O financiamento para este trabalho foi fornecido por uma Garnett Passe e Rodney Williams Memorial Foundation projeto de Subsídio à R. Lim e AJ Camp.

1. Animais <ol><li> Camundongos foram obtidas a partir do Centro de Rodent australiano (ARC, Perth, Austrália) e realizada na Universidade de Sydney Bosch Biotério em um claro / escuro ciclo normal de 12 horas, com enriquecimento ambiental. Todos os experimentos descritos foram aprovados pelo Comitê de Ética da Universidade de Sydney animal. </li><li> Os ratinhos macho e fêmea (C57/BL6) foram utilizadas para todas as experiências uma vez que esta estirpe são comumente usados ​​como pano de fundo para a manipulação genética, e podem ser consideradas equivalentes à estirpe selvagem 8-9. </li></ol> 2. Preparação de tecidos <ol><li> Preparar 300 ml de um fluido à base de glicerol cefalorraquidiano artificial (ACSF), constituído por (em mM): 26 de NaHCO3, 11 de glucose, 250 glicerol, 2,5 de KCl, 1,2 de NaH 2 PO 4, 1,2 MgCl 2, CaCl 2 e 2,5. Antes da adição de CaCl2, de gás a solução com carbogénio (95% O 2 e 5% de CO 2) para estabelecer apPrecipitação de cálcio de 7,4 H e evitar (nebulosidade). Congelar a solução num congelador -80 ° C durante 45 min de modo a que uma suspensão de gelo é formada. </li><li> Profundamente anestesiar os ratos com cetamina (100 mg / kg) (Parnell, Alexandria, Austrália) por meio de uma injecção intra-peritoneal. </li><li> Uma vez hind-membros reflexos pitada são ratos decapitar ausentes usando tesoura de aço inoxidável afiadas e fazer uma incisão na pele sagital utilizando uma lâmina de barbear (arredondado # 22) para expor o crânio. Neste ponto e em todo passos 2,3-2,8 a calota craniana, cérebro e aparelho vestibular subjacente deve ser mantido o mais fresco possível através da aplicação regular de ACSF gelada sobre o tecido. </li><li> Usando o braço pontas de tesouras padrão normal (FST, North Vancouver, Canadá), fazer uma pequena incisão no crânio, lambda e cortar ao longo da sutura sagital. Assegure-se que o cérebro não é &quot;arrastada&quot; pela lâmina de corte durante este passo. </li><li> Retire cuidadosamente os ossos parietais de distância lateralmente e do osso occipital posteriorly usando rasa curva Pearson fórceps (FST). </li><li> Uma pequena espátula de aço inoxidável, é, então, usado para levantar cuidadosamente o cérebro a partir da superfície das fossas craniana média e posterior, e o nervo vestibulocochlea exposta (CNVIII) cortada a meio caminho entre o ouvido interno e do tronco cerebral com um par de tesouras finas íris. Cortar esse nervo minimiza tensão indevida sobre os axônios dos aferentes primários e suas conexões com as células ciliadas. </li><li> A seguir à transecção de CN VIII do cérebro é removido in toto. </li><li> O labirinto vestibular é agora claramente visível na fossa craniana média, com a cóclea apontando ântero. Rongeur cada lado do labirinto vestibular antes da excisão suavemente segurando o canal semicircular anterior e puxando lateralmente. </li><li> Mergulhe os labirintos excisadas (Figura 1), em um prato de dissecação contém a gelada, ACSF continuamente gaseados descrito na seção 2.1. Sob um microscópio estereoscópico, segure o labirinto paraa base do prato de preensão da cóclea. Use uma pinça fina para raspar no osso que recobre a ampola do canal semicircular anterior. Uma vez que um pequeno buraco no osso é conseguida começam a apertar o osso ampulífuga. O cuidado deve ser tomado para não empurrar a pinça através do osso, pois isso pode causar danos ao labirinto membranoso subjacente e epitélio sensorial. Continuar este procedimento até que tanto a anterior e condutas membranosas semicirculares horizontais adjacentes e ampolas estão expostos (Figura 2). </li></ol><img alt="Figura 1" src="/files/ftp_upload/50471/50471fig1.jpg" /> Figura 1. O isolado do mouse labirinto