 소뇌 피질에서 하나의 등반 섬유를 해부하는 방법을 보여줍니다.

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 ...

Research

JoVE Journal

Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

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 ...

연구

신경과학

In vivo Laser Axotomy in C. elegans

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C로 생체내 레이저 Axotomy에서 엘레간스

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Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

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Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

공 초점 Macroscopy을 사용하여 출생 후의 소뇌 과립 세포 이동의 생체 이미징

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

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동시<em&gt; 생체</em두 망막의&gt; 기능 테스트로<em&gt; 생체</em&gt; 전위도 시스템

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

신경 죽음 및 마우스 기본 소 뇌과 립 신경에 변성을 모델링

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

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Vivo에서 복합 이미징 및 마우스 레이저 유도 안 Neovascularization 모델의 분석

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

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제자리에 Myelinated 축 삭에 스펙트럼 Reflectometric 현미경

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

성인 소뇌 슬라이스에 장기 우울증 유도의 평가

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...