나는 동물 행동의 통합 연구 (CISAB)의 센터에서 크리스토퍼 에반스와 대런 버크 감사드립니다.

재키 드레곤스 재키 도마뱀 (A. muricatus)는 기후 제어 포로 시설에서 개최되었습니다. 인클로저는 60cm 3 측정 가공 유리 terrariums의 건설되었다. 인클로저 또한 시드니 모래 기판, 기본 단풍, 자연 목재 날아 가득했습니다. 인클로저는 과목 모두보기 모니터의 중앙에 배치하는 허용되는 이동식 트롤리 위에 놓여 있었다. 시뮬레이션 여름 조건 중 10D 조명 사이클, 두 열 램프 (125W)와 적절한 자외선 (UV) 일광의 첫번째 반 시간 동안 조명 (300W)와 함께 제공되었습니다 도마뱀은 14L 아래 놓여 있었다. 도마뱀은 또한 물 광고 libitum, 자란 귀뚜라미 (Achetus 가정) 이외의 실험 세션 동안 비타민과 함께 찾아 봤어요, 그리고 정기적으로 이버멕틴의 희석 복용을 사용하여 기생충에 대한 wormed와 함께 제공되었습니다. 재생에 사용 장비 쓰리 동영상 재생 rigs는 자극 프리젠 테이션 (그림 1 참조)에 사용됩니다. 중앙 장비는 점 자극을 포함하는 아이맥 (애플 컴퓨터 주식 회사)로 구성되어 있습니다. 두 개의 추가 rigs 재생의 자극 (즉, 무척추 동물) 포함된 자극 프레 젠 테이션에 대한 최종 컷 프로 HD와 아이맥으로 구성되어 있습니다. 디지털 신호는 비디오 재생을위한 디지털 - 아날로그 변환에 대한 고급 디지털 비디오 컨버터 (카노푸스 © GT ADVC110, 멜버른, 빅토리아 호주)로 전송하고 소니 Trintron (소니 주식 회사,시나가와, 도쿄 일본) 모니터 (시리즈에 표시됩니다 Nos. PVM-14M2A/PVM-14L2/PVM-14N5A). 22X 광학 줌 (캐논 주식 회사, 일본)와 캐논 (모델 번호 MV650i) 디지털 캠코더 직접 시험 영역과 직접 소니 (소니 주식 회사,시나가와, 도쿄 일본) 비디오 카세트 레코더 (에 자극에 기록 응답 위에 장착할 수 있습니다 일련 번호 SLV - EZ717AS). 기록된 이미지는 컬러 뷰파인더 (NEC, 일련 번호 1892, 가나가와, 일본)에 발송됩니다. 도트 디스플레이의 시작부터 7.5 초, 전자 생성된 소리 마커가 VHS에 카노푸스 ® ADVC110 및 기록에 전송됩니다. 비디오 재생은 PAL - DV 표준 (; 수평 해상도 575 라인, 25 프레임 S - ​​1 5시 1분 압축)에 대한 조정됩니다. 랜덤 점 Kinematograms (RDKs) RDKs는 VPixx V1.79 (VPixx V1.79, VPixx 테크놀로지 주식 2002)를 사용하여 개발할 수 있습니다. 30 X 20도 필드와 함께 디자인 자극, 5 × 5 픽셀을 측정하는 각각의 점이 200 점 같이 입력합니다. 128 다른 자극을 생성하는 일관성 여덟 수준 및 두 방향과 결합 속도 8 변화와 도트 자극을 빌드합니다. 속도 매개 변수는 초당도 (° / s)로 설계되었습니다. 속도 차이는 0.5, 2.5, 5, 10, 20, 40, 80, 및 160 ° / s의되었습니다 일관성 비율도 왼쪽이나 오른쪽 방향 움직임에 0, 2.5, 5, 10, 20, 40, 80, 및 100 %되었습니다. 모션 JPEG - B 파일의 각 순서를 내보냅니다. 그러나 기능의 배열 RDKs을 디자인할 수 있습니다. 각 순서는 기간에 총 15 S입니다. 순서는 시험 속도 변수와 일치하는 0 % 기여도 2.5 S로 시작합니다. 이 기간은 현재 재판을 시작합니다. 이것은 속도와 일관성 조합의 변화 5의 다음이다. 순서는 적절한 방향으로 100 %의 7.5에 의해 완성되었다. 애플 파이널 컷 프로 HD (애플 컴퓨터 주식 회사)의 모든 모션 JPEG - B 시퀀스 및 편집을 가져옵니다. 각 간격으로 소리 마커를 추가합니다. 이 마커는 VHS에만 청취하고, 자극 프레 젠 테이션의 변화가 발생할 때 동작을 채점 때 큐로 사용됩니다. 파이널 컷 프로 영화로 128 시퀀스의 각 수출. 기본 reinforcers - Mealworms 실험하는 동안 차단 시험 당 다섯 시련 (16 실험)의 무작위 보강 일정 플레이스 과목. 기본 reinforcers은 mealworms (Tenebrio 몰리토)입니다. 보조 reinforcers - 애니메이션 무척추 동물 보조 reinforcers는 기본 reinforcer와 결합되어야하는 것입니다. 크리켓, 2) 진드기, 그리고 3) 스파이더 1) 같은 컴퓨터 애니메이션 무척추 동물의 세 가지 유형으로 생물 학적 의미 reinforcers를 만들었습니다. 초기 7.5은 여전히 배경으로 길이 15 s에 대한 Lightwave v8.3을 (NewTek 주식 회사, 샌안토니오, TX, 미국)를 사용하여 애니메이션을 생성하고, 해당 화면의 무척추 동물의 7.5의 다음. 포그래 클립에서 JPEG로 가져올 자연 식물의 배경을 tograph. 개별 JPEG 이미지 시퀀스 (375 프레임)에 각 순서를 수출하고, 비디오 재생에 대한 최종 컷 프로 HD를 사용하여 동영상 파일을 압축합니다. 에어컨 패러다임에 요법 이니을 방지하기 50 독특한 클립 디자인. 일반 절차 보는 모니터 사이 과목. 각 과목에 대한 RDK 자극의 프레 젠 테이션을 무작위. 팔일 동안 16 실험 단위로 자극을 표시합니다. 각각의 재판은 30-60에서 다양한 intertrial 간격 15 S 있습니다 S. 테스트 속도, 속도와 일관성을 테스트 5의, 그리고 속도와 일치하는 적절한 방향으로 100 % 7.5의 다음의 반사 0 % 기여도 2.5의 세그먼트로 각각의 시험 간격. 이 5의 중요한 - 기간 동안, 주제가 대응해야합니다. 답변을 왼쪽이나 오른쪽 모니터쪽으로 방향 및 지연과 같은 종속 조치를 기록합니다. 기본 reinforcer와 겨우 다섯 무작위 실험을 사용하여 애니메이션 무척추 각 재판을 강화. 각 블록 재판의 완성에서 테스트 영역에서 과목 케이스를 제거하고, 다른 인클로저를 소개합니다.

