Questo lavoro è stato sostenuto dal Premio IMPACT del Dipartimento di Radioterapia Oncologica, University of Alabama-Birmingham Comprehensive Cancer Center, Cancer Foundation i bambini da combattimento, e la Gabrielle Angelo Foundation (a ESY.).

1. Colture Cellulari <ol><li> Cellule Cultura neuronali e trattarli come necessario. </li><li> Cellule Harvest in provette da 15 ml: i media aspirato, sciacquare con tampone fosfato salino (PBS, calcio e magnesio libero), aggiungere tripsina, raccogliere in provette da 15 ml e neutralizzare tripsina con appropriati mezzi di siero contenenti. </li><li> Centrifugare a 1.000 xg per 5 min. </li><li> Aspirare media. </li><li> Risospendere le cellule in PBS. </li><li> Centrifugare a 1.000 xg per 5 min. </li><li> Aspirare media e Risospendere le cellule in PBS fresco. </li><li> Contare le celle utilizzando un emocitometro o il vostro contatore di cellule preferito. </li><li> Campioni cellulari dovrebbe essere preparata immediatamente prima di iniziare il test. </li><li> Tutti i campioni devono essere trattati alla luce scura o gialla per evitare danni del DNA dalla luce ultravioletta. </li></ol> 2. Comet Assay 1. Preparazione del vetrino <ol><li> Sciogliere 1% agarosio (1 g / 100 ml in 1X Tris base, acido borico, EDTA, TBE) in un microonde per 3 min finché tutti granuli scompaiono. </li><li> Immergere i vetrini nella agarosio fuso e pulire un lato pulire con un Kimwipe. Permettono l&#39;agarosio al aria-secco ad un film trasparente. Questo può essere fatto in anticipo e vetrini possono essere conservati. </li></ol> 2. Comet Assay Neutral <ol><li> Freddo soluzione di lisi (2,5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris Base, 1% sarcosinato di sodio lauril, e 1% Triton X-100, pH 10) a 4 ° C per almeno 1 ora prima dell&#39;uso. </li><li> 2.2.2. Fondere 1% basso punto di fusione agarosio (1 g / 100 ml in 1X Tris Base, acido borico, EDTA) in un forno a microonde per 3 min finché tutti i granuli scompaiono. L&#39;agarosio bisogno di essere raffreddato a 37 ° C in un bagno acqua per evitare induzione artificiale di tail cometa. Idealmente, l&#39;agarosio bisogno di essere raffreddato per mezz&#39;ora prima dell&#39;uso. </li><li> 2.2.3. Diluire la sospensione cellulare in modo che vi sono 100.000 cellule per ml. Combinare la sospensione cellulare con il punto di agarosio a bassa fusione (a 37 ° C) aun rapporto di 01:10 (v / v), brevemente vortice, e subito pipettare 50 pl nella diapositiva Comet. Utilizzare il lato della punta della pipetta per diffondere la sospensione cellulare uniformemente sulla superficie del campione. Ogni gruppo di trattamento deve essere almeno in triplicato. </li><li> Posto scorre appartamento in frigorifero per 30 minuti fino a un cerchio appare nella periferia della slitta. </li><li> Prechill la soluzione di lisi e Immergere vetrini in questa soluzione per 30 minuti al buio a 4 ° C (o in frigorifero). </li><li> Versare o aspirare la soluzione di lisi e aggiungere 1X tampone neutro elettroforesi (Tris base, acido borico, EDTA, 1X TBE). Lasciare vetrini in questo buffer per una mezz&#39;ora in frigorifero. </li><li> Aggiungi prechilled tampone di 1X per elettroforesi Neutral (TBE) nella camera elettroforesi, porre i vetrini in vassoio di elettroforesi diapositiva. Allineare diapositive in modo che siano equidistanti da elettrodi. </li><li> Versare 1X elettroforesi neutro buffer fino a 0,2 cm sopra le diapositive. Tampone in eccesso potrebbe, traFere con elettroforesi. </li><li> Impostare la tensione di alimentazione 1 V per cm (misurata elettrodo per elettrodo) e correre per 30 min a 4 ° C (o cella) al buio. </li></ol> 3. Comet Assay alcalino <ol><li> Freddo soluzione di lisi (2,5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris Base, 1% sarcosinato di sodio lauril, e 1% Triton X-100, pH 10) a 4 ° C per almeno 1 ora prima dell&#39;uso. </li><li> Fondere 1% basso punto di fusione agarosio (1 g / 100 ml in 1X Tris Base, acido borico, EDTA) in un forno a microonde per 3 min finché tutti i granuli scompaiono. Poi raffreddare in un bagno di acqua 37 ° C per almeno 30 min. </li><li> Combinare celle a 1 x 105 / ml con fuso punto agarosio low melting (a 37 ° C) ad un rapporto di 01:10 (v / v), brevemente vortice, e subito pipettare 50 pl nella diapositiva Comet. Utilizzare il lato della punta della pipetta per diffondere la sospensione cellulare uniformemente sulla superficie del campione. Ogni gruppo di trattamento deve essere almeno in triplicato. </li><li> Luogo scivolòes appartamento in frigorifero per 30 minuti fino a un cerchio appare nella periferia della slitta. </li><li> Immergere i vetrini in una soluzione di lisi prechilled e lasciare a 4 ° C per 1 ora per una notte al buio. </li><li> Rimuovere i vetrini