Questo lavoro è stato supportato da CNPq (Conselho Nacional de Desenvolvimento e Científico Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) e FAPERN (Fundação de Amparo un Pesquisa do Rio Grande do Norte).

All experiments involving live animals described in this protocol are conducted according to the National and International laws and were approved by the local University Animal Care and Use Committee (CEUA/UFRN), under the license 009/2014. The following protocol is performed in a sterile environment. Familiarity with basic cell culture is expected.
1. Dorsolateral Telencephalon Microdissection
<ol>
	<li>Prepare the dissection medium (100 mL): 98.5 mL Hank&#39;s Balanced Salt Solution (HBSS), 0.5 mL 5 M HEPES pH 7.4, 1 mL penicillin/streptomycin (10,000 units/mL and 10,000 &#181;g/mL).</li>
	<li>Filter-sterilize the dissection medium.</li>
	<li>Remove embryonic day 14 (E14) embryos from a pregnant mouse C57/Bl6 (Mus musculus) under anesthesia with isoflurane. 
	NOTE: Consider E0 as the day of vaginal plug detection. Dissection should be completed no later than 1 h after removal from the pregnant mouse. This protocol also applies for embryos E11-E17.</li>
	<li>Remove the embryos by hysterectomy under sterile conditions.</li>
	<li>Transfer the embryos to a Petri dish with cold dissection medium (4 &#176;C).</li>
	<li>Remove the brains from the skull by cutting along the longitudinal fissure. The number of embryos usually varies from five to ten. Each E14 brain yields approximately 2 x 106 cells, including progenitors and postmitotic neurons (1:1 ratio) 
	NOTE: Use the stereomicroscope for the following steps.</li>
	<li>Remove the meninges using dissection forceps. Split the telencephalon in the middle to separate the hemispheres. To isolate the dorsolateral telencephalon, cut along the dorsomedial curve and pallial-subpallial boundary using dissection forceps. Transfer the dorsolateral telencephalon to a 2 mL tube with cold dissection medium (4 &#176;C) until all brains are dissected.</li>
</ol>
2. Cell Dissociation and Plating
<ol>
	<li>Prepare the proliferation medium (50 mL of DMEM+10% FCS): 44 mL Dulbecco Modified Eagle&#39;s Medium (DMEM), 5mL Fetal Calf Serum (FCS), 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 &#181;g/mL). Filter-sterilize the proliferation medium.</li>
	<li>Prepare differentiation medium (50 mL of DMEM+2% B27): 48 mL DMEM, 1 mL B27, 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 &#181;g/mL). Filter-sterilize the differentiation medium.</li>
	<li>After micro-dissection of the dorsolateral telencephalon from E14 mice, centrifuge the tube with the collected tissue and cold dissection medium (for 5 min at 4 &#176;C, 340 x g) to precipitate the tissue.</li>
	<li>Remove the supernatant with a pipette and add 1 mL of pre-warmed (37 &#176;C) Trypsin-EDTA (0.05%) for chemical digestion. Incubate for 15 min at 37 &#176;C. Add 2 mL of proliferation medium to stop trypsin activity.</li>
	<li>Polish the tip of a glass Pasteur pipette over a gas burner or Bunsen burner for few seconds to slightly narrow the aperture. Wash the pipette by aspirating FCS to coat the tip. Avoiding bubbles, dissociate mechanically the cells with a fire-polished and FCS-coated Pasteur pipette.</li>
	<li>Centrifuge the cells (for 5 min at 4 &#176;C, 340 x g). Remove supernatant with a pipette. Add 1 mL of proliferation medium and resuspend the cells using a pipette. Repeat this step once.</li>
	<li>Prepare a 1:1 dilution of the cell suspension using a 0.4% Trypan Blue solution. Non-viable cells will be blue. Count the number of viable cells (unstained) using a Neubauer chamber.</li>
	<li>Dilute the cells in proliferation medium to get 106 cells/mL. Add 500 &#181;L of the cell suspension to each well of a 24 well tissue culture plate (approximately 2.5 &#215; 105&#160;cells/cm2). Incubate cells at 37 &#176;C and 5% CO2. 
	NOTE: Do not use glass coverslips, as they can move during the experiment and change the field of observation. Alternatively, use glass bottom tissue culture plates. 
	NOTE: If you are not using cell-culture treated multidished, it is important to pre-treat your plates with Poly-D-Lysin (50 &#181;g/ml) 2 h at 37 &#176;C. PDL-treated plates can be stored at 4 &#176;C after washing with distilled water.</li>
</ol>
3. Retroviral-mediated Transfection
NOTE: This is a simple method to transfect only progenitor cells. However, other viral vectors or chemical/electrical transfection can be used to insert genes of interest in cultured cells.
<ol>
	<li>To manipulate gene expression and label cells with fluorescent reporter proteins, add retroviruses carrying plasmids with genes of interest 2 h after cell plating. 
	NOTE: Retroviral vectors only integrate into the genome of dividing cells. Therefore, it is important to infect cells when proliferation is high. 
	NOTE: We used a plasmid containing the Beta-actin/CMV fusion sequence (CAG) and an internal ribosomal entry site (IRES) followed by the coding sequence for the green fluorescent protein (GFP), designated pCAG-IRES-GFP44. Genes of interest could be cloned between the CAG promoter and IRES sequence37.</li>
	<li>Dilute the retrovirus in differentiation medium to obtain the desired number of particles per mL. Add 500 &#181;L of differentiation medium with retrovirus to each well. 
	NOTE: The amount of retroviruses used is dependent on its titer and should be calculated to obtain 50 to 100 infected cells per well. This titer allows the identification of isolated GFP-expressing clones, whose relationship can be further confirmed by tracking phase contrast images (see section 6). The titration of retrovirus should be performed using the same cell population45.</li>
</ol>
4. Time-lapse Video-microscopy
NOTE: This step requires an inverted fluorescence microscope with incubation chamber (see Table of Materials).
<ol>
	<li>After the retrovirus infection, place the tissue culture plate into an incubator connected to temperature and CO2 controllers. 
	NOTE: Preheating the chamber for 3 h prior to starting imaging could help minimize drift and defocus during the acquisition. 
	NOTE: The chamber should maintain the cells at constant conditions of 37 &#176;C and 5% CO2.</li>
	<li>Select a position in the XY axis to serve as a zero-point (XY = 0). This will allow a reference to find the positions again in the future, for instance after post-imaging immunocytochemistry (see section 5). 
	NOTE: It is advisable to draw a sign in the plate that can be used as a reference to determine the zero-point.</li>
	<li>Select the positions to be imaged at 10X magnification. 
