Vorremmo ringraziare tutti i membri del nostro laboratorio per i loro consigli, utili discussioni e commenti sul manoscritto. Vorremmo inoltre particolarmente ringraziare Marta Koch e Bassem Hassan per il suggerimento eccellente che aspettiamo fino a 1,5 ore APF prima sezionare il cervello per la coltura in esperimenti di rimodellamento. Ringraziamo anche la possibilità di imaging NHGRI per l&#39;utilizzo del loro microscopio confocale. Questo lavoro è stato sostenuto dal Programma Neuroscienze di base del Programma di NINDS ricerca intramurale, NIH (NS003106 Z01).

A. Preparazione di Media <ol><li> Preparare mezzi completi da filtro sterilizzante Schneider Drosophila mezzo supplementato con 10% siero fetale bovino e 1% di penicillina / streptomicina. </li><li> Supporto deve essere preparato fresco ogni volta. In alternativa, una nuova aliquota di pre-congelati i media possono essere scongelati prima di ogni utilizzo. </li><li> Prima di coltura, aggiungere 10 mg / ml di insulina e 2 mg / ml ecdisone ai media. I farmaci sono stati preparati come scorte concentrati e congelata in piccole aliquote. Aliquote non erano scongelati ricongelato. </li><li> Tutte le soluzioni di lavoro deve essere mantenuta a 25 ° C. </li></ol> La coltivazione di cervelli larvali 1. Selezione e dissezione di cervelli larvali <ol><li> Aggiungere 500 microlitri di media per un inserto di 12 millimetri coltura cellulare Millicell. I cervelli sezionati saranno inseriti in questi inserti per facilitare immunostaining facile. </li><li> Aggiungere una piccola quantità di supporti preparati per un vetro da orologio dissezione. </li><li> Ucantare una piccola spatola, raccogliere vagare larve terzo stadio e metterli in vetro di orologio. (Si presume che il metodo di coltura ex vivo si applica altrettanto bene al primo o al secondo stadio le larve, ma non abbiamo testato questo.) </li><li> Tenendo l&#39;estremità posteriore della larva con una coppia di pinze, accuratamente aprire l&#39;estremità anteriore con una seconda coppia di pinze # 5. </li><li> Rapidamente sezionare il cervello, mantenendo il cordone nervoso ventrale intatta. </li></ol> 2. Ex vivo di coltura sezionati cervelli larvali <ol><li> Utilizzando pre-bagnato puntali, trasferire il cervello con cura in inserti di coltura cellulare, le pre-riempite con i media. </li><li> Cervelli di trasferimento, in media, a un incubatore a 25 ° C fino al punto temporale desiderato viene raggiunto. Questo metodo funziona in modo efficiente per un massimo di 48 ore nella cultura. </li><li> Sostituire il supporto a 8 ore. Oltre a ciò, sostituire la metà dei mezzi di ogni 5-6 ore. </li></ol> 3. La manipolazione farmacologica dellaBrains larvali Questo metodo di coltura ex vivo può essere utilizzato per corroborare farmacologicamente interazioni genetiche precedentemente dimostrato nel nostro laboratorio 1. <ol><li> Aggiungere 100 roscovitine uM e 100 micron a olomoucine supporti preparati. </li><li> Trasferimento cervelli sezionati a questo media. </li><li> Cervelli cultura a 25 ° C per 24 ore. </li><li> Sostituire il supporto a 8 ore. Oltre a questo, sostituire la metà dei media (con farmaci) ogni 5-6 ore. </li></ol> La coltivazione di Brains pupa 1. Selezione di Pupae per dissezione <ol><li> Segna pupe bianco su bottiglie / flaconi. </li><li> Scegliere solo pupe che sono completamente bianchi. Pupe con qualsiasi tipo di colorazione non dovrebbe essere incluso. </li><li> Questa fase è 0 ore dopo la formazione pupario (APF). </li><li> Questi pupe sarà pronto per dissezione 1,5 ore dopo la marcatura. Questo passo è fondamentale per assicurare che la potatura di sviluppo avviene in ex vivo </em&gt; cultura. </li></ol> 2. Dissezione di Pupae <ol><li> Aggiungere 500 microlitri di media per un inserto di 12 millimetri coltura cellulare Millicell. (I cervelli sezionati saranno inseriti in questi inserti per facilitare immunostaining facile). </li><li> Una volta che il desiderato punto temporale è stato raggiunto, aggiungere una piccola quantità di supporti preparati per un vetro da orologio dissezione. </li><li> Utilizzando una piccola spatola bagnata, pick up pupe precedentemente segnato per la dissezione e metterli in vetro di orologio. </li><li> Tenendo l&#39;estremità posteriore del pupe con una coppia di pinze, accuratamente aprire l&#39;estremità anteriore del caso pupa con una seconda coppia di pinze # 5. </li><li> Rapidamente sezionare il cervello, mantenendo il cordone nervoso ventrale intatta e allegato. </li></ol> 3. Coltura ex vivo di sezionati Brains pupa <ol><li> Trasferire il cervello con cura in inserti di coltura cellulare, le pre-riempite con i media. </li><li> Cervelli di trasferimento, in media, ad un incubatore a 25 ° C fino alla desiderata time-point viene raggiunto. Abbiamo usato questo metodo per un massimo di 10 ore in coltura per l&#39;esperimento illustrato, ma può essere esteso a lunghi periodi di osservazione e di trattamento, se necessario. </li><li> Per i punti temporali più di 8 ore APF, sostituire i supporti ogni 5-6 ore. </li></ol> Da questo passo, il protocollo rimane invariato sia per larve