Ringraziamo James Perna per l&#39;illustrazione grafica. Questo lavoro è stato sostenuto da sovvenzioni dal National Institute of Mental Health a YZ

Approvazione deve essere ottenuto da istituti d&#39;origine prima dell&#39;inizio della chirurgia e studio di imaging. Esperimenti descritti in questo manoscritto sono stati eseguiti in conformità con le linee guida e regolamenti della University of California, Santa Cruz Istituzionale cura degli animali e del Comitato Usa. 1. Chirurgia <ol><li> Sterilizzare in autoclave tutti gli strumenti chirurgici e sterilizzare l&#39;area di lavoro con il 70% di alcol accuratamente prima di un intervento chirurgico. </li><li> Anestetizzare il mouse intraperitoneale (IP) iniezione di soluzione anestetica KX (200 mg / ketamina kg e 20 mg / kg xilazina) in base al peso corporeo del topo. Nota: KX dosaggio può essere regolato a seconda del ceppo, età e lo stato di salute dei topi. Si raccomanda inoltre la consultazione veterinario per determinare la dose ottimale. </li><li> Eseguire un test toe-pinch periodicamente premendo le dita dei piedi del topo e di controllo per una risposta riflessiva per monitorare lo stato di anestesia.Assicurarsi che il mouse sia completamente anestetizzato prima di iniziare l&#39;intervento. Nota: Durante le sessioni di imaging prolungati, controllare lo stato di anestesia del topo periodicamente, amministrare ulteriore KX, se necessario. </li><li> Posizionare il mouse su una piastra elettrica per mantenere la temperatura corporea durante l&#39;intervento chirurgico. </li><li> Radere la testa del mouse usando una lama di rasoio per esporre il cuoio capelluto. </li><li> Sterilizzare l&#39;area rasata pulendo la pelle con alternanza di tamponi imbevuti di alcool e Betadine. Rimuovere eventuali ritagli di capelli residui. </li><li> Applicare delicatamente unguento occhio per lubrificare entrambi gli occhi, impedendo quindi danni permanenti causati dalla disidratazione durante l&#39;esperimento. </li><li> Effettuare una incisione retta lungo la linea mediana del cuoio capelluto, e spostare la pelle lateralmente verso i bordi del cranio. </li><li> Rimuovere il tessuto connettivo attaccato al cranio. </li></ol> 2. Diluito cranio-Preparazione <ol><li> Identificare la regione di imaging basato su ster. coordinate eotaxic Nota: Cercate di evitare grandi vasi sanguigni, che la penetrazione della luce di blocco e sfocare le strutture imaged. Vascolare è meglio osservata quando il cranio è umido con soluzione salina sterile. </li><li> Utilizzare il micro drill ad alta velocità per assottigliare una regione circolare del cranio (diametro 0,5-1,0 mm). Spostare il trapano parallelo alla superficie del cranio, piuttosto che tenendolo contro il cranio e premendo verso il basso. Drill fino a quando sia lo strato di osso compatto esterno e lo strato di osso spugnoso centrale vengono rimossi. Nota: Per prevenire i danni causati da surriscaldamento, evitare il contatto prolungato tra la punta e il cranio. </li><li> Continua assottigliamento dello strato di osso compatto interno con una lama microchirurgico nel centro della regione drill-assottigliato. Tenere la lama microchirurgico ad un angolo di circa 45 ° per raschiare il cranio senza premere contro il cranio finché un uniformemente diluiti piccola regione (200-300 micron di diametro) con uno spessore di approssimativaly 20-30 micron si ottiene. A causa della auto-fluorescenza del cranio, lo spessore può essere misurata attraverso la scansione la distanza tra la superficie superiore e inferiore del cranio sotto il microscopio a due fotoni. Nota: sebbene uno spessore cranio di meno di 20 micron fornisce una buona qualità delle immagini, non è consigliabile perché rende il processo di diradamento in sessioni di re-imaging successive difficile. Per evitare un eccessivo diradamento, lo spessore del cranio deve essere controllata periodicamente durante l&#39;intervento chirurgico (vedi Discussione). </li></ol> 3. Immobilizzazione <ol><li> Rimuovere con cautela detriti ossei. </li><li> Mettere una piccola goccia di colla cianoacrilato su ciascun bordo dell&#39;apertura centro della piastra di testa sterile, che è stato autoclavato (vedi Figura 1). Tenere la piastra di testa saldamente contro il cranio con la regione assottigliata situato nel centro dell&#39;apertura. Nota: Se la colla contamina il r assottigliataEgion per caso, rimuoverlo delicatamente con la lama microchirurgico. </li><li> Estrarre delicatamente la pelle dai lati del cranio ai bordi dell&#39;apertura centro della piastra di testa. </li><li> Attendere circa 10 minuti finché la piastra di testa è ben attaccato al cranio. </li><li> Posizionare