This work is supported by a Canadian Health Research Institute (CIHR) Team Grant to Dr. Bolduc (co-PI) and Dr. Guy Rouleau (PI). We would like to thank Dr. Oksana Suchowersky, Dr. Kathryn Todd, the members of the University of Alberta fly club and Dr. Clayton Dickson for help in development of the method and statistical analysis.

초파리 연구는 알버타의 연구 가이드 라인의 대학을 준수했다. 1. 플라이 컬렉션 <ol><li> 음식을 포함하는 25mm X 95mm 수집 유리 병에 CO 2 (g)을 마취과 장소를 사용하여 20 파리를 수집합니다. </li><li> 수평으로 파리를 포함하는 저장 유리 병은 유리 병 바닥에 축적 될 수있는 액체에 파리를 포획 방지 할 수 있습니다. </li><li> 약 15 시간 동안 인큐베이터에서 45 %의 습도에서 22 ℃에서 최소 21 시간 동안 파리를 품어. 12 시간 빛으로 인큐베이터를 설정 : 어두운주기를. </li></ol> 2. 등반 분석 <ol><li> 다음 날 아침, 250 ml의 유리에 하나의 유리 병에서 졸업 된 실린더 (20) 파리를 전송합니다. 그것을 일정하게 일상을 유지하기 위해 실린더의 위치를​​ 표시합니다. 유전자형 사이의 교차 오염을 방지하기 위해 하나의 유전자형 당 유리 실린더를 사용한다. 각 실험 및 RO의 끝에서 세탁유전자형 사이를 하므로. <ol><li> 온도 22 ° C와 40 %의 습도에서 각각 (비전의 잠재적 결함이있는 경우 또는 붉은 빛) 주변 광에 실험을 실시한다. 일 주기성 리듬 혼동을 피하기 위해 항상 날의 동일한 시간에 실험을 수행한다. </li></ol></li><li> 배리어 필름 (왁스 필름) 모든 파리의 탈출을 방지하기 위해 (그림 2)와 실린더의 상단을 밀봉합니다. </li><li> 삼각대에 비디오 카메라를 설정합니다. 250 ml를 190 ml의 라​​인에 초점 카메라는 실린더 (17.5 cm)를 졸업했다. </li><li> 실린더의 하단과 음식 바이알에서 죽은 파리의 수를 계산. 사망률로이 번호를 기록한다. </li><li> 아주 가볍게 내부 바닥면에 파리를 대체하기에 충분한 힘으로 반복적으로 폐쇄 셀 폼 패드에 대한 실린더를 누릅니다. 카메라에 기록을 눌러 다른 손을 사용하는 동안 10 배 - 5를 누릅니다. </li><li> 카메라의 &quot;녹음&quot;버튼을 누릅니다. </li><li> VID 시작EO 카메라 녹화와는 별개의 비 리듬 패턴으로 실린더를 여섯 번을 누릅니다. </li><li> 파리 마지막 도청되는 시간에서 2 분 동안 각각의 시험을 수행하고 선택된 각 시점에서 17.5 cm (190 ㎖) (매 10 초를 정량화)의 높이를 넘어 파리의 수를 기록한다. 참고 : 실린더에 표시 ml의 직경에 따라 하나의 실린더 모델마다 다를 것입니다. 오류를 방지하려면, 사용 된 각 실린더의 높이를 측정한다. </li><li> 시험이 종료되면, 95 % 에탄올 파리 폐기하십시오. </li><li> 모든 복제가 신선한 파리에 모든 시간을 테스트 할 때까지 반복 2.9에 2.1 단계를 반복합니다. 참고 : 5 복제가 돌연변이가 운동에 강한 영향을 충분히 할 수 있지만, (20) 파리 (200 파리)의 10 생물 복제는 작은 차이를 감지하는 것이 좋습니다. </li><li> 실험 종료 후, 실험실 세척기 건조 O에서 실린더 세척 / N이 다시 사용할 수있다. </li></ol> 3. 분석 <ol><li> Analy각각의 ZE 비디오는 시험 비행. 각각 10 초, 대상 회선을 통과 파리의 총 수를 기록한다. <ol><li> 파리는 다시 아래로 올라가서 또는 떨어지면, -1로 비행 기록은 아래로 다시 상승 또는 하락 비행과 같은 숫자로 목표 라인을 건너 다음 비행을 계산합니다. 15 일 플라이가 목표 선 이하로 떨어질 경우, 예를 들어, 다음 비행은 15 번째 비행이 아닌 16 일 고려 라인 (16 일 비행) 교차합니다. </li></ol></li><li> 재판에 남아있는 파리의 수를 얻기 위해 파리 (20)의 총 수에서 사망률을 뺍니다. 각 시점에서, 대상 회선 위의 파리의 분획을 얻었다. </li><li> 각 시점에서의 각각의 비율을 플롯 (도 3 참조). </li><li> 120 초의 데이터 점에서 성능을 분석하고 두 그룹이 존재하는 경우 또는 분산 분석과 페로 니 수정과 다중 비교를위한 사후 테스트 (계획되거나 계획되지 않은 협력을위한 Tukey에 대한 학생의 T-테스트를​​ 수행mparisons). 콜 모고 로프 - 스 미르 노프 (17)는 정상과 동일한 분산을 확인하기 위해 수행뿐만 아니라 컨트롤에 돌연변이 집단의 분포를 비교 테스트합니다. </li><li> 노화를 통해 데이터를 표시 진보적 인 적자 (그림 4)이 있는지 확인하기 위해 서로 다른 연령대의 파리 120 초에서 (2 일, 1 주, 2 주) 등반 파리의 비율을 플롯합니다. </li></ol>