e pupe. B. Fissaggio Brains sezionati <ol><li> Una volta che il desiderato punto temporale viene raggiunto, rimuovere i supporti e aggiungere 500 microlitri del 4% formaldeide in PBS 1x con 0,5% Triton X-100 (PBST) per 30 minuti, sostituendo con formaldeide fresco dopo 15 minuti. </li><li> Assicurarsi di non permettere il cervello per asciugare durante la sostituzione media con fissativo. </li><li> Lavare formaldeide con quattro lavaggi 10 minuti di 1x PBS con 0,5% Triton X-100 (PBST). </li></ol> C. colorazione Brains fissi <ol><li> Dopo formaldeide è stato lavato via, blocco cervello per 1 ora in una soluzione bloccante di 1x PBS con 1% normaleSiero di capra e l&#39;1% albumina di siero bovino. </li><li> Aggiungi anticorpi primari realizzati con la stessa soluzione bloccante. </li><li> Cervelli Incubare per una notte a 4 ° C. </li><li> La mattina dopo, lavare gli anticorpi primari con quattro lavaggi 10 minuti di PBST. </li><li> Aggiungere anticorpi secondari costituiti in soluzione bloccante. </li><li> Lasciare a temperatura ambiente per 4 ore. </li><li> Lavare anticorpi secondari con quattro lavaggi 10 minuti di PBST. </li></ol> D. Montaggio Brains fissate e colorate per la microscopia <ol><li> Equilibrare il cervello nei media Vectashield di montaggio per 1 ora. </li><li> Montare il cervello su vetrini utilizzando i mezzi di comunicazione Vectashield di montaggio. </li><li> Posizionare chip vetro su entrambi i lati del cervello prima di montare il vetrino coprioggetto. Ciò impedisce ai cervelli di essere schiacciato, mantenendo campione abbastanza sottile per analizzare tutti i piani focali con un microscopio confocale. Cervelli sono stati circa orientati su di montaggio per facilitare la visualizzazione delle caratteristiche di interesse. Se necessario,immagini del cervello erano alternate con il software Imaris per fornire una visione ottimale per la documentazione e la misurazione. </li><li> Guarnizione del coperchio scivola con smalto trasparente. </li><li> Conservare scivola al riparo dalla luce. </li></ol> E. rappresentativi Risultati La figura 1 mostra un pannello di cervelli larvali terzo instar di (A) wild-type mosche e (B) mosche che esprimono una posizione dominante-negativo derivato del Cdk5 (Cdk5DN) (Connell-Crowley et al., 2000). Entrambi esprimono actina-GFP (green) nei neuroni gamma MB. Come descritto in precedenza (Trunova et al., 2011), c&#39;è una regione negli assoni prossimali di wild-type neuroni gamma che non riesce ad accumulare actina-GFP, mostrando quindi come una &quot;zona chiara&quot; (A, staffa), e fasciclin due proteine ​​(rosso) si accumula nelle assoni solo fino al confine di questa zona ventrale (linea bianca orizzontale). La linea tratteggiata nel terzo pannello di figura 1 (A) indica la posizione del MB, evidenziando l&#39;attoin &quot;zona chiara&quot; e il suo margine ventrale. In mosche esprimono Cdk5DN, la zona chiara actina è assente (B) e Fasciclin 2 (FasII) immunoreattività dorsalmente diffonde attraverso quella regione. (Si noti che un numero variabile di FasII positivo progetto di assoni in orizzontale tutta la MB, queste non sono rilevanti per il fenotipo). Barra della scala rappresenta 20 micron. Utilizzando il nostro metodo di coltura ex vivo, abbiamo trattato wild-type mosche con una combinazione di roscovitine e olomoucine, inibitori farmacologici di Cdk5 attività. La figura 2 mostra un pannello di cittadini di instar cervelli larvali di controllo (A) e farmaco-trattata (B wild-type) mosche. Simile a mosche che esprimono un dominante negativo Cdk5 costrutto, cervelli trattati con inibitori Cdk5 per 24 ore, mostrano la perdita caratteristica della zona di actina chiaro e spostamento di confine FasII (parentesi quadre). Linee bianche indicano la posizione di wild-type confine FasII. Barra della scala rappresenta 20 micron. Figusull&#39;articolo 3, mostra una serie rappresentante degli enti di funghi Drosophila da 0-10 ore APF, etichettati con membrana mirati mCD8-GFP (verde). Parentesi indicano il calice, cioè, le proiezioni dendritiche del MB. Prima della metamorfosi, si possono vedere gli assoni MB scorrono dorso-ventrale in grossi fasci, così come una &#39;nebbia&#39; del segnale fluorescente derivante dalla dendritica arbor densa (staffa). Un pannello spettacoli cervelli sezionati al momento indicato. Si noti la potatura dei dendriti nella regione indicata dalle parentesi, in modo tale che entro 6-8 ore APF, la foschia del segnale dendritica è andato (anche se il segnale assonale è invariato). Brains nel pannello B sono stati sezionati a 0 ore APF, prima interna ecdisone spike (Truman e Riddiford, 2002), e coltivate ex vivo come indicato. Questi mostrano nessuna potatura di sviluppo dei dendriti, ma piuttosto il segnale dendritica persiste a 10 ore APF. Per ovviare a questo, le pupe sono contrassegnati APF a 0 ore, ma sezionato a 1,5 ore APF e colta. Vetrol C mostra una serie rappresentante degli enti di funghi queste Drosophila APF da 0-10 ore. Come nei non-coltura di campioni, il segnale dendritica non è rilevabile da 8 ore APF. Barra della scala rappresenta 20 micron.