la piastra di testa su due blocchi laterali della piastra di supporto e poi stringere le viti sui bordi della piastra di testa per immobilizzare il mouse sulla piastra di supporto (vedi Figura 1). Nota: Assicurarsi che non ci sia pelle o baffi tra la piastra di testa e blocchi prima di stringere le viti. Una buona preparazione immobilizzato dovrebbe mostrare alcun movimento osservabile del cranio al microscopio da dissezione quando la parte posteriore dell&#39;animale viene accarezzò delicatamente. </li><li> . Sciacquare il cranio esposto con soluzione fisiologica per rimuovere la colla non polimerizzato Nota: è importante per rimuovere eventuali residui di colla, come collante non polimerizzato offusca le immagini ei danni microscopio obiettivi &lt;./ Li&gt; </ol> 4. Imaging <ol><li> Prendere una foto del sistema vascolare del cranio esposta con la regione diluito mappa 1, che viene utilizzato per spostare la regione ripreso in sessioni di imaging successive (vedi Figura 2). </li><li> Posizionare il mouse sotto il microscopio imaging. Individuare la zona assottigliata sotto un obiettivo 10X aria usando epifluorescenza e spostare l&#39;area più sottile al centro della vista. </li><li> Aggiungere una goccia di soluzione salina sulla sommità del cranio e passare alla obiettivo 60X, sciacquato con acqua sterile. Seleziona una regione in cui le singole spine dendritiche sono chiaramente visualizzate lungo dendriti. Identificare ed etichettare la regione corrispondente Mappa 1 confrontando vascolare tra la vista epifluorescente e la foto vascolare. </li><li> Sintonizzare la lunghezza d&#39;onda del laser a due fotoni secondo i fluorofori. Per esempio, 920 nm per YFP; 890 nm per GFP; 1.000 nm per DsRed e tdTomato 24. </li><li> Acquisire pile di immagine con 2passi micron lungo l&#39;asse z usando l&#39;obiettivo 60X. Questo stack immagine copre circa 200 micron x 200 micron zona (512 x 512 pixel) e viene utilizzato come cartina 2 per trasferimento durante sessioni di imaging successive (vedi Figura 2). </li><li> Acquisire nove stack di immagini all&#39;interno Map 2 utilizzando lo zoom digitale 3X. Ogni stack immagine copre una superficie di circa 70 micron x 70 micron (512 x 512 pixel), con 0,7 micron passi lungo l&#39;asse z. Nota: L&#39;intensità del laser deve essere inferiore a 40 mW quando misurato a campioni di minimizzare fototossicità. </li></ol> 5. Recovery <ol><li> A seguito di imaging, staccare delicatamente la piastra di testa dal cranio. </li><li> Pulire accuratamente il cranio e la pelle per rimuovere tutti i residui di colla. Nota: Qualsiasi residuo di colla sulla pelle provoca irritazioni e rallentare la guarigione della pelle mentre l&#39;eventuale residuo di colla sul cranio causerà l&#39;erosione del cranio e di unngiogenesis nello strato osso appena cresciuti, rendendo il successivo riposizionamento e re-imaging difficile. </li><li> Sciacquare il cranio e la pelle con saline diverse volte. </li><li> Suturare il cuoio capelluto con sterile sutura chirurgica. </li><li> Tenere l&#39;animale su una piastra elettrica in una gabbia separata. Somministrare buprenorfina analgesico (0,1 mg / kg) per via sottocutanea per attenuare il dolore post-operatorio. Rispedire l&#39;animale verso la gabbia a casa dopo il pieno recupero. Monitorare l&#39;animale da vicino (controllare almeno una volta al giorno) fino a quando l&#39;incisione è guarita e punti di sutura vengono rimossi. Somministrare successive iniezioni di buprenorfina analgesico, se necessario. </li></ol> 6. Re-imaging <ol><li> Ripetere i passaggi 1,1-1,8. </li><li> Ripetere i passaggi 3,1-3,6 </li><li> Individuare regione già ripreso confrontando il pattern vascolare di Mappa 1 e il ramo modello dendritiche a Mappa 2. </li><li> Se reimaging viene effettuata entro 1 settimana, ripetere il passo 2.3 per rimuovere lo strato sottile di osso appena cresciuti in cima al re assottigliataregione con la lama microchirurgico. Se reimaging viene eseguita dopo più di 1 settimana, ripetere i passaggi sia 2.2 e 2.3. Nota: Lo strato osseo appena cresciuti costituito da una struttura meno condensata rispetto al livello originale osso compatto, con conseguente riduzione della qualità dell&#39;immagine. Pertanto, per acquisire la stessa qualità, è necessario sottile cranio leggermente superiore alla precedente sessione di imaging. </li><li> Regolare la posizione e l&#39;orientamento di ottenere pile di immagini con parametro precedentemente presi stack di immagini al microscopio a due fotoni. </li><li> Acquisire le immagini come al punto 4.6. </li><li> Ripetere i passaggi 5,1-5,5 dopo imaging. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

