automated-high-throughput-behavioral-analyses-in-zebrafish-larvae

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

Brown University 

The techniques used to label neural tissue for specific antigens can vary significantly. Some immunostaining methods use free-floating tissue sections, whereas others use tissue sections mounted on slides. Mounting sections on glass slides before labeling the tissue with antigens is preferred method for neonatal tissue; processing young tissue by free-floating methods often destroy it. Surprisingly optimal temperature for storing tissue can vary with age. This study describes parameters developed to obtain robust staining of both young and old tissue. Our results show the most robust staining was found in tissue that was (1) stored at very low temperatures (-20 degrees C and -80 degrees C), (2) pretreated with 0.01% peroxide, and (3) entirely immersed in the staining solutions during immunohistochemistry.

Temperature, peroxide concentration, and immunohistochemical staining method affects staining intensity, distribution, and background.

Les techniques utilis&eacute;es pour les tissus nerveux &eacute;tiquette pour des antig&egrave;nes sp&eacute;cifiques peuvent varier consid&eacute;rablement. Certaines m&eacute;thodes d&#39;immunostaining utilisent des sections de tissu flottant librement, tandis que d&#39;autres utilisent des sections de tissu mont&eacute;es sur des glissi&egrave;res. Montage des sections sur lames de verre avant le tissu avec des antig&egrave;nes d&#39;&eacute;tiquetage constitue la m&eacute;thode recommand&eacute;e pour le tissu n&eacute;onatal ; traitement des jeunes tissus par m&eacute;thodes flottants souvent d&eacute;truire. Temp&eacute;rature &eacute;tonnamment optimale pour le stockage de tissus peut varier avec l&#39;&acirc;ge. Cette &eacute;tude d&eacute;crit les param&egrave;tres mis au point pour obtenir la coloration robuste de tissus de jeunes et moins jeunes. Nos r&eacute;sultats montrent la coloration plus robuste a &eacute;t&eacute; trouv&eacute;e dans les tissus (1) stock&eacute; &agrave; tr&egrave;s basse temp&eacute;rature (-20 degr&eacute;s C et -80 degr&eacute;s C), (2) pr&eacute;trait&eacute;s avec 0,01 % de peroxyde et (3) enti&egrave;rement immerg&eacute; dans les solutions de coloration au cours de l&#39;immunohistochimie.

Temp&eacute;rature, concentration de peroxyde et immunohistochemical souillant m&eacute;thode affecte coloration intensit&eacute;, distribution et arri&egrave;re-plan.

Le tecniche utilizzate per tessuto neurale di etichetta per gli antigeni specifici possono variare significativamente. Alcuni metodi immunostaining utilizzano sezioni di tessuto libero di fluttuare, mentre altri usano montate su vetrini di sezioni di tessuto. Montaggio sezioni su vetrini prima di etichettare il tessuto con antigeni &egrave; il metodo preferito per il tessuto neonatale; elaborazione giovane tessuto dai metodi digalleggiante spesso distruggerlo. Temperatura sorprendentemente ottimale per la conservazione dei tessuti pu&ograve; variare con l&#39;et&agrave;. Questo studio descrive i parametri sviluppati per ottenere la colorazione robusto tessuto vecchi e giovani. Nostri risultati mostrano la colorazione pi&ugrave; robusto &egrave; stata trovata nel tessuto che &egrave; stato (1) conservato a temperature molto basse (-20 gradi C e -80 gradi C), (2) pretrattate con 0,01% perossido e (3) interamente immersi nelle soluzioni di colorazione durante l&#39;immunoistochimica.

Temperatura, concentrazione di perossido e metodo di colorazione immunoistochimica influisce sulla colorazione intensit&agrave;, distribuzione e sfondo.

The magnocellular division of the medial Preoptic nucleus (MPN mag) plays a critical role in the regulation of male sexual behavior in the hamster. Results from previous studies indicated that the number of neurons in the MPN mag is greater in males than females but failed to find significant differences in the volume of the nucleus suggesting that other elements in the nucleus may be greater in the female. The results of the present study, using NeuN to identify neurons, are in line with this hypothesis. The data show that (1) neurons in the MPN mag display two distinct phenotypes, those with a single nucleolus and those with multiple nucleoli; (2) the percentage of each phenotype is sex specific, differing over the course of development and (3) there is no sex difference in the number of glial cells at any age. Sex differences in the numbers of each type are correlated with developmental milestones and suggest that morphological changes are influenced by changes in circulating gonadal steroids during development.