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The authors have nothing to disclose.

초파리는 이미 파킨슨 병 (14) 및 다른 신경 퇴행성 조건 1, 2에 훌륭한 모델이 될 입증되었습니다. 초파리 가능한 유전 도구 외에도, 게놈이 높은 신경 질환 (19)에 관여하는 유전자를 위해 보존된다. (전체 엑솜 시퀀싱을 포함) 전체 게놈 유전자 스크리닝 방법의 출현은 인간의 운동 장애와 연관된 후보 유전자의 큰 목록을 계속 제공 할 가능성이있다. 이러한 ?...

파킨슨 병, 근 위축성 측삭 경화증, 및 유전성 강직성하지 마비와 같은 신경 퇴행성 장애 통렬한 점점 인식된다. 불행하게도, 이러한 신경 퇴행성 질환의 대부분은 치료없이 여전히. 이러한 유전자의 수가 증가하게되었다 전체 엑솜 시퀀싱으로 게놈 전체의, 편견 유전자 검사의 광범위한 임상 적 사용은 인간 기관차 장애에 연루된다. 이 진행에도 불구하고, 초기 말기에의 병리학 적 진행은, 이러한 장애의 어려운 남아있다. 초파리는 제어 된 공간 및 시간 방식으로 유전자 요구 사항을 공부에 대한 유전 적 도구를 하나 제공합니다. 또한, 초파리는 파킨슨 1, 알츠하이머 2, 지적 장애 3,4 및 다른 사람의 사이에서 간질 5,6 등의 신경 학적 상태에 대한 약물 스크리닝에 유용 입증되었습니다. 우리의 목표는 효과적인 비용을 개발하는 것이 었습니다및 신뢰성 분석 여전히 모터 성능의 작은 변화를 검출 할 정도로 민감 할 것이다 높은 처리량 분석을 허용 할 것이다. 초파리 등반 행동에 유전 적 변이 및 / 또는 환경 조건의 영향을 정량화하는 데 사용되는 여러 가지 분석이있다. 분석의 대부분은 부정적인 geotaxis, 또는 등반 분석으로 알려진 등반 파리의 자연적인 경향에 투자. Benzer 7 phototaxis의 연구에 사용되는 역류 장치도 gravitaxis을 연구하는데 사용될 수 있다고 1967 년 제안했다. 그 이후로, Ganetsky 8 및 많은 다른 사람 (9) -12은 초기 분석에 구축했다. 원리는 유리 병에 파리의 알려진 숫자를 배치하고 파리는 유리 병의 바닥에 떨어질 원인, 하드 표면에 강하게 유리 병을 활용할 수있다. 그것은 타고난 행동이기 때문에, 파리는 중력에 반대 유리 병의 정상에 오르려고 시도합니다. 이 분석은 정량적이다 measu얼마나 많은 파리 입술은 할당 된 시간 동안 유리 병에 마커 과거 올랐다. 속도 대신 파리 등반의 총 수의 측정은 신뢰할 수있는 매개 변수가되고 파리 기준의 수는 13으로 유의하지 않았다 경우에 결함을 보여 주었다. 등반 분석은 파킨슨 병 (14)를 포함하여 많은 신경 퇴행성 질환의 연구에 도움이 입증되었습니다. 그러나, 우리는 신경 퇴행이 이미 병리학 적 연구 (14)에서 볼 경우 기관차 결함이 시간에 감지하지 못할 수 있다고 지적했다. 따라서, 전통적인 분석의 사용은 질환의 발병 초기 ​​단계를 연구 할 수있는 능력을 제한 할 수있다. 병리의 나중 단계 동안 기관차 결함의 모양은 그 진행 완전한 구조가 너무 고급입니다 질병을 반영 할 수있다. 이것은 기존의 등반 분석의 감도와 잠재적 인 문제를 제기한다. tradi의 잠재적 인 무능력가벼운 기관차의 결함을 검출하는 분석을 등반적인는 파리가 상승해야되는 높이에 기인 할 수있다. 전통적인 분석 15,16 대책 파리의 수가 성공적으로 20 초에 10 2-5cm의 높이를 넘어야한다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Drosophila stocks</td>
			<td></td>
			<td></td>
			<td>The stocks are selected depending on the experiments. The temperature and humidity in the room and in the incubator must be controled and consistent to avoid flies being too staticky or too wet.</td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td></td>
			<td></td>
			<td>Any digital camcorder will do. Make sure they can focus on close object.</td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td>Kimble</td>
			<td>20028W</td>
			<td>Different models of graduated cylinder may have different diameter. It is therefore imporant to measure the height.</td>
		</tr>
		<tr>
			<td>computer</td>
			<td></td>
			<td></td>
			<td>Any model will do. We used the computer to monitor the climbing of the flies and record the number of flies at each time point</td>
		</tr>
	</tbody>
</table>

