Siamo grati a Lihong Yin per l&#39;assistenza tecnica. NVD è destinatario di un NARSAD Young Investigator Award ed è anche sostenuto da sovvenzioni dal NIH (5 K99 MH095825-02). La ricerca in laboratorio Fishell è sostenuto dal National Institute of Health, Istituto Nazionale di Salute Mentale (5 R01 MH095147-02, 5 R01 MH071679-09), Istituto nazionale dei disordini neurologici e Stroke (5 R01 NS081297-02, 1 P01 NS074972 -01A1) e la Fondazione Simons.

Tutti gli animali sono stati trattati in conformità con i regolamenti e le linee guida del Care and Use Committee animali istituzionale della Scuola di Medicina di NYU. Mouse Ceppi Swiss Webster topi femmina forniti da TACONIC sono stati utilizzati per questi esperimenti. Al fine di indirizzare specificamente interneuroni superficiali di livello, sono stati utilizzati embrioni E15.5. Nota: Il plasmide utilizzato in questo lavoro (Dlx5 / 6.eGFP plasmide 3 mg / mL) è stato generato utilizzando tecniche di clonazione standard. Il eGFP cDNA è stato clonato in un Dlx5 / 6-Pmin-polyA plasmide. Questo plasmide è disponibile su richiesta (demarn02@nyumc.org). 1 Preparazione di Microiniezione Pipettes <ol><li> Impostare l&#39;estrattore di calore # 2-860. </li><li> Posizionare e fissare il capillare di vetro in prossimità del filamento. Controllare che il diametro esterno (OD) della pipetta è di circa 30 micron. </li><li> Premere il tasto &quot;tirare&quot;. </li><li> Rimuovere con cautela il capillare. La parte inferiore è l&#39;ago. Eliminare la parte superiore. </li></ol> 2 Anestesia Procedura <ol><li> Preparare una camera contenente isoflurano. Nota: isoflurano è l&#39;anestetico preferito per la sua rapidità d&#39;azione e la bassa incidenza di effetti collaterali. Pentobarbital è un&#39;altra opzione; Tuttavia, la tolleranza di questo farmaco è variabile e può portare ad un tasso di mortalità superiore rispetto isoflurano. </li><li> Impostare il flusso di isoflurano al 4-5% in 0,5-1 L / min O 2. </li><li> Assicurarsi che la valvola per la camera è aperta, mentre la valvola per l&#39;adattatore testa è chiusa. </li><li> Posizionare il mouse incinta in camera. </li><li> Lasciare il mouse per andare sotto l&#39;effetto dell&#39;anestesia. Questo richiederà circa 2-3 min. Controllare la frequenza respiratoria. Se il mouse ha inizio iperventilazione, ridurre la dose di isoflurano. </li><li> Dopo che il mouse è anestetizzato, prontamente alla rimozione dalla camera. Posizionarlo lato ventrale sul riscaldamento pad e insert la testa nella maschera che continuerà a fornire isoflurano durante l&#39;intervento chirurgico. Assicurarsi di cambiare il flusso di isoflurano dalla camera al tubo che collega il pezzo adattatore testa. Mettere una goccia di lubrificante occhio in ogni occhio e chiudere gli occhi. </li><li> Impostare il pad di riscaldamento a 36 ° C gradi per ridurre al minimo l&#39;ipotermia. </li><li> Verificare l&#39;assenza di piede e di coda riflessi pizzicando le zampe posteriori e la coda. </li></ol> 3. Chirurgia <ol><li> Detergere la pelle che ricopre l&#39;addome con il 70% di etanolo. </li><li> Usare pinze per tirare la pelle verso l&#39;alto. Dopo ogni intervento chirurgico, lavare tutti gli strumenti con acqua e detergente, e sterilizzata a 72 ° CO / N. </li><li> Fai una piccola incisione verticale (circa 2 cm) lungo la linea mediana iniziando a metà strada tra il terzo e quarto paia di ghiandole mammarie. Usare le forbici chirurgia sottili. Fare attenzione a non danneggiare la parete addominale. </li><li> Separare delicatamente la pelle dal peritoneo. </li><li> Visualizza le arteriee vene sulla parete addominale. Fare un altro due centimetri incisione verticale attraverso il peritoneo (evitando i vasi) per esporre la cavità addominale. </li><li> Fissare la pelle e la parete addominale su ogni lato evitando il bloccaggio delle arterie. Nota: Se le arterie vengono accidentalmente perforati, sacrificare l&#39;animale immediatamente eseguendo dislocazione cervicale o overdose