dalla soluzione di lisi, drenare i vetrini e sciacquarli una volta con tampone di neutralizzazione fredda per 5 minuti per rimuovere detersivo residuo e sali prima alcali-svolgitore passo. </li><li> I vetrini in una camera riempita con elettroforesi su gel elettroforesi prechilled tampone appena fatto (300 mM NaOH, 1 mM EDTA,&gt; 13 pH) non superiore a 0,5 cm sopra le diapositive. Allineare diapositive in modo che siano equidistanti da elettrodi. </li><li> Lasciate diapositive sedersi in tampone alcalino per 30 minuti al buio per consentire lo svolgimento del DNA e l&#39;espressione di alcali-responsabili danni. </li><li> Impostare la tensione di alimentazione 1 V per cm (misurata elettrodo per elettrodo) e correre per 30 min a 4 ° C (o cella). </li></ol> 4. Di fissaggio e StaCellule ra zione <ol><li> Scolare il tampone in eccesso elettroforesi </li><li> Immergere i vetrini in pre-refrigerata acqua distillata per 5 minuti a temperatura ambiente. </li><li> Immergere i vetrini in pre-refrigerati etanolo al 70% per 5 minuti a temperatura ambiente. </li><li> Campioni a secco durante la notte. Non esporre i vetrini ad una luce forte. I campioni possono essere conservati per mesi a temperatura ambiente prima di segnare in questa fase. </li><li> Colorare i vetrini immergendoli in SYBR Green (1X diluito in PBS) per 20 minuti in frigorifero. </li><li> Estrarre i vetrini e permettere loro di asciugare completamente al buio. L&#39;agarosio diventerà trasparente quando è completamente asciutto. </li></ol> 4. Acquisizione di immagini e analisi <ol><li> Acquisire immagini con microscopio a fluorescenza impostato il filtro verde (Zeiss AxioVision) e analizzare con il software Comet Assay (Strumenti percettive). </li><li> Clicca sulla &quot;testa cometa&quot; (il nucleo) e il software calcola i parametri compreso il momento coda media e la quantità di DNA nelnucleo. </li><li> Analizzare almeno 200 cellule per il trattamento. </li><li> Esportare i dati in Microsoft Excel. </li><li> Calcola media momento coda per ogni gruppo di trattamento e dati del diagramma a seconda dei casi. </li></ol> 5. Risultati rappresentativi Un esempio di analisi Comet assay sul neuronali è mostrato in Figura 2 e Figura 3. In questo caso, l&#39;irradiazione delle cellule neuronali induce danno al DNA. Come le cellule vengono sottoposto al campo elettrico, l&#39;DNA migra a tassi differenti a causa di differenze di dimensioni che viene successivamente analizzati utilizzando il software Assay Comet. Più il danno al DNA, più lontano il DNA migra dal nucleo. Questo diminuisce l&#39;intensità fluorescente nel nucleo che viene successivamente raccolto dal software e risultati in momento coda superiore. Tabella 1 illustra una tabella rappresentante analisi cometa dosaggio e la Figura 4 mostra un rappresentantegraph sentante confrontando danno del DNA in cellule neuronali seguendo radiazione misurata dal test della cometa. <img src="/files/ftp_upload/50049/50049fig1.jpg" alt="Figura 1"/> Figura 1. Diagramma di flusso del test della cometa. <a href="https://www.jove.com/files/ftp_upload/50049/50049fig1large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img src="/files/ftp_upload/50049/50049fig2.jpg" alt="Figura 2"/> Figura 2. Immagini rappresentative di cellule neuronali (A) senza e (B) con la coda della cometa. <img src="/files/ftp_upload/50049/50049fig3.jpg" alt="Figura 3"/> Figura 3. Comet l&#39;analisi prova svolta utilizzando Assay il software Comet. (A) colpi di rappresentativi dello schermo di immagine acquisita utilizzando Carl microscopio a fluorescenza Zeiss e analizzati utilizzando CometAssay software. (B) Cliccando sul nucleo o la &quot;testa cometa&quot; (cerchiato in verde) genera una mappa fluorescente e grafico (cerchiato in giallo) e una (C) tabella di dati (cerchiato in rosso). <a href="https://www.jove.com/files/ftp_upload/50049/50049fig3large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img src="/files/ftp_upload/50049/50049fig4.jpg" alt="Figura 4"/> Figura 4. Grafico rappresentativa ottenuta riportando il momento medio di coda ottenuto analizzando danno al DNA in cellule neuronali irradiati e non irradiati mediante saggio neutro cometa. Come previsto, le radiazioni 3GY (x-ray) induce danno del DNA come illustrato dal momento media superiore coda nelle cellule irradiate neuronali. Mostrato è il momento coda media (+ / - errore standard), ** P &lt;0.01. <img src="/files/ftp_upload/50049/50049table1.jpg" alt="Tabella 1"/> Tabella 1. </strong&gt; tabella di rappresentativa ottenuta con l&#39;analisi assay cometa su cellule neuronali irradiate e non-irradiati. <a href="https://www.jove.com/files/ftp_upload/50049/50049table1large.jpg" target="_blank">Clicca qui per vedere più grande figura</a> .