vestibular. A. Painel esquerdo) Representação esquemática do isolado do mouse labirinto vestibular. Importantes pontos de referência para acessar o epitélio sensorial vestibular, a cóclea, anterior e horizontal canais semicirculares são laboratórioELED. Os asteriscos indicam a ampola do canal semicircular contendo o epitélio sensitivo vestibular. B. Painel) Fotomicrografia direito do labirinto vestibular isolado de um rato de 1 mês de idade. <img alt="Figura 2" src="/files/ftp_upload/50471/50471fig2.jpg" /> Figura 2. Exposição do labirinto vestibular Membranous. O osso que recobre a ampola do canal semicircular anterior e horizontal foram riscados para revelar as ampolas membranoso preto / marrom manchada e os nervos ampulares associados (CNVIII). O esquema no painel inferior mostra as estruturas da região em destaque da fotomicrografia e mostra a relação entre as condutas do canal semicircular para a ampola e CNVIII. <ol start="10"><li> Utilizando uma pinça fina, levante suavemente a ampolas e utrículo associado longe do labirinto ósseo, garantindo que a região central do ampolas (ContaIning o epitélio sensorial) não está danificado. Em alguns casos, a parte proximal da conduta membranoso semicircular pode ter de ser cortado com tesoura de íris para libertar as ampolas a partir do osso. </li><li> Transferir a tríade contendo os dois ampolas e utrículo em uma placa de Petri cheia de meio L-15 de cultura de Leibovitz (Sigma-Aldrich, St. Louis, MO). Use uma fibra óptica para &quot;backlight&quot; do tecido. Isto permite a visualização clara da crista contendo o epitélio sensorial dentro da ampola. </li><li> Cuidadosamente fazer uma incisão na salpicado &quot;telhado&quot; do utrículo com a tesoura de íris finas. Continuar desta incisão através do telhado do anterior e, em seguida, as ampolas horizontal. Adicione a incisão o mais próximo possível da borda do epitélio sensorial quanto possível, sem contacto que (Figura 3A). Certifique-se de que não existem pedaços de membranas que recobrem o epitélio sensorial (Figura 3B) </li><li> Transferir a preparação de semi isolado intacto a uma pequena glascâmara de gravação s de fundo cheio de L-15 mídia. Pesam-se a preparação utilizando uma grelha de fibras de nylon finas garantidos a um fio de platina em forma de U achatada (Figura 3B). Idealmente, as fibras da rede não deverá sobrepor-se ao epitélio sensorial, no entanto, em alguns casos em que é necessária uma maior estabilidade, uma única fibra atravessa em epitélio sensorial talvez preferida. </li></ol><img alt="Figura 3" src="/files/ftp_upload/50471/50471fig3.jpg" /> Figura 3. A preparação semi-isolado intacto do epitélio. A. Representação esquemática sensitivo vestibular da preparação semi-intactos e configuração do eléctrodo. A ampola que recobre a crista tem sido &quot;des-coberta&quot; para expor a superfície do epitélio sensorial (verde). B. Fotomicrografia da preparação semi-intacto &quot;tríade&quot; mostrando a anterior (AC) e horizontal (hc) crista (utrículo obscurecida sertraseiras ac). Note-se a fibra de nylon utilizados para garantir que a preparação da base da câmara de gravação. Barra de escala:. 100 mM C. Um eletrodo de registro posicionado em uma célula de cabelo vestibular individual. Barra de escala: 15 mM. 3. Eletrofisiologia <ol><li> Perfundir a preparação semi-intacta, com continuamente oxigenada L-15 médio. Os meios de comunicação contém um indicador de pH (vermelho de fenol), e de cor do meio devem ser monitorizados ao longo das gravações. O pH com o oxigénio deve ser 7,3-7,4 e corresponder a uma cor vermelha. </li><li> Prepare a gravação pipetas de 1,5 mm (1,19 milímetros ID) de vidro de borosilicato usando um protocolo de duas etapas (passo de calor 1: 72, e passo de calor 2: 50) em um extractor de micropipeta (Narishige, Japão, modelo PP-830) para alcançar um impedância final 3-4 