isoflurano. </li><li> Pre riscaldare la sterile PBS a 37 ° C. Coprire i morsetti con PBS umide Kim salviette. </li><li> Idratare la cavità addominale esposta con PBS. Ripetere questa operazione più volte durante l&#39;esecuzione della chirurgia. </li><li> Utilizzando pinze rotonde, tirare delicatamente la catena embrionale (E15.5) lontano dalla cavità. Lasciare riposare catena sulle salviettine umidificate. Inizia esponendo una metà dell&#39;utero prima. Una volta che gli embrioni in esso sono elettroporati, riportarlo dentro, poi lavorare sulla seconda metà. </li></ol> 4. elettroporazione <ol><li> Continuare a utilizzare pinze rotonde per manipolare gli embrioni. </li><li> Visualize ventricoli laterali. I ventricoli laterali corrono lungo la linea mediana (Figura 1b). </li><li> Aggiungi Veloce Verde (magazzino: 10% w / v) per la soluzione di DNA per una miscela 01:20 di visualizzare durante l&#39;iniezione. Preparare la soluzione di DNA sciogliendo il pellet ottenuto da un protocollo di purificazione maxi-prep serie in acqua distillata. </li><li> Utilizzare micropipette pre tirati con uno stantuffo per iniettare 1 ml di soluzione di DNA (Dlx5 / 6-eGFP, 3 mg / mL) con Fast verde. Tagliare la punta della micropipetta con un bel forcep # 5 (Dumont) lasciare 6 mm di lunghezza dall&#39;inizio della spalla alla fine della punta dell&#39;ago prima di caricare la pipetta. Tenere la testa di un embrione con una pinzetta rotonde. Immettere il cervello con la pipetta ad un angolo di 45 ° puntando il ventricolo laterale si trova vicino alla linea mediana. Se l&#39;ago penetra attraverso il ventricolo, ritirare lentamente la pipetta come si usa lo stantuffo per iniettare il DNA. Il ventricolo dovrebbe diventare verde quando ilsoluzione comincia a riempirlo. A questo punto, fermano la pipetta e iniettare 1 ml di DNA. Delicatamente, ritirare la pipetta. </li><li> Impostare il elettroporatore CUY21 a cinque 45V impulsi di 50 msec intervallate da 950 msec. </li><li> Utilizzare 5 millimetri elettrodi pagaia per gli embrioni E15.5. Immergere gli elettrodi in PBS per assicurare la trasmissione di corrente efficace. </li><li> Posizionare le piastre degli elettrodi intorno alla testa dell&#39;embrione con la pagaia positivo sul ventricolo contenente la soluzione di DNA. Leggermente abbassare la paletta positivo rispetto del negativo per creare una corrente ventrale attraverso eminenze gangliari. Questa manipolazione ridurrà espressione ectopica in cellule piramidali. </li><li> Dopo aver posizionato gli elettrodi, fornire un impulso premendo il pedale una volta. </li><li> Rimuovere gli elettrodi e pulire puliti. Ripetere i passaggi 4,6-4,9 con ogni embrione. </li><li> Dopo electroporating tutti gli embrioni, versare 3 ml di PBS nella cavità addominale e restituirli alla cavità addominale. </li><li>Prima sutura della parete addominale con un ago curvo e 5-0 sutura di seta e poi la pelle. Applicare lidocaina (4%) sulla ferita. Somministrare 120 mg / kg di Aspirina intra peritoneo per l&#39;analgesia. Nota: L&#39;intera procedura deve essere eseguita in 20 minuti o meno. E &#39;consigliabile che i principianti elettroporazione solo pochi embrioni per mantenere il tempo di funzionamento breve. Interventi chirurgici prolungati diminuirà il tasso di sopravvivenza degli embrioni. </li><li> Rimuovere l&#39;animale dal tubo dell&#39;anestesia e consentire il recupero sul pad di riscaldamento su una carta pulire. </li><li> Animale Ritorna alla gabbia, tenerlo sulla rampa di riscaldamento a 37 ° C e monitorare lo stato di salute. Gli animali in genere tollerano l&#39;intervento chirurgico bene e cominciano a muoversi lentamente subito dopo vengono rimossi dal tubo dell&#39;anestesia. Se si osserva un eccessivo sanguinamento o letargia, sacrificare immediatamente l&#39;animale eseguendo IACUC approvato le procedure di eutanasia come la dislocazione cervicale o overdose anestesia. </li><li> Gabbia Ritorna alla vivarium. Le femmine saranno partorire naturalmente. </li></ol>