	NOTE: Select 10-15 positions per well to increase the likelihood to observe retrovirally-transfected cells. 
	NOTE: Long-distance high magnification objectives (20X or 40X) can be used to gain higher resolution images. Depending on the working distance of objectives, glass-bottom plates may be required.</li>
	<li>Acquire phase contrast images every 5 min and fluorescence images every 3 h to decrease phototoxicity. Check and adjust the focus in the first 3 h of experiment, as it may change while the temperature equilibrates. Check and adjust the focus daily. 
	NOTE: We observe that the focus becomes stable after the first 24 h. However, it is advisable to monitor the focus.&#160;Alternatively, use a software-based autofocus system.</li>
	<li>After 7 days, stop acquisition and proceed to post-imaging immunocytochemistry (see section 5).</li>
	<li>After immunocytochemistry, replace the plate in the microscope incubator, find the zero-point, reset XYZ = 0 and reload the experiment with saved positions. Acquire the fluorescence images of each position using appropriate fluorescence filters.</li>
</ol>
5. Post-imaging Immunocytochemistry
<ol>
	<li>After 7 days in culture, fix the cell culture with 4% Paraformaldehyde (PFA) fixative for 15 min at room temperature. 
	CAUTION: PFA is toxic and should be handle in a chemical fume hood.</li>
	<li>Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.</li>
	<li>Incubate in primary antibodies overnight at 4 &#176;C in 0.5% triton and 10% of normal goat serum in PBS 0.1 M. 
	NOTE: Use anti-Microtubule Associated Protein 2 (MAP2) antibody to confirm the neuronal phenotype (mouse IgG1, 1:1,000) and anti-GFP to label the transduced cells (chicken antibody, 1:500). Other antibodies can be used to detect progenitor or glial cells.</li>
	<li>Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.</li>
	<li>Dilute secondary antibodies in 0.5% triton and 10% of normal goat serum in PBS 0.1 M and incubate for 2 h at room temperature. Recommended dilution: 1:1,000.</li>
	<li>Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.</li>
	<li>For nuclei staining, incubate the cells for 5 min with 0.1 &#181;g/mL 4&#8242;6&#8242;-diamino-2-phenylindone (DAPI) in PBS 0.1 M.</li>
	<li>Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.</li>
	<li>Keep the cells in PBS 0.1 M and protect from light (e.g., wrap the plate with foil).</li>
</ol>
6. Cell Tracking
NOTE: Here, we briefly describe the main steps to analyze time-lapse video-microscopy data using the software The Tracking Tool (tTt), which is available at the link provided in Table of Materials.
NOTE: If the image acquisition is performed with other software, import the image data with tTt Converter available in the tTt installer (link provided in Table of Materials). Only png, tif or jpg image formats are accepted. Follow the steps shown at Figure 1 A - B after starting the tTt Converter.
<ol>
	<li>Start the software tTt and choose a user name. Click on &#34;add user&#34; and &#34;continue&#34; button (Figure 2A). Browse and select the &#34;TTTWorkFolder&#34; (Figure 2B). Lineages trees, statistics and exported images will be saved in this folder. Next, select the experiment to be analyzed and load it (Figure 2B).</li>
	<li>If the images were converted previously by tTt Converter, click on &#34;LogFileConverter&#34; (Figure 3A) and specify the number of seconds between two consecutive time points in the LogFileConverter window. Click on &#34;Convert log files for whole experiment&#34; (Figure 1C).</li>
	<li>In a list of images, which appear in the tTt window, select the desired images that by loading manually or using the option displayed in the program. Load images (Figure 3).</li>
	<li>Select File &#62; Open &#62; New colony. Click on &#34;Tracking&#34;, followed by &#34;Start Tracking&#34; to start the tracking in a chosen position.</li>
	<li>A movie window will appear (Figure 4). Select the cell using the tracking circle and press the key 0. The software will go to the next frame. Keep on positioning the tracking circle around the tracked cell using the mouse and click 0 in each frame to add a track mark on the cell.</li>
	<li>Use the keys 1 and 3 to move to the previous or next frame, respectively. To delete a track mark, just click on the correct position to mark and press 0.</li>
	<li>If the cell divided, select the button &#34;division&#34; (red rectangle at Figure 4). Continue tracking one daughter cell by pressing Shift+D. To track the second daughter cell, select it in the cell editor and press F2.</li>
	<li>If the cell died during the video microscopy, select &#34;apoptosis&#34; (blue rectangle at Figure 4). 
	NOTE: Although the software labels the event as &#34;apoptosis&#34;, other mechanisms may be responsible for the cell death.</li>
	<li>If the cell leaves the field of observation or intermingles with neighboring cells precluding tracking, select &#34;lost&#34; (green rectangle at Figure 4).</li>
	<li>To stop tracking and continue later click on &#34;interrupt&#34; (purple rectangle at Figure 4). 
	NOTE: In the Cell editor window a tree will be generated during the tracking. To save the lineage tree, select File &#62; Save &#62; Current tree (in the Cell editor window).</li>
	<li>To start tracking a different cell, click on the cell using the right mouse button or select the cell using the left mouse button and click on Tracking &#62; Start Tracking.</li>
	<li>To export the movie with the tracking data, the software should not be in &#34;tracking mode&#34;. To turn off the tracking mode, execute step 6.10. In the Movie window select Export &#62; Movie. Set up the format, range of frames, number of frames per second and bitrate. Click on &#34;Start export&#34;.</li>
</ol>
7. Quantifications
NOTE: Several measurements can be performed using video microscopy data46. Here, we describe three possibilities that are exemplified later in the &#34;representative results&#34; section.
<ol>
	<li>Quantify the cell survival for each cell lineage by dividing the number of cells alive at different time points by the total number of cells generated before these time points were reached within individual clones37.</li>
	<li>Quantify the proportion of symmetric progenitor (SP, both daughter cells continue to proliferate), asymmetric (A, one daughter cell continues to proliferate and the other becomes postmitotic), or symmetric terminal (ST, both daughter cells become postmitotic)15,38.</li>
	<li>Measure the cell cycle length by calculating the time span of proliferating cells between their generation and division15.</li>
</ol>