mohms. </li><li> Encher a haste da pipeta com 3-4 mm de solução interno baseado em fluoreto de potássio contendo (em mM) 110 KF, 12 KCl, 27 de KOH, 1 de NaCl, 10 HEPES, 10 EGTA, 1,8 MgCl2,3 D-glicose, 2 e Na-ATP, pH 7,4 com KOH. </li><li> Enrole a haste da pipeta 2-3 vezes e, tanto para baixo até a ponta quanto possível, com uma tira fina de parafilme para isolar o eléctrodo e reduzir a capacitância pipeta. </li><li> Posicione a pipeta sobre o epitélio sensorial sob a ampliação de baixa potência (5X) em um microscópio vertical (Olympus BX51). Mudar para alta potência (40x) e visualizar as células ciliadas vestibulares individuais com uma câmera CCD anexo. </li><li> Posição pipeta sobre a membrana de uma célula de cabelo visualizado (figura 3C), utilizando um micromanipulador (Sutter Instruments, Califórnia, EUA). Uma vez que o selo é conseguida gigaohm, ruptura da membrana celular, com uma pequena quantidade de pressão negativa aplicada através de uma porta de sucção no suporte da pipeta. </li><li> As gravações de fixação de tensão de células inteiras, utilizando técnicas padrão de 8-9. </li></ol> 4. Dois fótons de microscopia <ol><li> Prepara-se uma solução salina a 0,9% contendo 5 mM de verde de Oregon488 BAPTA-1 (OGB-1; sal hexapotassium; Invitrogen, Alemanha). </li><li> Enquanto ainda submerso, coloque a preparação tríade semi-intacta &quot;des-coberta&quot; em um pequeno pedaço de papel de filtro (4x4 mm, 0,8 mm de espessura; Millipore, Alemanha), garantindo que o epitélio sensorial é desobstruída de cima. </li><li> Transferir o papel de filtro e a preparação para o eléctrodo de platina de base coberto salina (7 ul) de um electroporador que consiste na combinação de um gerador de impulsos e um amplificador de banda larga e tampa com mais 5 mL de solução a 5 mM do cálcio sintético Indicador de corante verde de Oregon 488 BAPTA-1 (Figura 4). </li></ol><img alt="Figura 4" src="/files/ftp_upload/50471/50471fig4.jpg" /> Figura 4. Massa de electroporação. A. esquemática, que mostra a secção transversal da configuração de electroporação. A preparação semi-intacta (*) é colocada sobre um pedaço de papel de filtro mergulhados em cálcio dye (verde Oregon 488 BAPTA-1) ea corrente aplicada entre os dois eléctrodos de platina. Adaptado de Briggman e Euler, 2011 10. <ol start="4"><li> Traga o início platina eléctrodo paralelo com o eléctrodo de base, a uma distância de cerca de 2 mm, tendo o cuidado de não perturbar a orientação da preparação quando em contato com o Verde 488-BAPTA uma solução Oregon é feita. </li><li> Passe um pulso de corrente em breve a preparação (Parâmetros para o atual, 13 V, 10 ms de largura de pulso, freqüência de 1 Hz, 10 pulsos de onda quadrada) 10-11. </li><li> Na parte inferior de uma câmara de registo com fundo de vidro cheio com meio L-15, colocar uma pequena pincelada de óleo de silicone e a posição da preparação semi-intacto na, novamente assegurar que o epitélio sensorial está desobstruído a partir de cima. Para garantir estabilidade em toda imagem, substituir a rede de nylon sobre a preparação como descrito acima (ver ponto 2.13). </li><li> Usando um microscópio de dois fotões fazer gravações ópticas espontaneamenteactividade cálcio percutânea no epitélio sensorial (Figura 7), utilizando protocolos padrão de imagem 10-11. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Os mecanismos subjacentes a nosso senso de equilíbrio têm recebido pouca atenção em comparação com outros sistemas sensoriais, por exemplo, os sistemas visual e auditivo. Dos estudos que investigaram alterações na função vestibular ou de equilíbrio, a maioria focada em medidas comportamentais, incluindo o reflexo vestíbulo-ocular, com conhecimento incompleto dos blocos de construção fundamentais da balança as próprias células ciliadas vestibulares. Esses estudos que se concentraram nas células...

