Hayvan Davranış Bütünleştirici Çalışması (CISAB) Merkezi Christopher Evans ve Darren Burke teşekkür etmek istiyorum.

Jacky Ejderhalar Jacky kertenkele (A. muricatus), iklim kontrollü esir tesislerinde yapıldı . Kutuları 60 cm 3 ölçüm fabrikasyon cam terraryumlar inşa edildi. Kutuları da Sydney kum substrat, doğal bitki örtüsü ve doğal ahşap tünemiş ile doluydu. Kutuları konularda tüm görüş monitörler ortasında yer için izin verdiğini hareketli arabaları üstüne yerleştirildi. Simüle yaz şartlarında 10D ışık döngüsü sırasında, hem de ısı lambaları (125W) ve yeterli ultraviyole (UV), ilk yarım saat boyunca gün ışığı aydınlatma (300W) ile verilmiştir: Kertenkeleler 14L altına yerleştirildi . Kertenkeleler de, su ad libitum beslenen cırcır (Achetus domesticus), deneysel olmayan oturumları sırasında vitaminleri ile tozunu ve rutin Ivermectin sulandırılmış dozlarda kullanılarak parazitler için wormed verildi. Oynatma için ikinci el ekipman Üç video oynatma kuleleri uyaran sunum (bkz. Şekil 1) için kullanılır. Merkezi teçhizat nokta uyaranlara içeren bir iMac (Apple Computer Inc.) Oluşur. Final Cut Pro HD oynatma uyaranlara (yani, omurgasızlar) içeren teşvik tanıtımı için bir iMac iki ek kulesi oluşur. Video oynatma için dijital-analog dönüşüm için gelişmiş bir dijital video dönüştürücü (Canopus © gt ADVC110, Melbourne, Victoria Avustralya) dijital sinyal gönderilir ve Sony Trintron (Sony Corporation, Şinagava, Tokyo Japonya) monitör (Seri görüntülenen Nos PVM-14M2A/PVM-14L2/PVM-14N5A). 22X optik zoom (Cannon A.Ş., Japonya) ile Canon (Model No MV650i) dijital video kamera, doğrudan test alanı ve doğrudan bir Sony (Sony Corporation, Şinagava Tokyo, Japonya) video kaset kaydedici (uyaranlara kayıtları tepkiler üzerine monte Seri No SLV-EZ717AS). Kaydedilen görüntüler, bir renk vizör (NEC, Seri No 1892, Kanagawa, Japonya) gönderilir . Nokta ekranın başından itibaren 7,5 saniye, elektronik olarak üretilen ses işaretçisi VHS üzerine Canopus ® ADVC110 ve kayıtları gönderilir. Video oynatma PAL-DV standart (05:01 sıkıştırma, yatay çözünürlük 575 satır, 25 kare s-1) için ayarlanır. Rasgele nokta Kinematograms (RDKs) RDKs VPixx V1.79 (VPixx V1.79, VPixx Teknolojileri A.Ş. 2002) kullanılarak geliştirilmiştir. Tasarım uyaranlara 30 x 20 derece görüş alanı, her nokta ile 5 x 5 piksel ölçümü, 200 nokta ile doldurun. 128 farklı uyaranlara üreten sekiz düzeyde tutarlılık ve iki yönde ile birlikte sekiz hız varyasyonlar, nokta uyaranlara oluşturun. Hız parametreleri saniyede derece (° / s) olarak dizayn edilmiştir. 