Neuronal composition of the magnocellular division of the medial preoptic nucleus (MPN mag) is sex specific in the Syrian hamster (Mesocricetus auratus).

La division magnocellulaire du noyau pr&eacute;optique m&eacute;dial (mag NPP) joue un r&ocirc;le essentiel dans la r&eacute;gulation du comportement sexuel des hommes chez le hamster. R&eacute;sultats d&#39;&eacute;tudes ant&eacute;rieures ont indiqu&eacute; que le nombre de neurones dans le mag de npp est plus grande chez les m&acirc;les que les femelles, mais pas de trouver des diff&eacute;rences significatives dans le volume du noyau, ce qui sugg&egrave;re que les autres &eacute;l&eacute;ments dans le noyau peuvent &ecirc;tre sup&eacute;rieure chez la femelle. Les r&eacute;sultats de cette &eacute;tude, &agrave; l&#39;aide de NeuN pour identifier les neurones, correspondent &agrave; cette hypoth&egrave;se. Les donn&eacute;es montrent que les neurones (1) dans le mag MPN affichent deux ph&eacute;notypes distincts, ceux avec un nucl&eacute;ole unique et ceux avec plusieurs nucl&eacute;oles ; (2) le pourcentage de chaque ph&eacute;notype est sexe sp&eacute;cifique, diff&eacute;rant au cours du d&eacute;veloppement et (3) il n&#39;existe aucune diff&eacute;rence de sexe dans le nombre de cellules gliales &agrave; tout &acirc;ge. Diff&eacute;rences entre les sexes dans les num&eacute;ros de chaque type sont corr&eacute;l&eacute;es aux &eacute;tapes importantes d&eacute;veloppementales et sugg&egrave;rent que des changements morphologiques sont influenc&eacute;es par des changements dans les st&eacute;ro&iuml;des gonadiques en circulation au cours du d&eacute;veloppement.

Composition neuronale de la division magnocellulaire du noyau pr&eacute;optique m&eacute;dial (mag NPP) est sexe sp&eacute;cifique chez le hamster syrien (Mesocricetus auratus).

La divisione magnocellulari del nucleo mediale Preoptic (mag MPN) svolge un ruolo critico nella regolazione del comportamento sessuale maschile nel criceto. Risultati da studi precedenti hanno indicato che il numero di neuroni nella mag MPN &egrave; maggiore nei maschi rispetto alle femmine, ma non &egrave; riuscito a trovare differenze significative nel volume del nucleo, suggerendo che altri elementi del nucleo possono essere maggiore nella femmina. I risultati del presente studio, utilizzando NeuN per identificare i neuroni, sono in linea con questa ipotesi. I dati mostrano che i neuroni (1) il mag MPN visualizzano due fenotipi distinti, quelli con un singolo nucleolo e quelli con pi&ugrave; nucleoli; (2) la percentuale di ogni fenotipo &egrave; sesso specifico, differendo nel corso di sviluppo e (3) non c&#39; non &egrave; alcuna differenza di sesso del numero di cellule gliali a qualsiasi et&agrave;. Le differenze di sesso nei numeri di ogni tipo sono correlate con pietre miliari dello sviluppo e suggeriscono che i cambiamenti morfologici sono influenzati da cambiamenti nella steroidi gonadici circolanti durante lo sviluppo.

Composizione neuronali della divisione magnocellulari del nucleo mediale preoptic (mag MPN) &egrave; sesso specifico nel criceto siriano (Mesocricetus auratus).

Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. The identification of these factors is facilitated by recent innovations in high-throughput screening. However, large-scale screening in whole organisms remains challenging, in particular when studying changes in brain function or behavior in vertebrate model systems. In this study, we present a novel imaging system for high-throughput analyses of behavior in zebrafish larvae. The three-camera system can image 12 multiwell plates simultaneously and is unique in its ability to provide local visual stimuli in the wells of a multiwell plate. The acquired images are converted into a series of coordinates, which characterize the location and orientation of the larvae. The developed imaging techniques were tested by measuring avoidance behaviors in seven-day-old zebrafish larvae. The system effectively quantified larval avoidance and revealed an increased edge preference in response to a blue or red 'bouncing ball' stimulus. Larvae also avoid a bouncing ball stimulus when it is counter-balanced with a stationary ball, but do not avoid blinking balls counter-balanced with a stationary ball. These results indicate that the seven-day-old larvae respond specifically to movement, rather than color, size, or local changes in light intensity. The imaging system and assays for measuring avoidance behavior may be used to screen for genetic and environmental factors that cause developmental brain disorders and for novel drugs that could prevent or treat these disorders.

A novel high-throughput imaging system for automated analyses of avoidance behavior in zebrafish larvae.

Le d&eacute;veloppement pr&eacute;coce du cerveau peut &ecirc;tre influenc&eacute; par de nombreux facteurs g&eacute;n&eacute;tiques et environnementaux, avec des effets durables sur le fonctionnement du cerveau et le comportement. L&#39;identification de ces facteurs est facilit&eacute;e par les r&eacute;centes innovations dans le criblage &agrave; haut d&eacute;bit. Toutefois, le d&eacute;pistage &agrave; grande &eacute;chelle dans tout l&#39;organisme reste ambitieuse, en particulier lorsque l&#39;on &eacute;tudie les changements dans le fonctionnement du cerveau ou comportement dans des syst&egrave;mes mod&egrave;les vert&eacute;br&eacute;s. Dans cette &eacute;tude, nous pr&eacute;sentons un nouveau syst&egrave;me d&#39;imagerie pour des analyses de haut d&eacute;bit de comportement chez les larves de poisson z&egrave;bre. Le syst&egrave;me de trois cam&eacute;ras peut l&#39;image 12 plaques multipuits simultan&eacute;ment et se distingue par sa capacit&eacute; de fournir des stimuli visuels les dans les puits d&#39;une plaque multipuite. Les images re&ccedil;ues sont convertis en une s&eacute;rie de coordonn&eacute;es, qui caract&eacute;risent l&#39;emplacement et l&#39;orientation des larves. Les techniques d&#39;imagerie avanc&eacute;s ont &eacute;t&eacute; test&eacute;s par la mesure des comportements d&#39;&eacute;vitement chez les larves de poisson z&egrave;bre &acirc;g&eacute; de sept jours. Le syst&egrave;me efficacement quantifi&eacute; &eacute;vitement larvaire et a r&eacute;v&eacute;l&eacute; une pr&eacute;f&eacute;rence accrue de bord en r&eacute;ponse &agrave; un stimulus &laquo; bouncing ball &raquo; bleu ou rouge. Larves aussi &eacute;viter un stimulus de balle qui rebondit quand il est &eacute;quilibr&eacute; avec une boule de stationnaire, mais n&#39;&eacute;vitent pas clignoter boules &agrave; contrepoids avec une boule de stationnaire. Ces r&eacute;sultats indiquent que les larves &acirc;g&eacute;es de sept jours r&eacute;pondent sp&eacute;cifiquement aux mouvement, plut&ocirc;t que de couleur, la taille ou les variations locales d&#39;intensit&eacute; lumineuse. Le syst&egrave;me d&#39;imagerie et les essais pour mesurer le comportement d&#39;&eacute;vitement peuvent &ecirc;tre utilis&eacute;s pour d&eacute;pister les facteurs g&eacute;n&eacute;tiques et environnementaux que cause cerveau developmental disorders and pour roman drogues qui pourrait emp&ecirc;cher ou traiter ces troubles.

Un roman haut d&eacute;bit syst&egrave;me d&#39;imagerie pour l&#39;analyse automatis&eacute;e des comportement d&#39;&eacute;vitement chez les larves de poisson z&egrave;bre.