<ol>
	<li>Fishell, G., Rudy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+inhibition+within+the+telencephalon:+&amp;#34;where+the+wild+things+are&amp;#34;.">Mechanisms of inhibition within the telencephalon: &amp;#34;where the wild things are&amp;#34;.</a> Annual review of neuroscience. 34, 535-567 (2011).</li><li>Stenman, J., Toresson, H., Campbell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+distinct+progenitor+populations+in+the+lateral+ganglionic+eminence:+implications+for+striatal+and+olfactory+bulb+neurogenesis.">Identification of two distinct progenitor populations in the lateral ganglionic eminence: implications for striatal and olfactory bulb neurogenesis.</a> J Neurosci. 23, 167-174 (2003).</li><li>Eisenstat, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DLX-1,+DLX-2,+and+DLX-5+expression+define+distinct+stages+of+basal+forebrain+differentiation.">DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation.</a> J Comp Neurol. 414, 217-237 (1999).</li><li>Batista-Brito, R., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+integration+of+cortical+interneurons+into+a+functional+network.">The developmental integration of cortical interneurons into a functional network.</a> Curr Top Dev Biol. 87, 81-118 (2009).</li><li>Miyoshi, G., Butt, S. J., Takebayashi, H., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologically+distinct+temporal+cohorts+of+cortical+interneurons+arise+from+telencephalic+Olig2-expressing+precursors.">Physiologically distinct temporal cohorts of cortical interneurons arise from telencephalic Olig2-expressing precursors.</a> J Neurosci. 27, 7786-7798 (2007).</li><li>Miyoshi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+fate+mapping+reveals+that+the+caudal+ganglionic+eminence+produces+a+large+and+diverse+population+of+superficial+cortical+interneurons.">Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons.</a> J Neurosci. 30, 1582-1594 (2010).</li><li>Bulfone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+Dlx-2+(Tes-1)+gene+is+expressed+in+spatially+restricted+domains+of+the+forebrain,+face+and+limbs+in+midgestation+mouse+embryos.">The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos.</a> Mechanisms of development. 40, 129-140 (1993).</li><li>Bulfone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+restricted+expression+of+Dlx-1,+Dlx-2+(Tes-1),+Gbx-2,+and+Wnt-3+in+the+embryonic+day+12.5+mouse+forebrain+defines+potential+transverse+and+longitudinal+segmental+boundaries.">Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries.</a> J Neurosci. 13, 3155-3172 (1993).</li><li>Robinson, G. W., Wray, S., Mahon, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+restricted+expression+of+a+member+of+a+new+family+of+murine+Distal-less+homeobox+genes+in+the+developing+forebrain.">Spatially restricted expression of a member of a new family of murine Distal-less homeobox genes in the developing forebrain.</a> The New biologist. 3, 1183-1194 (1991).</li><li>Close, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satb1+is+an+activity-modulated+transcription+factor+required+for+the+terminal+differentiation+and+connectivity+of+medial+ganglionic+eminence-derived+cortical+interneurons.">Satb1 is an activity-modulated transcription factor required for the terminal differentiation and connectivity of medial ganglionic eminence-derived cortical interneurons.</a> J Neurosci. 32, 17690-17705 (2012).</li><li>De Marco Garcia, N. V., Karayannis, T., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+is+required+for+the+development+of+specific+cortical+interneuron+subtypes.">Neuronal activity is required for the development of specific cortical interneuron subtypes.</a> Nature. 472, 351-355 (2011).</li><li>Petros, T. J., Rebsam, A., Mason, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+and+ex+vivo+electroporation+for+gene+expression+in+mouse+retinal+ganglion+cells.">In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells.</a> Journal of visualized experiments : JoVE. (31), (2009).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> Journal of visualized experiments : JoVE. (6), e239 (2007).</li></ol>

Gli autori non hanno nulla da rivelare.

LIMITI DELLA TECNICA Mentre questa tecnica permette di cella di analisi autonoma di processi cellulari, non è adatto per l&#39;analisi di popolazione. I electroporations sono molto scarse, con meno di un migliaio di cellule elettroporate al cervello. Di conseguenza, la tecnica non può essere utilizzata per valutare conseguenze comportamentali dovute alla manipolazione genetica degli interneuroni CGE-derivati. Mentre electroporations effettuate ai sottotipi bers...