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Osservazione in tempo reale delle cellule della corteccia cerebrale primaria permette l'analisi di proliferazione delle cellule, la modalità di divisione cellulare, durata del ciclo cellulare, differenziazione cellulare e cella sopravvivenza3,14,15,37. Ancora più importante, consente lo studio dei lignaggi unicellulare, consentendo l'identificazione delle fasi intermedie promulgata durante la progressione da NSC a neuroni3. Infine, la combinazione di questo sistema di coltura con strumenti di bioingegneria per manipolare l'espressione genica è una tecnica potente per studiare l'effetto delle cellule-autonoma di target selezionati5. Il metodo qui descritto può essere modificato per studiare la stirpe dei progenitori di corteccia cerebrale isolati a diversi stadi di sviluppo14, così come NSC isolate da altre fonti38,42. Metodi simili sono stati utilizzati anche per studiare stirpi delle cellule della retina in via di sviluppo41.
Qui, vi mostriamo un semplice esperimento mediante transfezione mediata retrovirale alle cellule progenitrici etichetta con una proteina fluorescente del reporter. Tuttavia, simili obiettivi possono essere raggiunti utilizzando altri vettori virali, chimico/elettrico transfezione o animali transgenici che esprimono le proteine fluorescenti sotto il controllo del tipo di cellula neurale specifici promotori38,39. Tutti questi metodi per controllare l'espressione genica possono essere applicati anche per indurre o sopprimere l'espressione di geni di interesse, permettendo lo studio dei meccanismi molecolari coinvolti nella stirpe delle cellule staminali neurali progressione15,18,39. Prevediamo inoltre che questo sistema può essere utilizzato per valutare come le diverse espressioni dei livelli di proteine specifiche regolano NSC comportamento e un neurone/macroglial differenziamento, simile a quello che è stato fatto nel sistema ematopoietico40. Inoltre, può aiutare a fare luce sulle potenzialità del singoli progenitori per generare distinti lignaggi neuronale.
Rispetto ad altre tecniche di imaging NSC di mammiferi in vivo-animali o nelle culture della fetta, il metodo qui descritto ha alcuni importanti vantaggi. In primo luogo, il basso costo del metodo è un beneficio significativo. Microscopi rovesciati semplice dotati di trasmessa e fluorescenza luci e una macchina fotografica controllata dal software basati su computer può essere utilizzati per acquisire immagini di culture 2D per fino a parecchie settimane. In secondo luogo, il numero di animali utilizzati per questi esperimenti è significativamente minore in altri metodi. In terzo luogo, il sistema permette un controllo preciso delle condizioni ambientali, permettendo così l'analisi degli effetti delle cellule-autonomi e non autonomi delle cellule di manipolazioni diverse. Infine, le singole celle possono essere osservate senza ambiguità per fino a 15 giorni, consentendo la ricostruzione precisa di alberi di grande lignaggio, che attualmente non è possibile sia nella corteccia cerebrale affettare culture o in vivo. D'altra parte, il sistema può presentare gli svantaggi connessi con la perdita di organizzazione del tessuto. Si consiglia, pertanto, che i cellulari comportamenti osservati in queste culture 2D dovrebbero essere idealmente confermati da altri esperimenti in vivo.
Dati precedenti utilizzando questo spettacolo di sistema che la cella ciclo allungamento del progenitore corticale in vivo48 sono riprodotta in vitro15. Il neurogena e gliogenic potenziale della corteccia cerebrale individuale progenitori sono anche imitati nel sistema 2D cultura descritto qui3. Infine, la proliferazione delle cellule e ciclo cellulare uscita rapporti osservati in vivo può anche essere imitato utilizzando questa cella cultura sistema15,18. Così, crediamo che vivere-imaging delle cellule della corteccia cerebrale primario coltivato nelle condizioni descritte in questo protocollo è un metodo potente e user-friendly per studiare i meccanismi cellulari e molecolari che controllano la proliferazione delle cellule progenitrici, differenziazione delle cellule neuronali e gliali e specifica di destino delle cellule.