Per ottenere un successo preparato assottigliato-teschio, diversi passaggi in questo protocollo sono cruciali. 1) Lo spessore del cranio. L&#39;osso cranico ha una struttura a sandwich, con due strati di alta densità dell&#39;osso compatto e uno strato intermedio di bassa densità dell&#39;osso spugnoso. Mentre il micro trapano ad alta velocità è adatto per rimuovere gli strati esterni di osso compatto e osso spugnoso, la lama microchirurgico è ideale per diluire lo strato interno di osso compatto. Poiché lo spesso...

La corteccia dei mammiferi partecipa a molte funzioni cerebrali, dalla percezione e il controllo del movimento per l&#39;elaborazione delle informazioni sensoriali astratta e cognizione. Varie funzioni corticali si basano sui diversi circuiti neurali, che sono costituiti da diversi tipi di neuroni che comunicano e scambiano informazioni su singole sinapsi. La struttura e la funzione delle sinapsi sono costantemente in corso di modifica in risposta alle esperienze e patologie. Nel cervello maturo, la plasticità sinaptica assume la forma di entrambe le modifiche di forza e l&#39;aggiunta / rimozione di sinapsi, che giocano un ruolo importante nella formazione e il mantenimento di un circuito neurale funzionale. Spine dendritiche sono le componenti postsinaptici della maggioranza delle sinapsi eccitatorie nel cervello dei mammiferi. Il fatturato costante e le variazioni morfologiche delle spine si crede per servire come un buon indicatore di modifiche nelle connessioni sinaptiche 1-7. Due fotoni scansione laser microscopia offre penetrazione profonda attraverso fitti, preparati opachi e bassa fototossicità, che lo rende adatto per l&#39;imaging dal vivo nel cervello intatto 8. In combinazione con marcatura fluorescente, imaging a due fotoni fornisce un potente strumento per sbirciare nel cervello vivente e seguire riorganizzazione strutturale a livello delle sinapsi individuali con alta risoluzione temporale e spaziale. Vari metodi sono stati utilizzati per preparare topi per l&#39;imaging dal vivo 9-13. Qui, descriviamo un preparato assottigliato-teschio in vivo di due fotoni imaging per studiare la plasticità strutturale postsinaptici spine dendritiche nella corteccia mouse. Usando questo approccio, i nostri studi recenti hanno rappresentato un quadro dinamico di cambiamenti spine dendritiche in risposta alle abilità motoria imparando Con la crescente disponibilità di animali transgenici con sottoinsiemi di neuroni fluorescente e rapido sviluppo di vivo tecniche in materia di etichettatura, procedure analoghe qui descritti possono essere applicati anche di indaginete altri tipi cellulari e regioni corticali, in combinazione con altre manipolazioni, così come utilizzato in modelli di malattia 16-23.

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		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Ketamine</td>
			<td style="width:181px;">Bioniche Pharma</td>
			<td style="width:108px;">67457-034-10</td>
			<td style="width:775px;">Mixed with xylazine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Xylazine</td>
			<td style="width:181px;">Lloyd laboratories</td>
			<td style="width:108px;">139-236</td>
			<td>Mixed with ketamine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Saline</td>
			<td style="width:181px;">Hospira</td>
			<td style="width:108px;">0409-7983-09</td>
			<td>0.9% NaCl for injection and imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Razor blades</td>
			<td style="width:181px;">Electron microscopy sciences</td>
			<td style="width:108px;">72000</td>
			<td>Double-edge stainless steel razor blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Alcohol pads</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">06-669-62</td>
			<td>Sterile alcohol prep pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Eye ointment</td>
			<td style="width:181px;">Henry Schein</td>
			<td style="width:108px;">102-9470</td>
			<td>Petrolatum ophthalmic ointment sterile ocular lubricant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">High-speed micro drill</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">18000-17</td>
			<td>The high-speed micro drill is suitable for thinning the outer layer of compact bone and targeting a small area</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Micro drill steel burrs</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">19007-14</td>
			<td>1.4 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Microsurgical blade</td>
			<td style="width:181px;">Surgistar</td>
			<td style="width:108px;">6961</td>
			<td>The microsurgical blade is suitable for thinning the inner layer of compact bone and middler layer of spongy bone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Cyanoacrylate glue</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">NC9062131</td>
			<td>Fix the head plate onto the skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Suture</td>
			<td style="width:181px;">Havard Apparatus</td>
			<td style="width:108px;">510461</td>
			<td>Non-absorbale, sterile silk suture, 6-0 monofilament</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Dissecting microscope</td>
			<td>Olympus</td>
			<td style="width:108px;"></td>
			<td>SZ61</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">CCD camera</td>
			<td>Infinity</td>
			<td style="width:108px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Two-photon microscope</td>
			<td style="width:181px;">Prairie Technologies</td>
			<td style="width:108px;"></td>
			<td style="width:775px;">Ultima IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">10X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 0.30, air</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">60X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 1.1, IR permeable, water immersion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Ti-sapphire laser</td>
			<td style="width:181px;">Spectra-Physics</td>
			<td style="width:108px;"></td>
			<td>Mai Tai HP</td>
		</tr>
	</tbody>
</table>

