Agradecemos James Perna para a ilustração gráfica. Este trabalho foi financiado por doações do Instituto Nacional de Saúde Mental para YZ

Aprovação deve ser obtida a partir de instituições de origem antes do início da cirurgia e estudo de imagem. Experimentos descritos neste manuscrito foram realizados de acordo com as diretrizes e regulamentos da Universidade da Califórnia, em Santa Cruz Institutional Animal Care e do Comitê Use. 1. Cirurgia <ol><li> Autoclave todos os instrumentos cirúrgicos e esterilizar o espaço de trabalho com 70% de álcool completamente antes da cirurgia. </li><li> Anestesiar o rato por intraperitoneal (IP) a injeção de solução anestésica KX (200 mg / kg de ketamina e 20 mg / kg de xilazina) de acordo com o peso corporal do rato Nota:. KX dose pode ser ajustada de acordo com a cepa, a idade eo estado de saúde dos ratos. Veterinária consulta para determinar a dose óptima é também recomendado. </li><li> Realizar um teste toe-pitada periodicamente, pressionando os dedos do rato e verificação de uma resposta reflexa para monitorar o estado de anestesia.Certifique-se de que o mouse está totalmente anestesiado antes de iniciar a cirurgia Nota:. Durante as sessões de imagem prolongados, verificar o estado de anestesia do rato periodicamente, administrar KX adicional se necessário. </li><li> Colocar o rato sobre uma almofada de aquecimento para manter a temperatura do corpo durante a cirurgia. </li><li> Raspar a cabeça do rato usando uma lâmina de barbear para expor o couro cabeludo. </li><li> Esterilizar a área raspada, limpando a pele com a alternância de compressas embebidas em álcool e betadine. Remova todos os recortes de cabelo residuais. </li><li> Gentilmente aplicar pomada para lubrificar os olhos, prevenindo danos permanentes causados ​​pela desidratação durante o experimento. </li><li> Fazer uma incisão em frente ao longo da linha média do couro cabeludo, e mover-se lateralmente para a pele das bordas do crânio. </li><li> Remover o tecido conectivo anexado ao crânio. </li></ol> 2. Diluído crânio-Preparação <ol><li> Identificar a região de imagem baseado em ster. coordenadas eotaxic Nota: Tente evitar grandes vasos sanguíneos, que a penetração da luz do bloco e esbater as estruturas fotografadas. Vasculatura é melhor observado quando o crânio é úmido com solução salina estéril. </li><li> Utilize o micro broca de alta velocidade para diluir uma região circular de crânio (0,5-1,0 mm de diâmetro). Mover a broca paralela à superfície do crânio, em vez de segurar o contra o crânio e pressionando para baixo. Broca até tanto a camada de osso compacto exterior ea camada de osso esponjoso meio são removidos Nota:. Para evitar danos causados ​​por superaquecimento, evitar o contato prolongado entre a broca eo crânio. </li><li> Continuar desbaste da camada interna do osso compacto com uma lâmina microcirúrgica no centro da região de desbaste-broca. Segurar a lâmina microcirúrgica a um ângulo de aproximadamente 45 ° para raspar o crânio sem pressionar contra o crânio até uma uniformemente diluído pequena região (200-300 mM de diâmetro) com uma espessura de aproximadamentely 20-30 mM é obtido. Devido à auto-fluorescência do crânio, a espessura pode ser medida por digitalizar a distância entre a superfície superior e inferior do crânio sob o microscópio de dois fotões Nota:. Embora uma espessura do crânio de menos de 20 mM proporciona uma boa qualidade de imagem, não é recomendado, porque torna o processo de afinamento em sessões de re-imagem subseqüentes difícil. Para evitar o excesso de desbaste, a espessura do crânio precisa ser verificado periodicamente durante a cirurgia (ver Discussão). </li></ol> 3. Imobilização <ol><li> Remova cuidadosamente os restos de osso. </li><li> Colocar uma pequena gota de cola de cianoacrilato sobre cada extremidade da abertura central da placa de cabeça estéril, que foi autoclavado (ver Figura 1). Manter a placa de cabeça firmemente contra o crânio com a região adelgaçada localizada no centro da abertura Nota:. Se a cola contamina o r diluídoegion por acidente, remova-a com cuidado com a lâmina de microcirurgia. </li><li> Suavemente puxar a pele a partir dos lados do crânio para os bordos da abertura central da placa de cabeça. </li><li> Espera aproximadamente 10 min, até que a placa de cabeça é também ligado ao crânio. </li><li> Coloque a placa de cabeça em dois blocos laterais da placa de retenção e, em seguida, aperte os parafusos ao longo das bordas da placa de cabeça para imobilizar o mouse sobre a placa de retenção (ver Figura 1) Nota:. Certifique-se de que não existe pele ou bigodes entre a placa de cabeça e os blocos antes de apertar os parafusos. Uma boa preparação imobilizada não deve apresentar movimento observável do crânio sob o microscópio de dissecação, quando a parte de trás do animal é afagou suavemente. </li><li> . Lavar o crânio exposto com solução salina para remover cola não polimerizada Nota: É importante remover qualquer cola restante, como cola não polimerizada borra imagens e danos microscópio objetivos &lt;./ Li&gt; </ol> 4. Imagem <ol><li> Tire uma foto da vasculatura do crânio exposta com a região diluído como Mapa 1, que é usado para mudar a região fotografada em sessões de imagem subseqüentes (ver Figura 2). </li><li> Posicione o mouse sob o microscópio de imagem. Localize a área diluído sob um objetivo ar 10X utilizando epifluorescência e mover a área mais fino do centro da vista. </li><li> Adicionar uma gota de solução salina no topo do crânio e mudar para o objectivo 60X, que tenha sido lavado com água esterilizada. Selecione uma região onde espinhas dendríticas individuais são claramente visualizado juntamente dendritos. Identificar e rotular a região correspondente no mapa 1, comparando vasculatura entre a visão de epifluorescência ea foto vasculatura. </li><li> Ajustar o comprimento de onda do laser de dois fotões de acordo com os fluoróforos. Por exemplo, 920 nm para YFP; 890 nm para GFP; 1.000 nm para DsRed e tdTomato 24. </li><li> Adquira pilhas de imagens com 2passos iM ao longo do eixo z com o objectivo de 60X. Esta pilha imagem cobre uma área de 200 x mM mM de aproximadamente 200 (512 x 512 pixels) e é usado como mapa 2 para o internamento durante as sessões de imagem subseqüentes (ver Figura 2). </li><li> Adquirir nove pilhas de imagens dentro de mapa 2 usando o zoom digital de 3X. Cada pilha de cobre uma área aproximada de 70 um x 70 um (512 x 512 pixels), com 0,7 mM etapas ao longo do eixo-z Nota:. A intensidade do laser deve ser inferior a 40 mW, quando medido em amostras de minimizar fototoxicidade. </li></ol> 5. Recuperação <ol><li> Na sequência de imagens, retire cuidadosamente a placa da cabeça do crânio. </li><li> Limpe bem o crânio ea pele para remover toda a cola restante Nota:. Qualquer cola remanescente na pele causará irritação e retardar a cicatrização da pele, enquanto que qualquer cola restante no crânio causará erosão do crânio e umangiogenesis na camada óssea recém-crescido, fazendo realocação subseqüente e re-imaging difícil. </li><li> Lavar o crânio ea pele várias vezes com solução salina. </li><li> Suture o couro cabeludo com sutura cirúrgica estéril. </li><li> Mantenha o animal em uma almofada de aquecimento em uma gaiola separada. Administrar analgésico buprenorfina (0,1 mg / kg) por via subcutânea para mitigar a dor pós-operatória. Devolver o animal para a gaiola para casa após recuperação completa. Monitorar o animal de perto (verifique pelo menos uma vez por dia) até que a incisão é curado e suturas são removidas. Administrar injeções subseqüentes de buprenorfina analgésico, se necessário. </li></ol> 6. Re-imagem <ol><li> Repita os passos de 1,1-1,8. </li><li> Repita os passos de 3,1-3,6 </li><li> Localize região anteriormente imaginado, comparando o padrão de vascularização ao Mapa 1 eo padrão dendrítico ramo ao Mapa 2. </li><li> Se recriação é realizada dentro de 1 semana, repetir o passo 2.3 para remover uma fina camada de tecido ósseo recém-cultivadas no topo da re afinadagião usando a lâmina microcirúrgica. Se recriação é realizada depois de mais de uma semana, repetir os passos tanto 2.2 e 2.3 Nota:. A camada de osso recentemente crescido consiste de uma estrutura menos condensada em comparação com a camada de osso original compacto, levando a uma redução da qualidade da imagem. Portanto, para obter a mesma qualidade, é necessário diluir o crânio ligeiramente mais do que a sessão de imagem anterior. </li><li> Ajuste a posição e orientação para obter pilhas de imagens que correspondem anteriormente tomadas pilhas de imagens sob o microscópio de dois fotões. </li><li> Adquirir imagens como no Passo 4.6. </li><li> Repita os passos de 5,1-5,5 após imagem. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Para se obter uma preparação bem sucedida diluído-crânio, várias etapas deste protocolo são cruciais. 1) A espessura do crânio. O osso do crânio tem uma estrutura de sanduíche, com duas camadas de osso compacto de alta densidade e uma camada intermédia de osso esponjoso de baixa densidade. Embora o micro-broca de alta velocidade é adequado para a remoção das camadas exteriores do osso compacto e osso esponjoso, a lâmina microcirúrgica é ideal para o desbaste da camada interna do osso compacto. À medida ...

