Vorrei ringraziare Christopher Evans e Darren Burke dal Centro per lo Studio del Comportamento Animale Integrativa (CISAB).

Jacky Dragons Lucertole Jacky (A. muricatus) sono stati trattenuti in strutture di prigioniero clima controllato. Recinti sono stati costruiti dei terrari in vetro realizzati da 60 cm 3. Recinzioni sono state riempite con substrato di sabbia Sydney, fogliame nativo, e posatoi in legno naturale. Recinti sono stati collocati in cima a carrelli mobili che ha permesso per i soggetti ad essere messi in mezzo tutti i monitor di visualizzazione. Lucertole sono stati posti sotto un 14L: 10D ciclo di luce in condizioni simulate estate, e sono stati forniti con entrambe le lampade di calore (125W) e ultravioletti adeguata (UV) illuminazione (300W) durante la prima mezz&#39;ora di luce. Lucertole sono stati forniti anche con acqua ad libitum, grilli nutriti (Achetus domesticus) spolverato con vitamine durante le sessioni non sperimentali, sverminati e regolarmente per i parassiti usando dosi diluite di Ivermectina. Usate per la riproduzione Tre impianti di riproduzione video vengono utilizzati per la presentazione dello stimolo (vedi Figura 1). L&#39;impianto centrale è costituito da un iMac (Apple Computer Inc.) contenente stimoli punto. I due impianti aggiuntivi consistono di un iMac con Final Cut Pro HD per la presentazione dello stimolo che contenevano la riproduzione di stimoli (per esempio, invertebrati). Il segnale digitale viene inviato a un convertitore video digitale avanzato (© gt Canopus ADVC110, Melbourne, Victoria, Australia) per il digitale-analogico di conversione per la riproduzione di video e visualizzati su un Trintron Sony (Sony Corporation, Shinagawa, Tokyo, Giappone), monitor (Serie nn. PVM-14M2A/PVM-14L2/PVM-14N5A). A (Modello n. MV650i) videocamera digitale Canon con zoom ottico 22X (Cannon Inc., Giappone) è montato direttamente sopra l&#39;area test e le risposte agli stimoli dati direttamente ad un (Sony Corporation, Shinagawa, Tokyo, Giappone) videoregistratore Sony ( Numero di serie SLV-EZ717AS). Le immagini registrate vengono inviati a un mirino a colori (NEC, Serial No. 1892, Kanagawa, Giappone). A 7.5 secondi dalla partenza del display punti, un marker del suono elettronico generato viene inviato ad un ® Canopus ADVC110 e registra su VHS. La riproduzione video è regolato per PAL-DV standard (5:1 di compressione, risoluzione orizzontale 575 linee, 25 fotogrammi s-1). Casuale-puntino Kinematograms (RDKs) RDKs possono essere sviluppate utilizzando VPixx V1.79 (VPixx V1.79, VPixx Technologies Inc. 2002). Stimoli di design con una x 30 20 gradi di campo, riempire con 200 punti, con ciascun punto di misura 5 x 5 pixel. Costruire stimoli punto con otto variazioni di velocità combinata con otto livelli di coerenza e di due direzioni, generando 128 diversi stimoli. Parametri di velocità sono stati progettati in gradi al secondo (° / s). Variazioni di velocità sono stati 0,5, 2,5, 5, 10, 20, 40, 80 e 160 ° / s. Coerenza percentuali erano 0, 2,5, 5, 10, 20, 40, 80 e 100% in un movimento a sinistra oa destra direzionale. Esportare ogni sequenza in formato JPEG-B file di Motion. Tuttavia, è possibile progettare il RDKs che una serie di funzioni. Ogni sequenza è un totale di 15 s di durata. La sequenza inizia con 2,5 s dello 0% corrisponde coerenza con la variabile velocità di prova. Questo periodo inizia il processo in corso. Questo è seguito da 5 s della variazione in combinazione di velocità e coerenza. La sequenza è stata completata da 7,5 s del 100% nella direzione appropriata. Importare tutti JPEG-B sequenze di movimento e modificare in Apple Final Cut Pro HD (Apple Computer Inc.). Aggiungi un marcatore suono ad ogni intervallo. Questo marcatore è solo udibile a VHS, ed è usato come spunto il punteggio comportamento quando le variazioni di presentazione dello stimolo si verificano. Esportati ciascuno dei 128 sequenze come i film di Final Cut Pro. Rinforzi primari - vermi della farina Soggetti luogo su un programma di rinforzo di cinque studi clinici randomizzati per blocco di prova (16 prove) durante l&#39;esperimento. Rinforzi primari sono vermi della farina (Tenebrio molitor). Rinforzi secondari - Invertebrati animati Rinforzi secondari devono essere accoppiato con il rinforzo primario. Creato rinforzi biologicamente significativo, ad esempio tre tipi di animazione computerizzata invertebrati: 1) grillo, 2) acaro, e 3) ragno. Creare animazioni utilizzando Lightwave V8.3 (NewTek Inc., San Antonio, TX, USA) per 15 s di lunghezza, con l&#39;iniziale di 7,5 s ancora in fondo, e seguita da 7,5 s di invertebrati nella schermata appropriata. PhoTOGRAFIA uno sfondo di vegetazione naturale di importare in formato JPEG in clip di eah. Esportare ogni singola sequenza in sequenze di immagini JPEG (375 fotogrammi), e comprimere in un file video utilizzando Final Cut Pro HD per la riproduzione video. Disegno 50 clip unica per evitare assuefazione al paradigma di condizionamento. Procedura generale Soggetti tra i monitor di visualizzazione. Randomize presentazione di stimoli RDK per ogni soggetto. Mostra gli stimoli in blocchi di 16 studi più di otto giorni. Ogni prova è di 15 s con intervalli tra le prove variando 30-60 s. Segmento di ogni intervallo di prova in 2,5 s di 0 coerenza% riflettente di velocità di prova, 5 s di test di velocità e coerenza, e seguita da 7,5 s del 100% in direzione appropriata abbinati con la velocità. Durante questo 5 s critico-periodo, i soggetti sono tenuti a rispondere. Record misure dipendenti come orientamento verso un monitor a sinistra oa destra e la latenza di rispondere. Utilizzare solo cinque prove casuale con un rinforzo primario e si rafforzano reciprocamente prova con un invertebrato animato. Al termine di ogni prova blocco, aprire le parti chiuse soggetto dalla zona di prova, e introdurre un altro recinto.