Apesar da contribuição significativa do sistema vestibular em nossas vidas cotidianas, um claro entendimento dos processos responsáveis ​​pelo declínio observado na função vestibular com a idade são ainda imperceptíveis. Uma das razões para essa falta de conhecimento é que o declínio da função vestibular foi quase exclusivamente explorado utilizando testes comportamentais, incluindo o reflexo vestíbulo-ocular (RVO), um indicador preciso da função vestibular extrínseca, mas fornece informações limitadas sobre as mudanças de componentes intrínsecos . Este é um grande impedimento para a nossa compreensão da função das células ciliadas vestibular na saúde, doença ou envelhecimento. Embora tenha havido muitos estudos sobre as células ciliadas vestibulares individuais, uma grande lacuna tem sido o recurso a preparações de células capilares agudas, onde as células do cabelo e terminais aferentes até cálice são removidos do seu ambiente normal através de tratamento mecânico e / ou enzimático. Tais abordagens inevitably perturbar a microarquitetura delicado entre células ciliadas e cálice, e os cabelos célula e célula de apoio. Com o desenvolvimento de preparações semi-intactas 1-5, e um isolado do rato de preparação de labirinto 6, existe agora uma oportunidade de estudar as diversas formas de comunicação sináptica sob condições que se aproximam mais in vivo. Na verdade, Lim et al. (2011) mostrou diferenças marcantes nas correntes de células inteiras gravadas de tipo agudo isolado I células ciliadas vestibulares em comparação com aqueles que permaneceram incorporado dentro do neuroepithelium. Especificamente, o potássio é pensado que se acumulam no espaço intercelular, entre a célula de cabelo e cálice aferentes, e altera significativamente a resposta das células ciliadas 7. Este tipo de informações pode ser impossível de obter, sem a preparação de semi-intacta do epitélio sensitivo vestibular descrito aqui. Nós demonstramos a preparação semi-intactas do mouse crista 3E mostram resultados representativos obtidos patch-célula inteira eletrofisiologia e de imagem de cálcio de dois fótons.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Leibovitz medium L-15</td>
 <td>Sigma Aldrich</td>
 <td>L4386-10X1L</td>
 <td></td>
 </tr>
 <tr>
 <td>BAPTA-1-oregon green</td>
 <td>Invitrogen</td>
 <td>O6806 </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT </td>
 </tr>
 <tr>
 <td>Stereo microscope</td>
 <td>Leica Microsystems</td>
 <td>A60S</td>
 <td></td>
 </tr>
 <tr>
 <td>Upright microscope</td>
 <td>Olympus</td>
 <td>BX51WI</td>
 <td></td>
 </tr>
 <tr>
 <td>Two-photon microscope</td>
 <td>Olympus/La Vision</td>
 <td>BX51WI/ TriMScope II</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont #5 SF Forceps</td>
 <td>FST</td>
 <td>11252-00</td>
 <td></td>
 </tr>
 <tr>
 <td>Friedman-Pearson Rongeurs</td>
 <td>FST</td>
 <td>16221-14</td>
 <td></td>
 </tr>
 <tr>
 <td>Standard Pattern Scissors</td>
 <td>FST</td>
 <td>14001-12</td>
 <td></td>
 </tr>
 <tr>
 <td>InstraTECH A-D converter</td>
 <td>HEKA</td>
 <td>ITC-18</td>
 <td></td>
 </tr>
 <tr>
 <td>Sutter Micromanipulator</td>
 <td>Sutter</td>
 <td>MP-225/M</td>
 <td></td>
 </tr>
 <tr>
 <td>multiclamp amplifier</td>
 <td>Axon Instruments</td>
 <td>700B</td>
 <td></td>
 </tr>
 <tr>
 <td>Data acquisition software (electrophysiology)</td>
 <td>Axograph</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>Imspector Data acquisition software (two-photon)</td>
 <td>Max Planck innovation</td>
 <td>N/A</td>
 <td></td>
 </tr>
 </tbody>
</table>