Cellule staminali neurali (NSC) generare neuroni e cellule macroglial durante lo sviluppo della corteccia cerebrale. Al primi-corticogenesis, NSC sottoporsi a diversi cicli di divisione cellulare simmetrica ed espandere il pool di cellule progenitrici. Quindi, NSC dividono asimmetricamente per generare neuroni direttamente o indirettamente attraverso mediatori1. Solo a metà - alla fine-corticogenesis, progenitori passare per generare gli astrociti e oligodendrociti2,3,4. Tuttavia, i completare i meccanismi che controllano la proliferazione delle cellule e differenziazione, come pure il contributo dei progenitori destino-limitato alla generazione di tipi unici di neuroni o cellule macroglial rimangono una questione di intenso dibattito4,5,6. Il potenziale delle singole CSN corticale per generare neuroni, astrociti e oligodendrociti è stato estesamente studiato in vitro e in vivo utilizzando una miriade di tecniche come: live imaging in cella singola culture7,8,9,10,11,12,13; live imaging ad alta densità culture culture3,14,15; live imaging fetta culture16,17,18; analisi clonale utilizzando virale mediata da vettore genetico etichettatura ad alta densità culture14,15,19,20,21; analisi clonale in vivo utilizzando retrovirus22,23,24,25,26,27,28,29,30,31,32,33; e analisi clonale in vivo usando animali transgenici34.
Ognuna di queste tecniche presenta vantaggi e svantaggi. Per esempio, in vivo lignaggio traccia è suscettibile al lumping e scissione errori3, che porti a conclusioni contrastanti circa il potenziale dei singoli progenitori corticali. Inoltre, sia gli studi in vitro ed in vivo basano sull'etichettatura delle cellule progenitrici a intervalli di tempo iniziale e posteriore analisi delle stirpi delle cellule possono essere influenzata dall'avvenimento inosservato di morte delle cellule durante il lignaggio-progressione35. Di conseguenza, un sistema adatto per analizzare le potenzialità del singolo NSCs dovrà consentire l'identificazione di tutte le cellule generate, così come la caratterizzazione appropriata dei destini di cella all'interno della stirpe. Combinazione di colture cellulari primarie e live imaging fornisce questa impostazione. Utilizzando cella singola cultura e time-lapse video microscopia, Tempio et al hanno dimostrato l'interruttore del lignaggio della corteccia cerebrale individuale progenitori da neurogenesi al gliogenesis11. Più tardi, hanno usato lo stesso sistema per mostrare che i diversi tipi di neuroni sono generati da un unico progenitore corticale12. Tuttavia, questo sistema presenta un importante avvertimento: solo 1% dei progenitori corticali isolati alle prime corticogenesis generare cloni di 4 o più progenie9. Dopo l'aggiunta di FGF2, la frequenza delle cellule generando 4 o più celle aumenta a 8-10%9. Tuttavia, questo numero è troppo piccolo considerando che praticamente tutti i progenitori corticali sono proliferativi in questa fase. Inoltre, i potenziali effetti di FGF2 il destino-specifica non possono essere esclusa36. Per ovviare a queste limitazioni, abbiamo usato colture ad alta densità cellulari che supportano la proliferazione di entrambi ventricolare (Pax6-esprimendo) e subventricolare (Tbr2-esprimendo) corticale progenitori15. L'osservazione in tempo reale di queste culture ha inoltre dimostrato che diverse caratteristiche della progressione del lignaggio di NSC sono riprodotte in queste condizioni, come la modalità di divisione cellulare, allungamento del ciclo cellulare, il potenziale delle singole cellule per generare neuroni e cellule gliali, tra gli altri3,15. Più recentemente, abbiamo utilizzato questo sistema anche a dimostrare che CREB-segnalazione colpisce la sopravvivenza delle cellule dei neuroni immaturi corteccia cerebrale in topi37. Così, crediamo che Time-lapse video-microscopia della corteccia cerebrale murino primario le cellule coltivate in ad alta densità è uno strumento potente e accessibile per studiare i meccanismi cellulari e molecolari della progressione del ciclo cellulare, la modalità di divisione cellulare, la sopravvivenza delle cellule e cellula destino specifica. Quest'ultima può essere eseguita utilizzando animali transgenici, permettendo l'identificazione di destini cellulari specifiche tempo reale38,39,40 o l'uso di post-imaging immunocytochemistry3,38,41,42.
Qui, forniamo un protocollo passo dopo passo per preparare la corteccia cerebrale primaria coltura cellulare supporto proliferazione delle CSN e la successiva generazione dei neuroni e delle cellule macroglial. Inoltre discutiamo l'uso di transfezione mediata retrovirale per manipolare l'espressione genica delle cellule individuali, che possono essere identificati e monitorata a livello di singola cellula usando la microscopia video time-lapse. Questo protocollo può essere utilizzato per studiare la corteccia cerebrale primario cellule isolate dall'inizio alla fine del corticogenesis nei roditori, ma qualche aggiustamento può essere richiesto secondo la fase14. NSC isolate da altre fonti può essere studiata anche usando la microscopia video time-lapse di culture 2D, ma il sistema di coltura appropriato dovrebbe essere determinato comparando i comportamenti delle cellule in vitro e in vivo38,43.