<ol>
	<li>Burghardt, G. M., Gans, C., Tinkle, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+processes+in+reptiles.">Learning processes in reptiles.</a> Biology of the reptilia. 7, 555-681 (1977).</li><li>VPixx, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Instruction Manual. VPixx Techniologies Inc. , 96 (2002).</li></ol>

자금은 맥쿼리 대학의 CISAB과 심리학과 대학원 수상하고, 호주 연구위원회에 의해 지원되었다. 연구는 맥쿼리 대학 동물 윤리위원회 (2003/014)와 뉴사우스 웨일즈 국립 공원과 호주의 야생 동물 서비스 (S11024)의 윤리 지침 내에서 실시했다.

도마뱀 종의 감각 능력을 테스트하는 것은 어려운되었으며, 대부분 지루하고 힘든 과정을 모두되었습니다. 이러한 어려움은 올바른 reinforcer를 찾거나 기능 프로토콜을 확립에서 줄기. 자, 내가 도마뱀의 모션 인식을 테스트하는 성공적인 프로토콜을 설립했습니다. 이 절차는 또한 다른 시각적 특성뿐만 아니라 다른 감각 메커니즘을 테스트 적용할 수 있습니다. 또한,이 절차는 다른 수종에 적용됩니다. 이 프로?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>iMac</td>
<td>Computer</td>
<td>Apple Computers, Inc.</td>
<td>&nbsp;</td>
<td>Three systems</td>
</tr>
<tr>
<td>Canopus&#174;</td>
<td>Digital-to-analog converter</td>
<td>Canopus&#174;</td>
<td>ADVC110</td>
<td>&nbsp;</td>
<tr>
<td>Sony Trintron</td>
<td>Colour viewfinder</td>
<td>Sony Corporation</td>
<td>PVM-14M2A, PVM-14L2, PVM-14N5A</td>
<td>Three monitors</td>
</tr>
<tr>
<td>Canon</td>
<td>Digital camcorder</td>
<td>Cannon Inc.</td>
<td>MV650i</td>
<td>Records behavior</td>
</tr>
<tr>
<td>NEC</td>
<td>Colour viewfinder</td>
<td>NEC</td>
<td>1892</td>
<td>View responses</td>
</tr>
<tr>
<td>Sony VCR</td>
<td>VHS recorder</td>
<td>Sony Corporation</td>
<td>SLV-EZ717AS</td>
<td>Record behavior to VHS</td>
</tr>
<tr>
<td>VHS tape</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Generic VHS</td>
<td>Recording media</td>
</tr>
<tr>
<td>VPixx</td>
<td>Software program</td>
<td>VPixx Technologies, Inc.</td>
<td>v1.79</td>
<td>To create RDKs</td>
</tr>
<tr>
<td>Final Cut Pro</td>
<td>Software program</td>
<td>Apple Computer, Inc.</td>
<td>HD</td>
<td>To create playback sequences</td>
</tr>
<tr>
<td>Lightwave&#174; 3D</td>
<td>Software program</td>
<td>NewTek Inc.</td>
<td>v8.3</td>
<td>To create secondary reinforcers</td>
</tr>
<tr>
<td>Mealworm</td>
<td>Tenebrio molitor</td>
<td>Pisces Enterprises LTD</td>
<td>&nbsp;</td>
<td>Primary reinforcer</td>
</tr>
<tr>
<td>Cricket</td>
<td>Achetus domesticus</td>
<td>Pisces Enterprises LTD</td>
<td>&nbsp;</td>
<td>Food for husbandry</td>
</tr>
<tr>
<td>Terrariums</td>
<td>Glass</td>
<td>Crystal Palace Aquariums</td>
<td>&nbsp;</td>
<td>Enclosure material for lizards (60 x 60 x 60 cm)</td>
</tr>
<tr>
<td>Globes</td>
<td>Spotone</td>
<td>Phillips</td>
<td>&nbsp;</td>
<td>125W, Heat lamps for lizards</td>
</tr>
<tr>
<td>Ultraviolet globes</td>
<td>Ultra-Vitalux</td>
<td>Osram</td>
<td>&nbsp;</td>
<td> 300W, UV lamps for lizards</td>
</tr>
<tr>
<td>Herptivite</td>
<td>&nbsp;</td>
<td>Rep-Cal Research Labs</td>
<td>&nbsp;</td>
<td>Vitamins: beta carotene, calcium, and vitamin D3</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