0.5, 2.5, 5, 10, 20, 40, 80, ve 160 ° / s Hız varyasyonlar Tutarlılık yüzdeleri ya sol ya da sağ yönlü hareket, 0, 2,5, 5, 10, 20, 40, 80, ve% 100 . Motion JPEG dosyası olarak her sıra ihracat. Ancak, bir dizi özellik RDKs tasarlayabilirsiniz. Her dizisi toplam süresi 15 sn. Sıra deneme hız değişkeni ile eşleşen 0% tutarlılık 2.5 sn ile başlar. Bu süre, mevcut çalışma başlatır . Bu değişim hız ve tutarlılık 5 s tarafından takip edilmektedir. Dizisi uygun yönde 7.5 s tarafından% 100 tamamlandı. Apple, Final Cut Pro HD (Apple Computers Inc.) İthalat tüm Motion JPEG-B dizileri ve düzenleyebilirsiniz. Her aralıkta bir ses işareti ekleyin. Bu marker, VHS için sadece sesli ve uyaran sunum değişiklik meydana geldiğinde davranış puanlama bir işaret olarak kullanılır. Final Cut Pro film gibi her biri 128 dizilerinin ihraç etti . İlköğretim reinforcers - mealworms Randomize bir deney sırasında bloğu deneme başına beş çalışma (16 çalışma) takviye programı yerleştirin konuları. İlköğretim reinforcers mealworms (Tenebrio Molitor). İkincil reinforcers - Hareketli Omurgasızlar İkincil reinforcers birincil güçlendiricidir ile eşleştirilmiş. 1): Böyle bir bilgisayar animasyonlu omurgasızlar üç çeşit biyolojik olarak anlamlı reinforcers, kriket, 2) akar, ve 3) örümcek düzenlendi. Ilk 7.5 sn hala arka plan, uzunluğu 15 s Lightwave v8.3 (Newtek Inc, San Antonio, Teksas, ABD) kullanılarak animasyonlar oluşturun ve uygun ekran omurgasız 7.5 s izledi. Phoeah klip JPEG olarak almak için doğal bitki örtüsünün bir arka plan tograph. Bireysel JPEG görüntü dizileri (375 kare) her dizisi İhracat ve Final Cut Pro HD video oynatımı için kullanarak bir film dosyası içine sıkıştırmak. Klima paradigma alışkanlık önlemek için 50 benzersiz klipleri tasarlayın. Genel prosedür Konularda görüş monitörler arasında yer. Her konu için RDK uyaranların sunum Rastgele. Sekiz gün içinde 16 çalışmaların bloklar halinde uyaranlara göster. Her deneme 30-60 arasında değişen intertrial aralıklarla 15 s s Segment 2.5 s 0% tutarlılık, test hızı, hız ve tutarlılık testi 5 s ve 7.5 s hız ile eşleşen uygun yönde% 100 takip yansıtıcı içine her deneme aralığı . Bu 5 kritik dönemde, konu, cevap vermek zorunda . Cevap için sol veya sağ monitör doğru yönelim ve gecikme gibi bağımlı önlemler kaydedin. Birincil güçlendiricidir sadece beş rasgele denemeler kullanın ve animasyonlu bir omurgasız her deneme güçlendirmek. Her blok deneme bitiminde test alanından tabi muhafazaları kaldırmak ve başka bir muhafaza tanıtmak .