<table width="953">
	<tbody>
		<tr height="40">
			<td height="40" style="width:320px;height:40px;">Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td style="width:123px;">Invitrogen Life Technologies</td>
			<td style="width:344px;">14175129</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">H3375-25G</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin/streptomycin</td>
			<td style="width:123px;">Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco Modified Eagle&#39;s Medium (DMEM)</td>
			<td style="width:123px;">Gibco</td>
			<td>12400-024</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal Calf Serum (FCS)</td>
			<td style="width:123px;">Gibco</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glucose</td>
			<td style="width:123px;">Gibco</td>
			<td>A2494001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B27</td>
			<td style="width:123px;">Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">trypsin-EDTA (0.05%)</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:344px;">25300054</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td style="width:344px;">16005</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:320px;height:40px;">Goat serum</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">69023</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:320px;height:40px;">Triton X-100</td>
			<td style="width:123px;">VWR International Ltd.</td>
			<td style="width:344px;">306324N</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane</td>
			<td>Sigma</td>
			<td>792632</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-MAP2, mouse</td>
			<td>Sigma</td>
			<td>M4403</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-GFP chicken</td>
			<td>Aves</td>
			<td>0511FP12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Goat anti-mouse alexa 594</td>
			<td>Invitrogen</td>
			<td>A11005</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Goat anti-chicken alexa 488</td>
			<td>Invitrogen</td>
			<td>A11039</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:320px;height:40px;">ImageJ</td>
			<td style="width:123px;">NIH</td>
			<td style="width:344px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">tTt</td>
			<td>ETH Zurich</td>
			<td style="width:344px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell observer microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pasteur pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">The Tracking Tool (tTt) software</td>
			<td></td>
			<td>https://www.bsse.ethz.ch/csd/software/ttt-and-qtfy.html</td>
			<td>download link</td>
		</tr>
	</tbody>
</table>