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<html> Croniche manipolazioni genetiche di struttura del tessuto e funzione, tra cui le mutazioni ed espressione dei transgeni, in genere producono una combinazione di effetti immediati e tardivi conseguenze indirette che possono essere molto difficili da separare. Integrare i metodi genetici con la farmacologia può facilitare l&#39;interrogatorio del corso tempo dietro un fenotipo. Ad esempio, questo è un approccio efficace per sezionare i contributi dei vari componenti citoscheletrici durante la spermatogenesi in <em...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Concentrazione finale </td></tr><tr><td> Schneider Drosophila media </td><td> Invitrogen </td><td> 21720-024 </td><td></td></tr><tr><td> Siero bovino fetale (inattivato al calore) </td><td> Invitrogen </td><td> 10082-147 </td><td> 10% </td></tr><tr><td> Penicillina / streptomicina </td><td> Invitrogen </td><td> 15140-122 </td><td> 1% </td></tr><tr><td> Insulina </td><td> Invitrogen </td><td> 12585-014 </td><td> 10μg/ml </td></tr><tr><td> Ecdisone </td><td> Sigma-Aldrich </td><td> H5142 </td><td> 2μg/ml </td></tr><tr><td> Roscovitine </td><td> Sigma-Aldrich </td><td> R7772 </td><td> 100μM </td></tr><tr><td> Olomoucine </td><td> Sigma-Aldrich </td><td> O0886 </td><td> 100μM </td></tr><tr><td> Siero di capra normale </td><td> Invitrogen </td><td> 50062Z </td><td> 1% </td></tr><tr><td> Albumina di siero bovino </td><td> Fisher Scientific </td><td> M9501 </td><td> 1% </td></tr><tr><td> Formaldeide </td><td> Sigma-Aldrich </td><td> D4551 </td><td> 4% </td></tr><tr><td> Triton X-100 </td><td> Fisher Scientific </td><td> BP151 </td><td> 0,5% </td></tr><tr><td> Soluzione tampone fosfato </td><td> Invitrogen </td><td> 70011-044 </td><td> 1X </td></tr><tr><td> Vectashield </td><td> Vector Laboratories </td><td> H1000 </td&gt; <td></td></tr><tr><td> Millicell colture cellulari Inserti </td><td> Millipore </td><td> PI8P01250 </td><td></td></tr><tr><td> Multidish, 4 e </td><td> Thermo Scientific </td><td> 176740 </td><td></td></tr><tr><td> Dumont # 5 Pinze </td><td> Strumenti di Scienze Belle </td><td> 11251-20 </td><td></td></tr><tr><td> Vetrini </td><td> Fisher Scientific </td><td> 12-544-7 </td><td></td></tr><tr><td> Vetro di copertura </td><td> Fisher Scientific </td><td> 12-548-5M </td><td></td></tr></tbody></table>