<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>

Finansman CISAB Macquarie Üniversitesi Psikoloji Bölümü Yüksek Lisans Ödülü ve Avustralya Araştırma Kurumu (TÜBİTAK) tarafından desteklenmiştir. Araştırma Macquarie Üniversitesi Hayvan Etik Kurulu (2003/014) ve Yeni Güney Galler Milli Parklar ve Yaban Hayatı Hizmetleri Avustralya (S11024) etik kurallar içinde yapılmıştır.

Test kertenkele türü duyusal kapasiteye zor olmuştur ve büyük ölçüde, hem de yorucu ve zorlu bir süreç olmuştur. Bu zorluklar, doğru güçlendiricidir bulma ya da fonksiyonel bir protokol kuran kök. Burada, kertenkele hareket algılama test etmek için başarılı bir protokol kurdu. Bu işlem aynı zamanda diğer görsel özelliklerin yanı sıra, diğer duyusal mekanizmaları test geçerli olabilir. Buna ek olarak, bu yordamı, diğer türler için geçerli olabilir. Bu protokolün yararlılığı sadece bize doğal davranı?...

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		<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

Bir tür görsel duyarlılık testi, davranış, evrim ve ekoloji ile ilgili temel bilgiler sağlar. Ancak, görsel sistem belirli özellikleri test, fonksiyonel uygulamalar için daha fazla ampirik kanıt. Duyu sistemi soruşturma duyusal kapasitesi, öğrenme ve hafıza yeteneği hakkında bilgi sağlar ve sapmaları ölçmek için bilinen temel davranış (Burghardt, 1977) kurar. Ancak, memeli ve kuş sistemlerin aksine, bir sürüngen türü, öğrenme ve hafıza için test zordur. Ayrıca, duyu yeteneği psikofiziksel tedbir olarak aynı şekilde bir edimsel paradigma kadar zor. Tarihsel olarak, sürüngen türleri, motivasyon, fizyoloji, metabolizma, ve temel biyolojik özellikleri ile ilgili sorunlar nedeniyle klima çalışmalarda yetersiz yanıt verdiler. Burada, edimsel paradigma bir roman model kertenkele türü göstermek Jacky ejderha (Amphibolurus muricatus) ve çevresel duyarlılık göze çarpan hız ve hareket özellikleri test etmek için nasıl tarif. Bu yöntem, öğrenme ve kertenkeleler duyusal kapasite değerlendirilmesi yenilikçi bir yaklaşım kullanır. Ben hassasiyetini hızı ve sinyal gücü düzeyi, tutarlı bir yönde hareket eden noktalar oranı değiştirerek işlemek için ölçmek için rasgele nokta kinematograms (RDKs) kullanımı istihdam etmektedir. Biyolojik olarak anlamlı bir uyaran RDKs temsil, görsel sistem yürütmektedir, insanlar ve diğer hayvanlar duyarlılığını ölçmek için kullanılan klasik bir psikofiziksel aracı yoktur. Burada, RDKs üç video oynatma sistemlerini kullanarak kertenkele görüntülenir. Kertenkeleler hareket eden noktalar olarak algıladığımız hangi yönü (sol veya sağ) seçin. Bilgisayar animasyonlu omurgasızlar tarafından simüle biyolojik olarak önemli av uyaranlara uygun yönde seçimi ile takviye edilmiştir.

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

Edimsel koşullanma paradigması kullanarak rasgele nokta kinematograms ve bilgisayar tarafından oluşturulan omurgasızlar video oynatma kullanan bir kertenkele görsel duyarlılık testi.

Kertenkeleler Hareket Hız ve Yön Visual Hassasiyet Test

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 Görüntüleme.  Macquarie University. Edimsel koşullanma paradigması kullanarak rasgele nokta kinematograms ve bilgisayar tarafından oluşturulan omurgasızlar video oynatma kullanan bir kertenkele görsel duyarlılık testi.

Video: 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.

Mikroenjeksiyon kullanarak Yaprak bitleri Viral İletim Fizyolojik Engelleri Test

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

Biyoloji

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.

Yetişkin Fare Olfaktör Davranış Testi

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.

Tek hücreli fare Koni fotoreseptörlerin Emiş Kayıtlar

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.

Zebrafish görme fonksiyonu çalışma optokinetik yanıt

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.

İnsülin Enjeksiyon ve Yetişkin İnsülin Hassasiyet Tedbir hemolimf Ekstraksiyon 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.

Drosophila'da Sıcaklık Tercih Davranışının ve Sirkadiyen Ritminin Tasarımı ve Analizi

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.

Fare Modeli Doğrulanması Hücre-spesifik şekilde LINC kompleksler bozacak

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.

Dinamik Hücresel Protrüzyonlarda Protein Konsantrasyonunun Yazılım Destekli Takibi için Grafiksel Bir Kullanıcı Arayüzü

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.

Yüksek hızlı süper çözünürlük hızı mikroskobu canlı birincil Cilium içinde uygulanması

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.

Sivrisinekler için roman böcek kimyaları toksisite testleri için iletişim kuralları

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