La maggior parte delle corticali interneuroni GABAergici provengono da due strutture embrionali transitori denominate le eminenze gangliari mediale e caudale (MGE e CGE, rispettivamente) 4. Parvalbumina e somatostatina esprimere interneuroni sono originari del MGE, mentre Calretinin (Cr), Vasointestinal peptide (VIP) e Reelin (Re) che esprime interneuroni provengono dalla CGE. Questi sottotipi di interneuroni possono essere distinte dalle loro date di nascita. MGE sottotipi derivati ​​sono nati tra il giorno embrionale 9.5 (E9.5) e e16.5 5,6. Al contrario, gli interneuroni CGE derivati ​​nascono da E12.5 attraverso e18.5 con il loro picco produttivo di E15.5 6. L&#39;orientamento genetico di questa popolazione tardi nato, tuttavia, rimane sfuggente. Le distale-meno (Dlx) geni murini sono espressi esclusivamente in via di sviluppo proencefalo ventrale 3. Interneuroni GABAergici e neuroni striatali di proiezione, ma cellule piramidali corticali non esprimono Dlx1, 2,5, e 6 geni nelle prime fasi di sviluppo 3. Infatti, i geni Dlx sono espressi in zona MGE e CGE subventricolare (SVZ) in tutti i progenitori GABAergici. L&#39;espressione di questi geni si restringe per selezionare sottotipi nelle fasi post-mitotici 7-9. Evidenza sperimentale precedente ha mostrato che l&#39;elemento Dlx5 / 6 enhancer permette di targeting selettivo di lignaggi GABAergici in modelli di topi transgenici 2. Abbiamo testato l&#39;uso di uno di questi elementi enhancer nel contesto di espressione episomiale nel cervello di topo in via di sviluppo. Abbiamo sub clonato l&#39;elemento Dlx5 / 6 enhancer insieme ad un promotore minimo e la proteina fluorescente verde potenziata (eGFP) in un Bluescript (BS) backbone plasmide (Figura 1). Abbiamo introdotto il plasmide mediante elettroporazione in utero a E15.5 di mirare selettivamente Cr, VIP e Re- sottotipi 3,8,10. La nostra tecnica permette di elettroporazione sparse, che facilitala ricostruzione delle caratteristiche morfologiche delle cellule singe. Inoltre, eccezionalmente alti livelli di espressione genica in neuroni corticali GABAergici permette di studi funzionali. Abbiamo effettuato perdita e guadagno di studi di funzione utilizzando diversi wild type e geni dominanti negativi 11.

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			<td height="21" style="height:21px;">5-0 Silk Black Braid 18&#34; C-1 Box 36&#160;</td>
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subtype-selective electroporation of cortical interneurons

Lo studio del sistema nervoso centrale (CNS) maturazione si basa su di targeting genetica delle popolazioni neuronali. Tuttavia, il compito di limitare l&#39;espressione di geni di interesse per specifici sottotipi neuronali si è dimostrato straordinariamente difficile a causa della relativa scarsità di specifici elementi promotori. Interneuroni GABAergici costituiscono una popolazione neuronale con una vasta diversità genetica e morfologica. Infatti, più di 11 differenti sottotipi di interneuroni GABAergici sono stati caratterizzati nella corteccia mouse 1. Qui vi presentiamo un protocollo adatto per il targeting selettivo delle popolazioni GABAergici. Abbiamo raggiunto sottotipo selettivi mirati interneuroni GABAergici utilizzando l&#39;elemento enhancer della trascrizione homeobox fattori Dlx5 e Dlx6, omologhi del distale-less (Dll) gene Drosophila 2,3, per guidare l&#39;espressione di geni specifici mediante elettroporazione in utero.

Abbiamo adattato la tecnica di elettroporazione in utero per raggiungere tipo cellulare specifico orientamento dei neuroni in scadenza. Per guidare l&#39;espressione di eGFP in interneuroni CGE-derivati, abbiamo utilizzato l&#39;elemento enhancer Dlx5 / 6 e limitati i nostri iniezioni per E15.5, la fase in cui la maggior parte degli interneuroni CGE-derivati ​​vengono generati. Abbiamo effettuato l&#39;analisi a P8 e P15 11 (figure 1 e 2). Abbiamo confer...

Watch this Scientific Journal Video about Subtype-selective Electroporation of Cortical Interneurons at JoVE.com

Subtype-selective Electroporation of Cortical Interneurons

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Sottotipo-selettivo elettroporazione della corticale interneuroni

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

subtype-selective-electroporation-of-cortical-interneurons

Research

JoVE Journal

Neuroscience

8.7K Views.  New York University School of Medicine. This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Video: Subtype-selective Electroporation of Cortical Interneurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In utero Elettroporazione seguita da primaria Cultura neuronale per studiare la funzione dei geni in sottoinsieme di neuroni corticali

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

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Neuroscienze

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

Calcio Imaging funzionale nello sviluppo di reti corticali

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Isolamento e la coltura di astrociti corticali mouse

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Neonatale Elettroporazione zona subventricolare

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Ex utero elettroporazione e Espianti intero emisfero: un semplice metodo sperimentale per gli studi del primo sviluppo corticale

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Neonatale Pial elettroporazione Surface

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Whole-cell patch-clamp registrazioni da Morphologically- e neurochimico-identificati interneuroni dell&#39;ippocampo

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Una guida per<em&gt; In vivo</em&gt; Singola unità di registrazione da Optogenetically Identificato corticale inibitorio interneuroni

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Metodo elettrofisiologiche per la registrazione intracellulare di tensione Risposte di<em&gt; Drosophila</em&gt; Fotorecettori e Interneuroni a luce stimoli<em&gt; In Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

Visualizzazione dei moduli corticali nelle cortecce dei mammiferi appiattite

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...