Lo sviluppo iniziale del cervello pu&ograve; essere influenzato da numerosi fattori genetici e ambientali, con effetti duraturi sulla funzione del cervello e comportamento. L&#39;identificazione di questi fattori &egrave; facilitata dalle recenti innovazioni in high-throughput screening. Tuttavia, lo screening su larga scala in tutto organismi rimane impegnativo, in particolare quando si studia il comportamento in sistemi modello vertebrato o cambiamenti nella funzione del cervello. In questo studio, vi presentiamo un sistema di imaging romanzo per high-throughput di analisi del comportamento nelle larve di zebrafish. Il sistema del tre-fotocamera pu&ograve; immagine 12 piastre trasportatrici simultaneamente ed &egrave; unico nella sua capacit&agrave; di fornire stimoli visivi locali nei pozzetti di una piastra di vetrini. Le immagini acquisite sono convertite in una serie di coordinate, che caratterizzano la posizione e l&#39;orientamento delle larve. Le tecniche di imaging sviluppate sono state testate da misurare i comportamenti di evitamento in larve di zebrafish di sette giorni di et&agrave;. Il sistema efficacemente quantificato larvale evasione e ha rivelato una preferenza maggiore edge in risposta ad uno stimolo &#39;bouncing ball&#39; blu o rosso. Le larve anche evitare uno stimolo palla rimbalza quando &egrave; controbilanciato con una palla ferma, ma non evitare lampeggiante palle bilanciati con una palla ferma. Questi risultati indicano che le larve di sette giorni di et&agrave; rispondono specificamente a movimento, piuttosto che colore, dimensione o modifiche locali nell&#39;intensit&agrave; della luce. Il sistema di imaging e saggi per misurare il comportamento di evitamento possono essere utilizzati a schermo per fattori genetici e ambientali che causa dello sviluppo cervello disturbi e per il romanzo di droga che potrebbe prevenire o curare questi disturbi.

Un romanzo ad alta velocit&agrave; sistema di imaging per l&#39;analisi automatiche del comportamento di evitamento in larve di zebrafish.

Neurobehavioral disorders such as anxiety, autism, and attention deficit hyperactivity disorders are typically influenced by genetic and environmental factors. Although several genetic risk factors have been identified in recent years, little is known about the environmental factors that either cause neurobehavioral disorders or contribute to their progression in genetically predisposed individuals. One environmental factor that has raised concerns is chlorpyrifos, an organophosphate pesticide that is widely used in agriculture and is found ubiquitously in the environment. In the present study, we examined the effects of sub-chronic chlorpyrifos exposure on anxiety-related behavior during development using zebrafish larvae. We found that sub-chronic exposure to 0.01 or 0.1 Î¼M chlorpyrifos during development induces specific behavioral defects in 7-day-old zebrafish larvae. The larvae displayed decreases in swim speed and thigmotaxis, yet no changes in avoidance behavior were seen. Exposure to 0.001 Î¼M chlorpyrifos did not affect swimming, thigmotaxis, or avoidance behavior and exposure to 1 Î¼M chlorpyrifos induced behavioral defects, but also induced defects in larval morphology. Since thigmotaxis, a preference for the edge, is an anxiety-related behavior in zebrafish larvae, we propose that sub-chronic chlorpyrifos exposure interferes with the development of anxiety-related behaviors. The results of this study provide a good starting point for examination of the molecular, cellular, developmental, and neural mechanisms that are affected by environmentally relevant concentrations of organophosphate pesticides. A more detailed understanding of these mechanisms is important for the development of predictive models and refined health policies to prevent toxicant-induced neurobehavioral disorders.

Developmental sub-chronic exposure to chlorpyrifos reduces anxiety-related behavior in zebrafish larvae.