quantitative analysis of climbing defects in a drosophila model of neurodegenerative disorders

신경 퇴행성 질환으로 인한 기관차 결함은 무증상 변성 년 다음, 질병의 늦은 발병 증상이 될 수 있으며, 따라서 현재의 치료 치료 전략은 치료되지 않습니다. 전체 엑솜 서열의 사용을 통해 유전자의 증가는 인간의 보행에 역할을하는 것으로 확인되었다. 이 유전자를 식별에도 불구하고,이 유전자가 정상 기관차의 기능에 매우 중요 얼마나 알려져 있지 않다. 따라서, 새로운 치료 적 관심의 대상을 식별하기 위해 이들 유전자의 역할을 규명하기위한 모형 유기체를 이용하는 신뢰성있는 분석은, 어느 때보 다 필요하다. 우리는 이전 온화한 결함의 검출이 가능하며 시간이 지남에 따라 이들 결함을 평가하는 능력을 가지고 음극 geotaxis 에세이 민감화 버전을 설계했다. 유리에서 수행되는 분석은 왁스 배리어 막에 의해 밀봉되는 실린더를 졸업. 임계 거리를 증가시킴으로써, 17.5 ℃로 상승 될m은 우리가 가벼운 이동성 장애를 감지하는 민감도를 관찰 한 2 분에 실험 기간을 증가시키고. 분석은 효과적인 비용과 높은 재현성 결과를 얻기 위해 광범위한 훈련을 필요로하지 않습니다. 이것은 운동의 결함 초파리 돌연변이 체에서 후보 약물 스크리닝을위한 우수한 기술한다.

등반은 강력하고 재현 동작입니다. (- 30 초 25) 실제로, 햇 야생형 파리 빠르게 목표 거리 등반 성능에 도달한다. 돌연변이 파리는 대상에 오르는 무능력을 완료하는 가벼운 (또는 지연)에서 성능의 범위를 제시한다. 우리는 두 개의 서로 다른 돌연변이 대립 유전자와 여기를 보여줍니다. 첫번째는 spastin 유전자 (온천 5.75) (18)의 완전한 결실로 인한 ​​유전자 spastin의 심한 대립 유전자이다...

Watch this Scientific Journal Video about Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders at JoVE.com

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

퇴행성 신경 질환의 초파리 모델에서 등반 결함의 정량 분석

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

quantitative-analysis-climbing-defects-drosophila-model

Research

JoVE Journal

Neuroscience

18.3K 조회수.  University of Alberta. We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets. 

비디오: Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

전체의 전의 생체내의 Culturing, 개발 Drosophila의 뇌는

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

연구

신경과학

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

에 신약 표현형을 평가<em&gt; 초파리</em&gt; 등산 분석 실험에 의한 도파민 뉴런과 뇌 전체의 면역 염색

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

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.

글루타민산 염과 GABA의 빠른 마이크로 이온 도입 : 시냅틱 통합을 조사하는 유용한 도구

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

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

망막 리본 시냅스에서 막 수용체의 고해상도 양이 Immunogold 분석

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

의 시냅틱 변조 분석<em&gt; 노랑 초파리</em&gt; 장시간 빛에 노출 후 광수

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

태아 알코올 스펙트럼 장애의 쥐 모델에서 Hippocampal 기능 장애를 평가 하기 위해 추적 Eyeblink 클래식 컨디셔닝의 사용

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

FITC 표시 된 알 부 민 신경 질환의 마우스 모델에서의 정 맥 주입에 의해 혈액-뇌 장벽 침투성의 평가

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

초파리 에서 신경 돌기 Arborization 복잡성의 정량 분석

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

ImageJ를 사용 하 여 붙일 태그가 단백질의 편견된 분석 통해 초파리 에서 Neurodegeneration의 양적 세포 생물학

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

대규모 병렬 단일 핵 RNA 시퀀싱에 대 한 성인 척수 핵의 절연

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...