O córtex dos mamíferos participa em muitas funções do cérebro, desde o controle da percepção e movimento sensorial abstrair processamento de informação e cognição. Várias funções corticais construir sobre diferentes circuitos neurais, que são feitas de diferentes tipos de neurônios se comunicando e trocando informações nas sinapses individuais. A estrutura e função das sinapses são constantemente a ser modificados em resposta a experiências e patologias. No cérebro maduro, plasticidade sináptica toma a forma de ambas as mudanças de força e adição / remoção de sinapses, jogando um papel importante na formação e manutenção de um circuito neural funcional. As espinhas dendríticas são as componentes pós-sinápticos da maioria das sinapses excitatórias no cérebro de mamíferos. O volume de negócios constante e alterações morfológicas de espinhos são acreditados para servir como um bom indicador de modificações nas conexões sinápticas 1-7. Dois fótons micro varredura a laserscopy oferece penetração profunda através, preparações espessas e opacas baixo fototoxicidade, o que o torna adequado para imagens ao vivo no cérebro intacto 8. Em combinação com marcação fluorescente, dois fótons de imagem fornece uma ferramenta poderosa para espreitar para o cérebro vivo e siga reorganização estrutural nas sinapses individuais com alta resolução espacial e resolução temporal. Vários métodos têm sido usados ​​para preparar os ratos para imagens ao vivo 9-13. Aqui, descrevemos uma preparação diluída crânio-in vivo de dois fótons de imagem para investigar a plasticidade estrutural das espinhas dendríticas pós-sinápticos no córtex mouse. Usando essa abordagem, os nossos estudos recentes têm representado uma imagem dinâmica de alterações da coluna dendríticas em resposta a habilidade motora aprendendo Com o aumento da disponibilidade de animais transgênicos com subconjuntos de neurônios marcados com fluorescência e rápido desenvolvimento de técnicas in vivo de rotulagem, procedimentos semelhantes descritos aqui também pode ser aplicado para investigaçõesoutros tipos de células e de te regiões corticais, combinadas com outras manipulações, bem como os utilizados em modelos de doenças 16-23.