ex vivo culturing of whole, developing drosophila brains

Noi descriviamo un metodo per la coltura ex vivo di interi cervelli Drosophila. Questo può essere usato come un contrappunto al croniche manipolazioni genetiche per lo studio della biologia cellulare e dello sviluppo delle strutture cerebrali centrali, consentendo acuti interventi farmacologici e immagini dal vivo di processi cellulari. Come esempio della tecnica, prima lavorare dal nostro laboratorio 1 ha dimostrato che un vano precedentemente non subcellulare compreso tra i vani assonale e somatodendritic di assoni del cervello Drosophila centrale. Lo sviluppo di questo vano, indicato come il segmento assone iniziale (AIS) 2, è stato dimostrato geneticamente dipendere dalla chinasi neurone-specifica ciclina-dipendente, Cdk5. Mostriamo qui che il trattamento ex vivo delle wild-type cervelli larvali di Drosophila con la Cdk5-inibitori farmacologici specifici roscovitine e olomoucine 3 produce modifiche acute nell&#39;organizzazione actina enella localizzazione della proteina Fasciclin superficie cellulare 2, che imitano i cambiamenti visti in mutanti che mancano Cdk5 attività geneticamente. Un secondo esempio di tecnica di coltura ex vivo è previsto per il rimodellamento dei collegamenti del corpo fungo embrionale (MB) neuroni gamma durante la metamorfosi da larva ad adulto. Il corpo fungo è il centro di apprendimento e la memoria olfattiva in volo 4, e questi neuroni gamma potare i rami assonali e dendritiche durante lo sviluppo pupale e poi ri-rami si estendono in un timepoint tardi per stabilire il modello di adulto innervazione 5. Potatura di questi neuroni del MB è stato verificato tramite degenerazione locale di rami neuriti 6, da un meccanismo che viene attivato da ecdisone, un ormone steroideo, agendo sul recettore B1 ecdisone 7, e che dipende dall&#39;attività del sistema ubiquitina-proteasoma 6. Il nostro metodo di coltura ex vivo possono be utilizzato per interrogare ulteriormente il meccanismo di rimodellamento sviluppo. Abbiamo trovato che l&#39;impostazione in coltura ex vivo, neuroni gamma di MB ricapitolato il processo di potatura di sviluppo con un percorso simile a quello che in vivo. È essenziale, tuttavia, attendere 1,5 ore dopo la formazione pupario prima espianto il tessuto affinché le cellule ad impegnarsi irreversibilmente a metamorfosi; dissezione degli animali durante l&#39;insorgenza di pupariation portato a metamorfosi poco o nessun in coltura. Così, con opportune modifiche, l&#39;approccio coltura ex vivo può essere applicato allo studio dinamico così come aspetti allo stato stazionario di biologia cervello centrale.

Watch this Scientific Journal Video about Ex vivo Culturing of Whole, Developing Drosophila Brains at JoVE.com

Ex vivo Culturing of Whole, Developing Drosophila Brains

Questo articolo descrive un metodo con il quale si può imitare<em&gt; In vivo</em&gt; Sviluppo del<em&gt; Drosophila</em&gt; Corpo funghi in un<em&gt; Ex vivo</em&gt; Sistema di coltura.

Ex coltura vivo di Whole, in via di sviluppo Brains Drosophila

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

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Neuroscience

Ex vivo Culturing of Whole, Developing Drosophila Brains

13.0K Views.  National Institute of Neurological Disorders and Stroke. Questo articolo descrive un metodo con il quale si può imitare In vivo Sviluppo del Drosophila Corpo funghi in un Ex vivo Sistema di coltura.

Video: Ex vivo Culturing of Whole, Developing Drosophila Brains

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Visualizzazione del sistema nervoso embrionale in Whole-mount Drosophila Embrioni

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

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Neuroscienze

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In elettroporazione in vivo di Retina topo in via di sviluppo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

Ex vivo Imaging in diretta di divisioni cellulari singoli in mouse neuroepitelio

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Tutta Monte immunomarcatura di recettore olfattivo neuroni nel<em&gt; Drosophila</em&gt; Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultaneo<em&gt; Ex vivo</em&gt; Test funzionale di due retine per<em&gt; In vivo</em&gt; Sistema Elettroretinogramma

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Ex Vivo optogenetic dissezione di circuiti paura nel cervello fette

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Un semplice passo-dissezione Protocollo per tutto il montaggio Preparazione per adulti<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Ex Utero Elettroporazione e colture Organotypic fetta di cervelli embrionali del Mouse per vivere-Imaging di migrazione interneuroni GABAergici

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Calcio Imaging per visualizzare le risposte del cervello alla segnalazione endocrina in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

Analisi metabolica di Drosophila melanogaster Larval e cervello adulto

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Ex coltura vivo di Whole, in via di sviluppo Brains Drosophila

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