two-photon in vivo imaging of dendritic spines in the mouse cortex using a thinned-skull preparation

Nella corteccia dei mammiferi, i neuroni formano reti estremamente complicate e scambiare informazioni a livello delle sinapsi. I cambiamenti della concentrazione sinaptica, nonché aggiunta / rimozione di sinapsi, si verificano in modo dipendente dall&#39;esperienza, fornendo la base strutturale di plasticità neuronale. Come componenti postsinaptici delle sinapsi più eccitatori nella corteccia, spine dendritiche sono considerati un buon indicatore di sinapsi. Prendendo vantaggi della genetica del topo e tecniche di etichettatura fluorescenti, i singoli neuroni e le loro strutture sinaptiche possono essere etichettati nel cervello intatto. Qui vi presentiamo un protocollo di imaging transcranica con due fotoni microscopia a scansione laser per seguire fluorescente post-sinaptici spine dendritiche nel tempo in vivo. Questo protocollo utilizza un preparato assottigliato-teschio, che mantiene il cranio intatto ed evita effetti infiammatori provocati dall&#39;esposizione delle meningi e la corteccia. Pertanto, le immagini possono essere acquisite subito dopo suviene eseguita rgery. La procedura sperimentale può essere eseguita ripetutamente su vari intervalli temporali da ore ad anni. L&#39;applicazione di questa preparazione può anche essere esteso per indagare diverse regioni corticali e strati, così come altri tipi di cellule, in condizioni fisiologiche e patologiche.

In YFP-H topi linea 25, proteina fluorescente gialla esprime in un sottoinsieme di neuroni piramidali strato V, che proiettano loro dendriti apicali agli strati superficiali della corteccia. Attraverso la preparazione assottigliato-teschio, i segmenti dendritiche fluorescente possono essere ripetutamente ripreso sotto il microscopio a due fotoni su vari intervalli di imaging, che vanno da ore a mesi. Qui mostriamo un esempio di rappresentazione quattro volte le stesse dendriti oltre 8 giorni in corteccia moto...

Watch this Scientific Journal Video about Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation at JoVE.com

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

Two-Photon<em&gt; In vivo</em&gt; Imaging di spine dendritiche nella corteccia mouse Uso un teschio Diluito-Preparazione

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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Research

JoVE Journal

Neuroscience

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

17.8K Views.  University of California, Santa Cruz. Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

Video: Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

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 ...

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Neuroscienze

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

Imaging in vivo del midollo spinale di topo Utilizzo di due fotoni Microscopia

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

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. ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

Un lucido e rinforzato Assottigliato cranio-Window a lungo termine Imaging del cervello di topo

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

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 ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

Diluito-cranio Tecnica finestra corticale per<em&gt; In Vivo</em&gt; Tomografia a coerenza ottica Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In Vivo a due fotoni di imaging dei cambi molecolari esperienza-dipendenti nei neuroni corticali

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l&#39;integrazione Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Due fotoni Imaging di Cellular Dynamics nel midollo spinale del mouse

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

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

Imaging in vivo del midollo spinale di topo Utilizzo di due fotoni Microscopia

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

Un lucido e rinforzato Assottigliato cranio-Window a lungo termine Imaging del cervello di topo

Visualizzazione e manipolazione genetica di dendriti e spine nella corteccia cerebrale e nell'ippocampo mouse utilizzando

Diluito-cranio Tecnica finestra corticale per

In Vivo a due fotoni di imaging dei cambi molecolari esperienza-dipendenti nei neuroni corticali

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l'integrazione Synaptic

Due fotoni Imaging di Cellular Dynamics nel midollo spinale del mouse

Simultanea a due fotoni