an isolated semi-intact preparation of the mouse vestibular sensory epithelium for electrophysiology and high-resolution two-photon microscopy

Compreender as células ciliadas vestibulares funcionar em condições normais, ou como trauma, doenças e envelhecimento interromper esta função é um passo vital para o desenvolvimento de abordagens preventivas e / ou estratégias terapêuticas. No entanto, a maioria dos estudos com vista a função vestibular anormal não ter sido a nível celular, mas focado principalmente em ensaios comportamentais de disfunção vestibular, tais como análises de marcha e desempenho reflexo vestíbulo-ocular. Embora este trabalho rendeu dados valiosos sobre o que acontece quando as coisas dão errado, pouca informação é recolhida sobre as causas da disfunção. Dos estudos que incidem sobre os processos celulares e subcelulares que fundamentam função vestibular, a maioria têm contado com agudamente células ciliadas isoladas, desprovidas de suas conexões sinápticas e meio ambiente da célula de apoio. Por isso, um grande desafio técnico tem sido o acesso às células ciliadas vestibulares extremamente sensíveis em uma preparaçãoparação que é menos interrompido, fisiologicamente. Aqui demonstramos uma preparação semi-intactas do mouse vestibular epitélio sensorial que mantém o micro-ambiente local, incluindo células ciliadas / complexos aferentes primários.

As propriedades electrofisiológicas de células ciliadas vestibulares são dependentes da micro-arquitectura complexa dentro do qual estão incorporados 7. Figura 5 mostra que a preparação epitélio vestibular semi-intactos podem ser usados ​​para diferenciar entre células ciliadas do tipo I (Figura 5A), as células ciliadas do tipo II (Figura 5B), e o cálice aferente primário (Figura 5C), com base em condutâncias de células intei...

Watch this Scientific Journal Video about An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy at JoVE.com

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Análise da função das células ciliadas vestibular é complicada por sua localização profunda na parte mais difícil do crânio, no osso temporal petroso. A maioria dos estudos com células capilares funcionais têm utilizado intensamente as células ciliadas isoladas. Aqui nós descrevemos uma preparação semi-intactos do epitélio vestibular rato para estudos eletrofisiológicos e de microscopia de dois fótons.

Uma preparação semi-intactas isolada do Rato Vestibular epitélio sensorial de Eletrofisiologia e de alta resolução Microscopia de dois fótons

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

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Research

JoVE Journal

Neuroscience

11.8K visualizações.  University of Sydney. Análise da função das células ciliadas vestibular é complicada por sua localização profunda na parte mais difícil do crânio, no osso temporal petroso. A maioria dos estudos com células capilares funcionais têm utilizado intensamente as células ciliadas isoladas. Aqui nós descrevemos uma preparação semi-intactos do epitélio vestibular rato para estudos eletrofisiológicos e de microscopia de dois fótons.

Vídeo: An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

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 ...

Neurociência

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Preparação do tecido e imunocoloração de Fibras nervosas sensoriais que inervam rato da Pele e Ossos Limb

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

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 ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Um<em&gt; In-vitro</em&gt; Preparação de neurônios entéricos isolados e células gliais do plexo mioentérico do Rato Adulto

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

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

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

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 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 ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Quantificação de Vascular Cerebral Arquitetura usando dois fótons Microscopia em um modelo de mouse Neuroinflammation induzida por HIV

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

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 ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Imagem transpupilar de dois fótons in vivo da retina do rato

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.