testing visual sensitivity to the speed and direction of motion in lizards

어떤 종류의 시각적 감도를 테스트하는 것은 행동, 진화 및 생태에 관한 기본적인 정보를 제공합니다. 그러나, 시각 시스템의 특정 기능을 테스트하는 기능을 응용 프로그램에 대한 더 많은 경험적인 증거를 제공합니다. 감각 시스템에 대한 조사는 감각 능력, 학습 및 기억 능력에 대한 정보를 제공하고, 편차를 측정에 알려진 기본 동작 (Burghardt, 1977)를 설정합니다. 그러나, 포유 동물이나 조류 시스템과는 달리, 파충류 종의 학습과 기억에 대한 테스트를하는 것은 어렵습니다. 또한, 감각 능력의 psychophysical 조치로 operant 패러다임을 사용하면 마찬가지로 어렵습니다. 역사적으로, 파충류 종의 때문에 동기 부여, 생리학, 신진 대사, 기본적인 생물 학적 특성과 관련된 문제의 조화 실험에 제대로 대응합니다. 여기 내가 operant 패러다임 소설 모델 도마뱀 수종을 사용 증명, 재키 드래곤 (Amphibolurus muricatus) 및 돌출 속도와 움직임의 특성에 주변 감도를 테스트하는 방법에 대해 설명합니다. 이 방법은 평가 학습과 도마뱀의 감각 능력에 대한 혁신적인 접근 방식을 사용합니다. 나는 감도 속도와 일관된 방향으로 이동 점의 비율을 변경하여 신호 강도의 수준을 조작할 수를 측정하는 임의의 점 kinematograms (RDKs)의 사용을 사용합니다. RDKs은 생물학적으로 의미있는 자극을 대표, 시각 시스템을 종사하고, 인간과 다른 동물의 감도를 측정하는 데 사용되는 고전적인 psychophysical 도구입니다하지 않습니다. 여기 RDKs 세 비디오 재생 시스템을 사용하여 도마뱀에 표시됩니다. 도마뱀은 그들이 점이 이동하는 인식하는 방향 (왼쪽 또는 오른쪽)을 선택하는 것입니다. 적절한 방향의 선택은 컴퓨터 애니메이션 무척추 동물에 의해 시뮬레이션 생물학적으로 중요한 먹이의 자극에 의​​해 강화됩니다.

Watch this Scientific Journal Video about Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards at JoVE.com

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

임의의 점 kinematograms과 컴퓨터 - 생성 무척추 동물의 비디오 재생을 고용 op​​erant 조절 패러다임을 사용하는 도마뱀의 시각적 감도를 테스트.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

testing-visual-sensitivity-to-speed-direction-motion

Research

JoVE Journal

Biology

11.4K 조회수.  Macquarie University. 임의의 점 kinematograms과 컴퓨터 - 생성 무척추 동물의 비디오 재생을 고용 op​​erant 조절 패러다임을 사용하는 도마뱀의 시각적 감도를 테스트.

비디오: Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

Microinjection을 사용 Aphids에 바이러스성 전송 생리 장벽을 테스트

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

연구

생물학

Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

성인 마우스의 후각 행동 테스트

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

마우스 콘 Photoreceptors에서 단일 셀 석션 레코딩

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

zebrafish의 시각 기능을 연구하기 위해 optokinetic 응답을 사용하여

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

인슐린 주입 및 성인에서 인슐린 민감도를 측정하는 Hemolymph 추출 Drosophila melanogaster</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

드로소필라의 온도 선호도 행동과 Circadian 리듬의 설계 및 분석

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

마우스 모델의 유효성 검사는 세포 고유의 방식으로 LINC 단지를 방해

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

동적 세포 돌출부에서 소프트웨어를 이용한 단백질 농도 추적을위한 그래픽 사용자 인터페이스

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

모기에 소설 Insecticidal 화학 물질의 독성을 테스트 하기 위한 프로토콜

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...