<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>

Il finanziamento è stato supportato dal premio post-laurea CISAB e Dipartimento di Psicologia dell&#39;Università Macquarie University, e il Consiglio di ricerca australiana. La ricerca è stata condotta secondo le linee guida etiche dell&#39;Università Macquarie comitato etico degli animali (2003/014) e il New South Wales National Parks e servizi naturalistici (S11024) dell&#39;Australia.

Test della capacità sensoriale di specie di lucertola è stata difficile, ed è stata in gran parte sia un processo noioso e faticoso. Queste difficoltà derivano da trovare il rinforzo giusto o stabilire un protocollo funzionale. Qui, ho stabilito un protocollo di successo in cui sperimentare la percezione del movimento in lucertole. Questa procedura può essere applicata anche in altri test caratteristiche visive così come altri meccanismi sensoriali. Inoltre, questa procedura può essere applicabile ad altre specie. L&#39;utilità di q...

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

Test di sensibilità visiva in qualsiasi specie fornisce informazioni di base sul comportamento, l&#39;evoluzione, e l&#39;ecologia. Tuttavia, i test caratteristiche specifiche del sistema visivo fornire prove più empirica per applicazioni funzionali. Indagine sul sistema sensoriale fornisce informazioni sulla capacità sensoriale, l&#39;apprendimento e la capacità di memoria, e stabilisce il comportamento di base noto in cui misurare le deviazioni (Burghardt, 1977). Tuttavia, a differenza dei sistemi di mammifero o aviaria, i test per l&#39;apprendimento e la memoria in una specie di rettili è difficile. Inoltre, utilizzando un paradigma operante come misura di capacità psico sensoriali come è altrettanto difficile. Storicamente, specie di rettili hanno risposto male ai processi di condizionamento a causa di problemi legati alla motivazione, la fisiologia, il metabolismo, e caratteristiche biologiche di base. Qui, dimostrare un paradigma operante utilizzato un romanzo specie di lucertole modello, il drago Jacky (Amphibolurus muricatus) e descrivere come testare la sensibilità periferica alla velocità e le caratteristiche salienti del movimento. Questo metodo utilizza un approccio innovativo per l&#39;apprendimento e la capacità di valutazione sensoriale nelle lucertole. Io impiegare l&#39;uso di casuale-puntino kinematograms (RDKs) per misurare la sensibilità alla velocità, e manipolare il livello di potenza del segnale, modificando la percentuale di punti in movimento in una direzione coerente. RDKs non rappresentano uno stimolo biologicamente significativo, coinvolge il sistema visivo, ed è uno strumento classico psicofisico utilizzato per misurare la sensibilità negli esseri umani e altri animali. Qui, RDKs vengono visualizzati lucertole con tre sistemi di riproduzione video. Lucertole sono per selezionare la direzione (destra o sinistra) in cui percepiscono punti per essere in movimento. Selezione di direzione appropriata è rafforzata da preda stimoli biologicamente importanti, simulato dal computer-animated invertebrati.

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

Test di sensibilità visiva in lucertole utilizzando un paradigma di condizionamento operante che si avvale di riproduzione video di casuale-puntino kinematograms e generati dal computer invertebrati.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

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. Test di sensibilità visiva in lucertole utilizzando un paradigma di condizionamento operante che si avvale di riproduzione video di casuale-puntino kinematograms e generati dal computer invertebrati.

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.

Test della barriere fisiologiche di trasmissione virale in Afidi Utilizzando Microiniezione

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

Ricerca

Biologia

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.

Olfattivo test comportamentali nel topo adulto

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.

Unicellulari registrazioni di aspirazione da Fotorecettori Cone mouse

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.

Utilizzando la risposta optocinetico per studiare la funzione visiva del pesce zebra

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.

L&#39;insulina per iniezione ed estrazione emolinfa per misurare la sensibilità all&#39;insulina nei adulti 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.

Progettazione e analisi del comportamento delle preferenze di temperatura e del suo ritmo circadiano 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.

La convalida di un modello di topo per Disrupt LINC Complessi in modo specifico-Cell

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.

Un&#39;interfaccia utente grafica per il monitoraggio assistito della concentrazione di proteine ​​in protrusioni cellulari dinamiche

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.

Applicazione di microscopia di velocità ad alta velocità super-risoluzione in Live cilio primario

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.

Protocolli per testare la tossicità di romanzo chimiche insetticide per le zanzare

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