Chciałbym podziękować Christopher Evans i Darren Burke z Centrum Integracyjne Studium Animal Behaviour (CISAB).

Jacky Dragons Jacky jaszczurki (A. muricatus) odbyły się w klimatyzowanym niewoli obiektów. Obudowy zostały wykonane z gotowych terrariów miarka 60 cm 3. Obudowy zostały również wypełnione podłoża Sydney piasku, liści rodzimych, okonie i naturalnych drewnianych. Obudowy zostały umieszczone w górnej części ruchome wózki, które pozwoliły na tematy, które będą umieszczone w środku wszystkie monitory widzenia. Jaszczurki zostały objęte 14L: 10D światło cyklu podczas symulowanych warunkach letnich i zostały dostarczone zarówno z lampami ciepła (125W) i odpowiednie ultrafioletowego (UV) oświetlenie (300W) w pierwszym pół godziny światła dziennego. Jaszczurki również zaopatrzone w wodę ad libitum, świerszcze karmione (Achetus domesticus) posypana witamin podczas sesji nieeksperymentalnych i rutynowo odrobaczone pasożytów przy użyciu rozcieńczonych dawek Iwermektyna. Sprzęt używany do odtwarzania Trzy platformy odtwarzanie wideo są wykorzystywane do prezentacji bodźca (patrz rys. 1). Centralna platformę składa się z iMac (Apple Computer Inc) zawierające bodźce kropka. Dwóch dodatkowych platform składa się z iMac z Final Cut Pro HD prezentacji bodźca, który zawierał bodźce odtwarzania (np. bezkręgowce). Sygnał cyfrowy jest przesyłany do zaawansowanych cyfrowych konwerter wideo (Canopus © gt ADVC110, Melbourne, Victoria Australia) dla cyfrowo-analogowej do odtwarzania plików video i wyświetlane na Sony Trintron (Sony Corporation, Minato, Tokio, Japonia) monitor (Series nr PVM-14M2A/PVM-14L2/PVM-14N5A). Canon (Nr kat. MV650i) kamera cyfrowa z 22x zoomem optycznym (Cannon Inc, Japonia) jest montowany bezpośrednio nad obszarem badań i zapisy reakcji na bodźce bezpośrednio do (Sony Corporation, Minato, Tokio, Japonia) Sony magnetowid ( Nr seryjny SLV-EZ717AS). Zarejestrowane obrazy są wysyłane do kolorowy wizjer (NEC, numer seryjny 1892, Kanagawa, Japonia). Na 7,5 sekundy od rozpoczęcia wyświetlania punktów, elektronicznie generowanych marker dźwięk jest wysyłany do Canopus ® ADVC110 i nagrywa na VHS. Odtwarzanie wideo jest dostosowana do PAL-DV (5:1 kompresji, rozdzielczość pozioma 575 linii, 25 klatek na s-1). Random-dot Kinematograms (RDKS) RDKS mogą być opracowane z wykorzystaniem VPixx V1.79 (VPixx V1.79, VPixx Technologies Inc 2002). Bodźce Design z 30 x 20 pól stopnia wypełnienia z 200 punktów, z każdego punktu pomiarowego 5 x 5 pikseli. Zbuduj bodźce dot z ośmioma zmiany prędkości w połączeniu z ośmiu poziomów spójności i dwóch kierunkach, tworząc 128 różnych bodźców. Parametry prędkości zostały zaprojektowane w stopniach na sekundę (° / s). Zmiany prędkości były o 0,5, 2,5, 5, 10, 20, 40, 80, i 160 ° / s. Procentowe spójności wynosiły 0, 2,5, 5, 10, 20, 40, 80, i 100% albo w lewo lub w prawo ruch kierunkowy. Eksport każdej sekwencji w formacie JPEG-B Motion pliku. Można jednak zaprojektować RDKS które wiele funkcji. Każda sekwencja jest w sumie 15 s czas trwania. Sekwencja zaczyna się od 2,5 s od 0% spójności dopasowane do zmiennej prędkości procesu. Okres ten rozpoczyna bieżącego procesu. To jest po 5 s zmienności w połączeniu szybkości i spójności. Sekwencja została zakończona w 7,5 s 100% w odpowiednim kierunku. Import wszystkich Motion JPEG-B sekwencji i edytować w programie Apple Final Cut Pro HD (Apple Computers Inc.) Dodawanie dźwięku marker w każdym przedziale. Znacznik ten jest słyszalny na VHS, i jest używany jako sygnał, gdy punktacji zachowania, gdy zmiany w prezentacji bodźca występuje. Oddana każdego z 128 sekwencje jak Final Cut Pro filmów. Primary wzmocnień - Mealworms Przedmiotów miejsce na losowo harmonogramu wzmocnienia pięciu prób na próby bloku (16 prób) w czasie trwania eksperymentu. Primary wzmocnień są mealworms (Tenebrio Molitor). Wtórnych wzmocnień - Animowane Bezkręgowce Wtórnych wzmocnień mają być połączone z głównym wzmocnieniem. Utworzono biologicznie znaczących wzmocnień, takich jak trzy typy bezkręgowców animowanego: 1) krykiet, 2) roztocza, i 3) pająka. Tworzenie animacji za pomocą Lightwave v8.3 (NewTek Inc, San Antonio, TX, USA) przez 15 s długości, z początkowego 7,5 s nadal w tle, a po 7,5 s od bezkręgowców na odpowiednim ekranie. Photograph tle naturalnej roślinności na import w formacie JPEG w pliku eah. Eksport każdej sekwencji na poszczególne sekwencje obrazów JPEG (375 klatek), i skompresować do pliku filmowego za pomocą Final Cut Pro HD do odtwarzania plików video. Projekt 50 unikalne klipy, aby zapobiec przyzwyczajeniu do paradygmatu klimatyzacji. Procedura ogólna Badanych miejsce między monitorami widzenia. Losowe prezentacji RDK bodźce dla każdego przedmiotu. Pokaż bodźców w blokach z 16 prób w ciągu ośmiu dni. Każda próba jest 15 s z intertrial różnych odstępach od 30-60 s. Segment każdego przedziału próby w 2,5 s od 0% spójności odzwierciedlają prędkość testowania, 5 s testów szybkości i spójności, a po 7,5 s 100% w odpowiednim kierunku dopasowane prędkości. W tym 5 s krytycznego okresu, przedmioty są wymagane na odpowiedź. Rekord zależy środki ukierunkowanie monitorowania w lewo lub prawo i opóźnienie reakcji. Użyj tylko pięć losowo prób z podstawowym wzmocnieniem i wzmacniają proces z animowanym bezkręgowców. Po zakończeniu każdej próby bloku, usuń temat obudowy z obszaru badań i wprowadzenia innej obudowie.