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Nessun conflitto di interessi dichiarati.

Il test della cometa ha la capacità unica di analizzare cellule singole. Ciò è vantaggioso in identificazione di sottopopolazioni di cellule che dimostrano risposta differenziale ad agenti citotossici. Alcune limitazioni pratiche devono essere presi in considerazione. Il numero di celle che possono essere valutati singolarmente possono variare a seconda dell&#39;individuo. La dimensione del campione deve essere aumentato se vi sono variazioni nei danni al DNA all&#39;interno di una popolazione. Vitale cella singola s...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> 1,5 ml provette </td><td> Santa Cruz Biotechnology, Inc </td><td> Sc-200271 </td><td></td></tr><tr><td> 10X TBE (Tris base, acido borico, EDTA) </td><td> Fisher Scientific </td><td> BP13331 </td><td></td></tr><tr><td> Provetta da 15 ml </td><td> Fisher Scientific </td><td> 0553851 </td><td></td></tr><tr><td> Agarosio </td><td> Sigma </td><td> A5093 </td><td></td></tr><tr><td> Alluminio foil </td><td> Fisher Scientific </td><td> 01213101 </td><td></td></tr><tr><td> Coppa </td><td> Fisher Scientific </td><td> FB102300 </td><td></td></tr><tr><td> Centrifuga </td><td> Thermo Scientific </td><td> 75004261PR </td><td></td></tr><tr><td> Comet Assay software </td><td> Comet Assay IV System Image Analysis </td><td></td><td></td></tr><tr><td> Cilindro </td><td> Fisher Scientific </td><td> 08555F </td><td></td></tr><tr><td> EDTA </td><td> GIBCO </td><td> 15575 </td><td></td></tr><tr><td> Elettroforesi camera di </td><td> Thermo Scientific </td><td> 09528101 </td><td></td></tr><tr><td> Etanolo </td><td> Fisher Scientific </td><td> A407P4 </td><td></td></tr><tr><td> Microscopio a fluorescenza </td><td> Zeiss Axio Vision </td><td></td><td> Ogni microscopio a fluorescenza con filtro verde sarà sufficiente </td></tr><tr><td> Emocitometro </td><td> Fisher Scientific </td><td> 0267152 </td><td></td></tr><tr><td> Basso punto di fusione agarosio </td><td> Promega </td><td> V2111 </td><td></td></tr><tr><td> Microonda </td><td> Sears </td><td></td><td></td></tr><tr><td> Soluzione tampone fosfato, calcio libero, magnesio libero </td><td> HyClone </td><td> SH3025601 </td><td></td></tr><tr><td> Alimentazione </td><td> BioRad </td><td> 1645050 </td><td></td></tr><tr><td> Frigorifero </td><td> Sears </td><td></td><td></td></tr><tr><td> Righello </td><td> Staples </td><td></td><td></td></tr><tr><td> Scorrere </td><td> Fisher Scientific </td><td> 12550143 </td><td></td></tr><tr><td> Cloruro di sodio </td><td> Sigma </td><td> S7653 </td><td></td></tr><tr><td> Soda caustica </td><td> Sigma </td><td> S5881 </td><td></td></tr><tr><td> Sodio lauril sarcosinato </td><td> Fisher Scientific </td><td> S529 </td><td></td></tr><tr><td> SYBR Green </td><td> Invitrogen </td><td> S7585 </td><td></td></tr><tr><td> Vassoio </td><td> Fisla scientifica </td><td> 15242B </td><td></td></tr><tr><td> Tris Base </td><td> Sigma </td><td> T6066 </td><td></td></tr><tr><td> Triton-X 100 </td><td> Sigma </td><td> T8787 </td><td></td></tr><tr><td> Tripsina </td><td> HyClone </td><td> SH3023601 </td><td></td></tr><tr><td> Vortice </td><td> Fisher Scientific </td><td> 02216108 </td><td></td></tr><tr><td> Bagnomaria </td><td> Fisher Scientific </td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