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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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			<td height="21" style="height:21px;width:189px;">CCD camera</td>
			<td>Infinity</td>
			<td style="width:108px;"></td>
			<td></td>
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		<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>
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		<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>
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two-photon in vivo imaging of dendritic spines in the mouse cortex using a thinned-skull preparation

No córtex dos mamíferos, os neurônios formam redes extremamente complicadas e troca de informações nas sinapses. As alterações na concentração sináptica, bem como a adição / remoção de sinapses, ocorrem de uma forma dependente da experiência, fornecendo a base estrutural de plasticidade neuronal. Como componentes pós-sinápticas das sinapses excitatórias mais no córtex, espinhas dendríticas são consideradas como um bom indicador de sinapses. Tirar vantagens da genética e técnicas de rotulagem fluorescentes, neurônios individuais e suas estruturas sinápticas podem ser rotuladas no cérebro intacto. Aqui apresentamos um protocolo de imagem transcraniana por microscopia de varredura a laser de dois fótons de seguir fluorescente etiquetado espinhas dendríticas pós-sinápticos ao longo do tempo in vivo. Esse protocolo utiliza uma preparação diluída de caveira, que mantém o crânio intactos e evita efeitos inflamatórios provocados pela exposição das meninges e o córtex. Assim, as imagens podem ser adquiridas imediatamente após surgery é realizada. O procedimento experimental pode ser realizado repetidamente durante vários intervalos de tempo variando de horas a anos. A aplicação desta preparação também pode ser expandido para investigar diferentes regiões e camadas corticais, bem como outros tipos de células, em condições fisiológicas e patológicas.

Em YFP-H linha de camundongos 25, proteína fluorescente amarela expressa em um subconjunto de neurônios piramidais da camada V, que projetam seus dendritos apicais para as camadas superficiais do córtex. Através da preparação diluída de caveira, os segmentos dendríticas fluorescentemente marcadas podem ser repetitivamente trabalhada sob microscópio de dois fotões durante vários intervalos de imagem, variando de horas a meses. Aqui, mostramos um exemplo de uma imagem de quatro vezes as mesmas dendri...

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; Imagem de espinhas dendríticas no Rato Cortex Usando um desbaste-crânio Preparação

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

two-photon-vivo-imaging-dendritic-spines-mouse-cortex-using-thinned

Research

JoVE Journal

Neuroscience

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

17.8K visualizações.  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.

Vídeo: 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.

Imagem crônica de rato Visual Cortex usando uma preparação diluída crânio-

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

Neurociência

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.

Em Imaging vivo do Cordão do rato Spinal Usando Two-photon 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.

Detecção de hipóxia microrregional no Cortex Cerebral mouse por imagem de dois fótons de fluorescência endógena 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.

A polido e reforçado Janela afinado crânio para longo prazo de imagens do cérebro de camundongo

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.

Visualização e manipulação genética de dendritos e espinhas no córtex cerebral e hipocampo do rato usando<em&gt; In utero</em&gt; Eletroporação

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.

Diluído crânio-técnica de janela cortical para<em&gt; Em Vivo</em&gt; Tomografia de coerência óptica

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.

Vivo em dois fótons de imagem de experiência dependentes de alterações moleculares em neurônios corticais

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 ...

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.

Dois fótons de imagem da Cellular Dynamics na Medula Espinhal Rato

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.

Simultânea de dois fótons<em&gt; In Vivo</em&gt; Imagem de Synaptic Entradas e metas pós-sinápticos no córtex do rato retrosplï¿½ico

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Na Vivo Imagem de dois fotões de neurônios Cortical em camundongos Neonatais

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...