<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>

Finansowanie zostało poparte Award Podyplomowe CISAB i Zakład Psychologii Uniwersytet Macquarie, i Australian Research Council. Badania przeprowadzono w ramach etycznych z Macquarie University Zwierząt Komisji Etyki (2003/014) i New South Wales National Parks and Wildlife Services (S11024) z Australii.

Badanie sensoryczne zdolności gatunków jaszczurek było trudne, i był w dużej mierze zarówno nudny i żmudny proces. Trudności te wynikają z znalezienie właściwego wzmocnieniem lub ustanowieniem funkcjonalnego protokołu. Tutaj założyłem udanej protokół, w którym do badania percepcji ruchu u jaszczurek. Procedura ta może mieć zastosowanie również w badaniach innych cechach wizualnych, a także inne zmysły mechanizmów. Ponadto, procedura ta może być stosowana do innych gatunków. Użyteczność tego protokołu nie tylk...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>iMac</td>
<td>Computer</td>
<td>Apple Computers, Inc.</td>
<td>&nbsp;</td>
<td>Three systems</td>
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<td>Canopus&#174;</td>
<td>Digital-to-analog converter</td>
<td>Canopus&#174;</td>
<td>ADVC110</td>
<td>&nbsp;</td>
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<td>Sony Trintron</td>
<td>Colour viewfinder</td>
<td>Sony Corporation</td>
<td>PVM-14M2A, PVM-14L2, PVM-14N5A</td>
<td>Three monitors</td>
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<td>Canon</td>
<td>Digital camcorder</td>
<td>Cannon Inc.</td>
<td>MV650i</td>
<td>Records behavior</td>
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<td>NEC</td>
<td>Colour viewfinder</td>
<td>NEC</td>
<td>1892</td>
<td>View responses</td>
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<td>Sony VCR</td>
<td>VHS recorder</td>
<td>Sony Corporation</td>
<td>SLV-EZ717AS</td>
<td>Record behavior to VHS</td>
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<td>VHS tape</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Generic VHS</td>
<td>Recording media</td>
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<td>VPixx</td>
<td>Software program</td>
<td>VPixx Technologies, Inc.</td>
<td>v1.79</td>
<td>To create RDKs</td>
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<td>Final Cut Pro</td>
<td>Software program</td>
<td>Apple Computer, Inc.</td>
<td>HD</td>
<td>To create playback sequences</td>
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<td>Lightwave&#174; 3D</td>
<td>Software program</td>
<td>NewTek Inc.</td>
<td>v8.3</td>
<td>To create secondary reinforcers</td>
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<td>Mealworm</td>
<td>Tenebrio molitor</td>
<td>Pisces Enterprises LTD</td>
<td>&nbsp;</td>
<td>Primary reinforcer</td>
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<td>Cricket</td>
<td>Achetus domesticus</td>
<td>Pisces Enterprises LTD</td>
<td>&nbsp;</td>
<td>Food for husbandry</td>
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<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>
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<td>Globes</td>
<td>Spotone</td>
<td>Phillips</td>
<td>&nbsp;</td>
<td>125W, Heat lamps for lizards</td>
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<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>
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testing visual sensitivity to the speed and direction of motion in lizards

Testowanie wrażliwości wizualnej w każdym gatunku zawiera podstawowe informacje dotyczące zachowania, ewolucji i ekologii. Jednak testy specyficzne cechy układu wzrokowego dostarczyć więcej dowodów empirycznych na funkcjonalne aplikacje. Śledztwo w sprawie systemu sensorycznego zawiera informacje na temat zdolności sensoryczne, uczenia się i zdolność pamięci, i ustanawia znane zachowanie podstawowych, w których do oceny odchyleń (Burghardt, 1977). Jednak w przeciwieństwie do systemów ssaków lub ptaków, testy do nauki i zapamiętywania w gatunki gadów jest trudne. Ponadto, za pomocą operant paradygmat jako psychofizycznej miarą zdolności zmysłowych jest również jako trudne. Historycznie, gatunków gadów słabo reagowały na próby klimatyzowania ze względu na kwestie związane z motywacją, fizjologii, metabolizm i podstawowych cech biologicznych. Tutaj pokażę operant paradygmatu używane powieści gatunków jaszczurek modelu, Jacky smoka (Amphibolurus muricatus) i opisać jak testować obwodowej wrażliwości na najistotniejsze szybkość i cech ruchu. Metoda ta wykorzystuje innowacyjne podejście do oceny zdolności uczenia się i czuciowych u jaszczurek. I zatrudnić stosowanie losowych dot kinematograms (RDKS) do pomiaru wrażliwości na szybkość i manipulować poziom siły sygnału, zmieniając część punktów ruchu w spójny kierunek. RDKS nie stanowią biologicznie znaczący bodziec, angażuje układu wzrokowego i jest klasycznym psychofizycznej narzędzie do pomiaru wrażliwości u ludzi i innych zwierząt. Tutaj RDKS są wyświetlane za pomocą trzech jaszczurki odtwarzania video. Jaszczurek, aby wybrać kierunek (lewo lub w prawo), w którym dostrzec kropki się poruszać. Wybór odpowiedniego kierunku jest wzmocnione przez biologicznie ważne bodźce drapieżne, symulowane przez bezkręgowce animowanego.

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

Testowanie wrażliwości wizualnej u jaszczurek przy operant paradygmat klimatyzacji, która zatrudnia odtwarzania wideo losowych punktów, kinematograms i generowanych komputerowo bezkręgowców.

Badania wizualne Wrażliwość na prędkość i kierunek ruchu w Jaszczurki

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 Views.  Macquarie University. Testowanie wrażliwości wizualnej u jaszczurek przy operant paradygmat klimatyzacji, która zatrudnia odtwarzania wideo losowych punktów, kinematograms i generowanych komputerowo bezkręgowców.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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