Un certo numero di farmaci indirizzare i percorsi di riparazione del DNA e indurre cellule uccidere con la creazione di danno al DNA. Così, i processi per misurare direttamente il danno al DNA sono stati ampiamente valutati. I metodi tradizionali sono in termini di tempo, costosa, molte risorse e richiedono cellule replica. In contrasto, il test della cometa, un unico gel test cellulare elettroforesi, è più veloce, non invasivo, economico, misura diretta e sensibile di danno e riparazione del DNA. Tutte le forme di danno al DNA e riparazione del DNA possono essere visualizzate a livello di singola cellula utilizzando questa tecnica potente. Il principio alla base del test della cometa è che il DNA intatto è altamente ordinato che i danni del DNA sconvolge questa organizzazione. Le sorgenti di DNA danneggiato nella matrice di agarosio e quando sottoposto ad un campo elettrico, il DNA carico negativamente migra verso il catodo, che è caricato positivamente. Le grandi filamenti di DNA non danneggiati non sono in grado di migrare lontano dal nucleo. Danno al DNAcrea frammenti di DNA più piccole che viaggiano oltre la DNA intatto. Comet Assay, un software di analisi delle immagini, misure e confronta l&#39;intensità complessiva fluorescente del DNA nel nucleo con il DNA che è migrata dal nucleo. Segnale fluorescente dal DNA migrato è proporzionale al danno del DNA. Coda DNA più lungo luminoso indica il danno al DNA è aumentato. Alcuni dei i parametri che vengono misurati sono momento tail che è una misura della sia la quantità di DNA e distribuzione del DNA nella lunghezza tail, tail e percentuale di DNA nella coda. Questo test permette di misurare la riparazione del DNA e da risoluzione del danno al DNA significa riparazione ha avuto luogo. Il limite di sensibilità è di circa 50 rotture di filamenti per 1,2 cellula diploide mammiferi. Cellule trattati con un qualsiasi agenti DNA dannosi come etoposide, possono essere utilizzato come controllo positivo. Così il test della cometa è una procedura rapida ed efficace per misurare la danno al DNA.

Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Il test della cometa è un modo efficiente di rilevare rotture a singolo e doppio filamento, compresi alcali-labili siti e DNA-DNA/DNA-protein legami incrociati sul DNA in tutte le cellule, compresi i neuroni dell&#39;ippocampo. Il metodo sfrutta la migrazione differenziale di DNA in un campo elettrico dovuto a differenze nelle quantità di danno al DNA.

Il saggio danneggiare il DNA in neuroni ippocampali Utilizzo del Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

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Research

JoVE Journal

Neuroscience

27.1K Views.  University of Alabama-Birmingham. Il test della cometa è un modo efficiente di rilevare rotture a singolo e doppio filamento, compresi alcali-labili siti e DNA-DNA/DNA-protein legami incrociati sul DNA in tutte le cellule, compresi i neuroni dell'ippocampo. Il metodo sfrutta la migrazione differenziale di DNA in un campo elettrico dovuto a differenze nelle quantità di danno al DNA.

Video: Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Saggio β-amiloide tossicità utilizzando un transgenici C. elegans Modello

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Ricerca

Neuroscienze

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulazione e mitocondriale di trasporto: Imaging Live in neuroni dell&#39;ippocampo oltre durate a lungo

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Isolamento e la coltura di neuroni ippocampali da topi prenatali

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Fosfato di calcio trasfezione di primaria neuroni dell&#39;ippocampo

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Imaging spine dendritiche di Rat Primaria neuroni dell&#39;ippocampo utilizzando Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Fluorescenza e bioluminescenza di subcellulare Ca<sup&gt; 2+</sup&gt; In Aged neuroni dell&#39;ippocampo

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Stripe Assay per studiare l&#39;attività attrattiva o repulsiva di un substrato proteico Utilizzando dissociata neuroni dell&#39;ippocampo

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Valutazione della complessità dendritica ippocampale nei topi invecchiati utilizzando il metodo Golgi-Cox

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Endocitosi delle vescicole sinaptiche in colture di neuroni ippocampali di misura

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...