live imaging of primary cerebral cortex cells using a 2d culture system

Durante lo sviluppo della corteccia cerebrale, cellule progenitrici sottoporsi a diversi cicli di divisioni cellulari simmetriche e asimmetriche per generare nuovi progenitori o neuroni postmitotic. Più tardi, alcuni progenitori passare ad un destino di gliogenic, aggiungendo alle popolazioni astrociti e oligodendrociti. Utilizzo di time-lapse video-microscopia di colture di cellule della corteccia cerebrale primario, è possibile studiare i meccanismi cellulari e molecolari che controllano la modalità di divisione cellulare e i parametri del ciclo cellulare delle cellule progenitrici. Allo stesso modo, il destino delle cellule postmitotic può essere esaminato utilizzando proteine reporter fluorescenti cellula-specifico o post-imaging immunocitochimica. Ancora più importante, tutte queste caratteristiche possono essere analizzate a livello di singola cellula, permettendo l'identificazione di progenitori impegnati per la generazione di tipi cellulari specifici. Manipolazione dell'espressione genica può essere eseguita anche mediante transfezione mediata virale, permettendo lo studio dei fenomeni di cella-autonomi e non autonomi delle cellule. Infine, l'utilizzo di proteine fluorescenti di fusione permette lo studio della distribuzione simmetrica e asimmetrica delle proteine selezionate durante la divisione e la correlazione con il destino di cellule figlie. Qui, descriviamo il metodo time-lapse video-microscopia per immagine cellule murine di corteccia cerebrale primaria per diversi giorni e analizzare la modalità di divisione cellulare, durata del ciclo cellulare e destino delle cellule appena generate. Inoltre descriviamo un metodo semplice per transfect cellule progenitrici, che possono essere applicate a manipolare i geni di interesse o semplicemente marcare le cellule con proteine reporter.

Primary cultures of cerebral cortex cells isolated from embryos E14 contain both progenitor and neuronal cells. During the period of imaging, progenitors undergo several rounds of cell division, increasing the number of cells (Figure 5 and Video Figure 1).
Retroviral-mediated transfection of a few progenitor cells facilitates the identification of cell clones (Figure 6). Note that GFP expression is detectable after 24 h. At this time-point, it is possible to identify GFP-expressing cells in phase contrast images and track them back to complete the lineage. The GFP images included here do not have a good contrast because: 1) endogenous GFP expression was observed a few days after retroviral transduction; 2) images were taken using a 10X long-distance objective through the plastic bottom of a 24-well plate; and 3) the fluorescence exposure time is set to the minimum detection of GFP signal without damaging cells (phototoxicity).
The tracking of single progenitor cells generates lineage trees revealing important information about their lineages (Figure 7). Based on these lineage trees, we can assess the clonal connection among differentiated cells, measure the cell cycle length, evaluate the mode of cell division based on the proliferative behavior of daughter cells (Symmetric Proliferative-both daughter cells undergo new cycle of cell division; Asymmetric - one daughter cell undergoes a new cycle of cell division and the other becomes postmitotic; Symmetric Terminal - both daughter cells become postmitotic), and quantify cell survival, cell speed and cell growth rate3,15,37,47.
Post-imaging immunocytochemistry using antibodies against the neuronal marker MAP2 and the reporter protein GFP shows differentiated neurons and non-neuronal cells generated from cortical progenitors that have been tracked (Figure 8).