Neurocomportementales des troubles comme l&#39;anxi&eacute;t&eacute;, autisme et hyperactivit&eacute; troubles d&eacute;ficitaires de l&#39;attention sont g&eacute;n&eacute;ralement influenc&eacute;s par des facteurs g&eacute;n&eacute;tiques et environnementaux. Bien que plusieurs facteurs de risque g&eacute;n&eacute;tiques ont &eacute;t&eacute; identifi&eacute;es au cours des derni&egrave;res ann&eacute;es, on en sait peu sur les facteurs environnementaux qui causent des troubles neurocomportementaux ou contribuent &agrave; leur progression chez les individus g&eacute;n&eacute;tiquement pr&eacute;dispos&eacute;s. Un facteur environnemental qui a soulev&eacute; des pr&eacute;occupations est le chlorpyrifos, un pesticide organophosphor&eacute; qui est largement utilis&eacute; dans l&#39;agriculture et se trouve partout dans l&#39;environnement. Dans la pr&eacute;sente &eacute;tude, nous avons examin&eacute; les effets de l&#39;exposition subchronique chlorpyrifos sur le comportement li&eacute; &agrave; l&#39;anxi&eacute;t&eacute; au cours du d&eacute;veloppement &agrave; l&#39;aide de larves de poisson z&egrave;bre. Nous avons constat&eacute; que l&#39;exposition subchronique &agrave; 0,01 ou 0,1 &mu;M chlorpyrifos durant le d&eacute;veloppement induit des d&eacute;fauts comportements sp&eacute;cifiques chez les larves de poisson z&egrave;bre &acirc;g&eacute;s de 7 jours. Les larves affichent la diminution de la vitesse de nage et thigmotaxie, mais aucun changement dans le comportement d&#39;&eacute;vitement ont &eacute;t&eacute; observ&eacute;s. Exposition &agrave; 0,001 &mu;M chlorpyrifos n&#39;affecte pas le comportement de nage, thigmotaxie ou &eacute;vitement et exposition &agrave; 1 &mu;M chlorpyrifos induit des anomalies comportementales, mais aussi induit des d&eacute;fauts dans la morphologie des larves. Thigmotaxie, une pr&eacute;f&eacute;rence pour le bord, &eacute;tant un comportement li&eacute; &agrave; l&#39;anxi&eacute;t&eacute; chez les larves de poisson z&egrave;bre, nous proposons que l&#39;exposition subchronique chlorpyrifos interf&egrave;re avec le d&eacute;veloppement de comportements li&eacute;s &agrave; l&#39;anxi&eacute;t&eacute;. Les r&eacute;sultats de cette &eacute;tude fournissent un bon point de d&eacute;part pour l&#39;examen des m&eacute;canismes mol&eacute;culaires, cellulaires, d&eacute;veloppementaux et neurones qui sont affect&eacute;es par des concentrations de pesticides organophosphor&eacute;s. Une meilleure compr&eacute;hension de ces m&eacute;canismes est importante pour le d&eacute;veloppement de mod&egrave;les pr&eacute;dictifs et les politiques de sant&eacute; raffin&eacute; pour pr&eacute;venir les troubles neurocomportementaux induite par la substance toxique.

L&#39;exposition subchronique d&eacute;veloppementale de chlorpyrifos r&eacute;duit le comportement li&eacute; &agrave; l&#39;anxi&eacute;t&eacute; chez les larves de poisson z&egrave;bre.

Neurocomportamentali disturbi come l&#39;ansia, l&#39;autismo e disturbi dell&#39;iperattivit&agrave; deficit attenzione in genere sono influenzati da fattori genetici e ambientali. Anche se diversi fattori di rischio genetici sono stati identificati negli ultimi anni, poco &egrave; noto circa i fattori ambientali che causano disturbi neurocomportamentali o contribuiscono alla loro progressione in individui geneticamente predisposti. Un fattore ambientale che ha sollevato preoccupazioni &egrave; clorpirifos, un pesticidi organofosfati che sono ampiamente utilizzato in agricoltura ed sono trovato ubiquitously nell&#39;ambiente. Nel presente studio, abbiamo esaminato gli effetti dell&#39;esposizione di clorpirifos subcronica sul comportamento correlato all&#39;ansia durante lo sviluppo utilizzando larve di zebrafish. Abbiamo trovato che l&#39;esposizione subcronica a 0,01 o 0,1 &mu;M clorpirifos durante lo sviluppo induce specifici difetti comportamentali in larve di zebrafish di 7 giorni di et&agrave;. Le larve visualizzato diminuisce in velocit&agrave; di nuotata e tigmotassia, tuttavia sono stati osservati cambiamenti nel comportamento di evitamento. L&#39;esposizione a 0,001 &mu;M clorpirifos non influisce sulla piscina, tigmotassia o evitare il comportamento e l&#39;esposizione a 1 &mu;M clorpirifos indotta da difetti comportamentali, ma anche indotto difetti nella morfologia larvale. Poich&eacute; tigmotassia, una preferenza per il bordo, &egrave; un comportamento correlato all&#39;ansia in larve di zebrafish, proponiamo che l&#39;esposizione subcronica clorpirifos interferisce con lo sviluppo di comportamenti di ansia. I risultati di questo studio forniscono un buon punto di partenza per l&#39;esame dei meccanismi molecolari, cellulari, dello sviluppo e neurali che sono affetti da ecologicamente rilevanti concentrazioni di pesticidi organofosfati. Una pi&ugrave; dettagliata comprensione di questi meccanismi &egrave; importante per lo sviluppo di modelli predittivi e politiche sanitarie raffinato per prevenire disturbi neurocomportamentali tossico-indotta.