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56063/56063fig1.jpg" /> 
Figure 1. Using &#34;tTt Converter&#34; to analyze time-lapse imaging acquired from another software. (A) Browse the image data to add them directly into the input folder (red rectangle). Activate the &#34;Parse meta-information from image file names&#34; for different positions, imaging channels or z-indexes (red arrow). tTt Converter automatically converts 16-bit images to 8-bit, however to specify the black- and white-point for each imaging channel, it is necessary to enable the &#34;Set 16-bit to 8-bit black/whitepoint&#34; option. More information about this step is available at &#34;How to set bp/wp for each channel&#34; bottom (red arrow). After editing the &#34;Experiment meta information&#34; section, browse the destination folder (blue rectangle) and click on &#34;Convert x images&#34; (orange rectangle). (B) Window of completed conversion. (C) Definition of seconds as fixed interval at &#34;tTt Log File Converter&#34; window (red arrow) before loading the images for tracking. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56063/56063fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56063/56063fig2.jpg" /> 
Figure 2. Selection of experiment at tTt. (A) In the User query window, click on &#34;Add User&#34; and enter user initials followed by selecting the &#34;Continue&#34; option. (B) Browse the folder for analysis at &#34;TTTWorkFolder&#34; section (red rectangle) and select the experiment at &#34;experiment folder&#34; section (blue rectangle). Next load the experiment (red arrow). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56063/56063fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56063/56063fig3.jpg" /> 
Figure 3. Selection of images. (A) Select the position to be analyzed (red rectangle). If the images were converted click on &#34;LogFileConverter&#34; and proceed as shown in Figure 1C. (B) Select the &#34;all&#34; bottom (red rectangle) and load the images (blue rectangle). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56063/56063fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56063/56063fig4.jpg" /> 
Figure 4. Tracking window. (A) Adjust the brightness and contrast using the &#34;adjust gamma&#34; button and select the &#34;Division&#34; (red rectangle), &#34;Apoptosis&#34; (blue rectangle), &#34;Lost&#34; (green rectangle) or &#34;Interrupt&#34; (purple rectangle) option for cell division, cell death, cell lost or to stop tracking, respectively. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56063/56063fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56063/56063fig5.jpg" /> 
Figure 5. Phase contrast images obtained by time-lapse video-microscopy at different time points. (A-H) Photomicrographs showing phase contrast images of a primary cerebral cortex cell culture at (A) Day 0, 04 h 15 min 20 s, (B) Day 0, 12 h 20 min 29 s, (C) Day 0, 20 h 27 min 09 s, (D) Day 1, 04 h 41 min 26 s, (E) Day 1, 12 h 45 min 57 s, (F) Day 1, 20 h 50 min 33 s, (G) Day 2, 04 h 54 min 52 s, and (H) Day 2, 12 h 59 min 15 s. Note the significant increase in cell density over time. Scale bar: 50 &#181;m <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56063/56063fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56063/56063fig6.jpg" /> 
Figure 6. GFP expression in retrovirally-transduced cells. Phase contrast images (A-G) and GFP fluorescent images (A&#39;-G&#39;) at the same time point. Note that GFP expression is absent during the first hours of imaging and starts to increase over time. (A-A&#39;) Day 0, 16 h 01 min 05 s, (B-B&#39;) Day 0, 21 h 49 min 26 s, (C-C&#39;) Day 1, 06 h 29 min 33 s, (D-D&#39;) Day 1, 15 h 35 min 00 s, (E-E&#39;) Day 2, 00 h 40 min 10 s, and (F-F&#39;) Day 2, 09 h 44 min 28 s. (F&#39;&#39;) High magnification of the dashed box in F&#39;. Yellow arrows (B-F) indicate the phase contrast image of a cell expressing GFP (B-F`). Scale bar: 50 &#181;m <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56063/56063fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56063/56063fig7.jpg" /> 
Figure 7. Cell lineage of single progenitor cells. (A-C) Phase contrast images showing an example of cell division. (A) Progenitor cell round up. (B) Cell membrane constriction. (C) Mitosis completion. (D) Example of a single-cell lineage tree generated in the software tTt. Colored arrows indicate different modes of cell division: Symmetric Proliferative (yellow arrow); Asymmetric (blue arrow); Symmetric Terminal (red arrow). &#34;X&#34; indicates cell death. Numbers indicate the cell cycle length of the progenitor cells. Scale bar: 50 &#181;m <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56063/56063fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56063/56063fig8.jpg" /> 
Figure 8. Post-imaging immunocytochemistry identifies neuronal cells. Cerebral cortex cells immunostained with antibodies against GFP (A) and the neuronal marker MAP2 (B). Merged images in (C) and higher magnification of merged images in (C&#39;). Yellow arrows point to GFP+ cells expressing MAP2. Tracking these cells back to the beginning of the experiment allows the identification of sibling neurons in the culture. Scale bars: 100 &#181;m <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56063/56063fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/56063/56063movie1.jpg" /> 
Movie 1. Time-lapse video microscopy of primary cerebral cortex cell culture. Phase contrast images acquired every 20 min displaying the detailed behavior of the cells in vitro. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56063/Movie_1.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System at JoVE.com

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

La formazione immagine dal vivo è un potente strumento per studiare i comportamenti cellulari in tempo reale. Qui, descriviamo un protocollo per time-lapse video-microscopia delle cellule della corteccia cerebrale primario che consente un esame dettagliato delle fasi promulgata durante la progressione di lignaggio da cellule staminali neurali primarie a differenziato neuroni e cellule gliali.

Immagini di corteccia cerebrale primario celle utilizzando un sistema 2D cultura dal vivo

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

live-imaging-primary-cerebral-cortex-cells-using-2d-culture

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.0K Views.  Federal University of Rio Grande do Norte. La formazione immagine dal vivo è un potente strumento per studiare i comportamenti cellulari in tempo reale. Qui, descriviamo un protocollo per time-lapse video-microscopia delle cellule della corteccia cerebrale primario che consente un esame dettagliato delle fasi promulgata durante la progressione di lignaggio da cellule staminali neurali primarie a differenziato neuroni e cellule gliali.

Video: Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral line-widths and allows high-resolution spectra to be obtained from extracts, intact cells, cell cultures, and more importantly intact tissue to investigate relationships between metabolites and cellular processes. In vivo HRMAS 1H-MRS studies have yet to be reported in the live fruit fly Drosophila melanogaster. Drosophila, as a simpler genetic organism, allows the multiple biological functions and various evolutionarily conserved signaling pathways to be examined at the whole organism level and it is a useful model for investigating genetics and physiology. To this end, we developed and implemented an in vivo HRMAS 1H-MRS method to investigate live Drosophila at 14.1 T. Here, we outline an HRMAS 1H-MRS protocol for the molecular characterization of Drosophila with a conventional MR spectrometer equipped with an HRMAS probe. This technique is a novel, in vivo, non-destructive Drosophila metabolite measurement approach, which enables the identification of disease biomarkers and thus may contribute to novel therapeutic development.

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

This technique enables the use of high-resolution magic angle spinning proton MR spectroscopy (HRMAS 1H-MRS) for molecular characterization of live Drosophila melanogaster with a conventional 14.1 tesla spectrometer equipped with an HRMAS probe.

Spettroscopia di Risonanza Magnetica dal vivo Drosophila melanogaster Utilizzando Magic Angle Spinning

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral ...

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Neuroscienze

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

Imaging cronica di mouse corteccia visiva Utilizzando un Assottigliato-teschio di preparazione

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Live-imaging di traslocazione della PKC in SF9 cellule e nei neuroni sensoriali Aplysia

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Live-cellula Imaging di cellule sensoriali Precursore in Organo intatto Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

Rilevamento di ipossia microregionale nella corteccia cerebrale di topo Imaging a due fotoni di fluorescenza endogena NADH

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualizzazione e manipolazione genetica di dendriti e spine nella corteccia cerebrale e nell&#39;ippocampo mouse utilizzando<em&gt; In utero</em&gt; Elettroporazione

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Imaging funzionale di Auditory Cortex in gatti adulti utilizzando ad alto campo fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Immagini dal vivo della mitosi in via di sviluppo embrionali di topo Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Immagini dal vivo di<em&gt; Drosophila</em&gt; larvali neuroblasti

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Imaging di attività neurale nella corteccia somatosensoriale primaria utilizzando Thy1-topi transgenici GCaMP6s

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...