Developmental esposizione subcronica di clorpirifos riduce il comportamento correlato all&#39;ansia in larve di zebrafish.

The magnocellular division of the medial preoptic area (MPN mag) integrates pheromonal and hormonal signals to play a critical role in the expression of male typical sex behavior. The MPN mag contains two morphologically distinct neuronal populations; the percentage of each type within the nucleus is sex specific. Males have more neurons with a single nucleolus whereas females have more with multiple nucleoli. To determine which neuronal subtype mediates pheromonal induction of copulation, tissue from male and female hamsters exposed to female pheromones was immunolabeled for the immediate early protein (EGR-1). Subsequently the tissue was counterstained and the number of ERG-1 neurons with one or two nuclei was determined. The results indicate that pheromones stimulate neurons with single nucleoli in males but fail to stimulate either neuronal subtype in females suggesting that synaptic input to the MPN mag is sexually differentiated.

Exposure to female pheromones stimulates a specific type of neuronal population in the male but not female magnocellular division of the medial preoptic nucleus (MPN mag) of the Syrian hamster.

La division magnocellulaire de l&#39;aire pr&eacute;optique m&eacute;dial (mag NPP) int&egrave;gre un signal ph&eacute;romonale et hormonaux jouent un r&ocirc;le essentiel dans l&#39;expression du comportement masculin sexe typique. Le mag MPN contient deux populations neuronales morphologiquement distinctes ; le pourcentage de chaque type dans le noyau est sexe sp&eacute;cifique. Les hommes ont plus de neurones avec un nucl&eacute;ole unique tandis que les femelles ont plus avec plusieurs nucl&eacute;oles. Pour d&eacute;terminer quel sous-type neuronale m&eacute;die ph&eacute;romonale induction de la copulation, les tissus des hamsters m&acirc;les et femelles expos&eacute;s aux ph&eacute;romones femelles ont &eacute;t&eacute; immunomarqu&eacute;es pour la prot&eacute;ine d&eacute;but imm&eacute;diate (EGR-1). Par la suite le tissu &eacute;tait Eosine et le nombre de neurones de l&#39;ERG-1 avec un ou deux noyaux a &eacute;t&eacute; d&eacute;termin&eacute;. Les r&eacute;sultats indiquent que les ph&eacute;romones stimulent les neurones avec des nucl&eacute;oles unique chez les m&acirc;les mais ne parviennent pas &agrave; stimuler chaque sous-type neuronale chez les femelles, ce qui sugg&egrave;re que les entr&eacute;e synaptique &agrave; la mag npp est sexuellement diff&eacute;renci&eacute;e.

Exposition aux ph&eacute;romones femelles stimule un type sp&eacute;cifique de la population neuronale chez le m&acirc;le, mais la division non femelles magnocellulaire du noyau pr&eacute;optique m&eacute;dial (mag NPP) du hamster syrien.

Holly Richendrfer

Department of Molecular Biology,
Cell Biology and Biochemistry,
Department of Molecular Biology, Cell Biology and Biochemistry

Brown University

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

Holly Richendrfer

Department of Molecular Biology,Cell Biology and Biochemistry,Department of Molecular Biology, Cell Biology and Biochemistry

Brown University

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

Department of Molecular Biology,
Cell Biology and Biochemistry,
Department of Molecular Biology, Cell Biology and Biochemistry