We would like to acknowledge the significant contribution Dr. Shiwei Huang (Australian National University) in validating the method. Furthermore, we would like to thank Ms. Kasia Pietrajtis for helpful comments regarding Golgi cells and Mr. Vitaly Lerner for the cortex experimental data. This work was supported by PITN-GA-2009-238686 (CEREBNET), FP7-ICT (REALNET), ELSC and ISF.

Todos os procedimentos experimentais descritos neste protocolo foram aprovados pelo Animal Care da Universidade Hebraica e do Comitê Use. 1. Preparar as soluções e ferramentas para corte <ol><li> Prepare 1 litro de solução fisiológica padrão (SPS) que contém os iões descritas na Tabela 1. <ol><li> Prepara-se uma solução de estoque contendo os sais em 10 vezes a concentração final previamente em uma garrafa de vidro de armazenamento preenchido com 1000 ml de água desionizada (condutividade de 0,055 mS / cm). Adicionar sais (NaCl, KCl, KH 2 PO 4, e MgSO4), e agita-se até estar completamente dissolvido. Guardar a solução estoque em 4 ° C. </li><li> Adicione a solução final SPS no dia do experimento. Acrescentam-se 100 ml da solução de estoque de 700 ml de água purificada, dissolver a glicose (3,6 g) e NaHCO3 (2,18 g) usando uma barra magnética e, em seguida, adicionar água purificada até 1 L. </li><li> Equilibrar as solutisobre ele por gaseamento com 95% O2 / 5% de CO 2 durante ~ 20 min. Após isto, adicionar 2 ml de uma solução M de CaCl2. </li></ol></li><li> Reúna as seguintes ferramentas (Figura 1). <ol><li> Prepare grandes tesoura ou outro instrumento de decapitação, pequenas tesouras cirúrgicas para a abertura do crânio, uma pinça de ponta fina para elevação do crânio para expor o cérebro, bisturi com lâmina para dissecar a parte desejada do cérebro para ser cortada e uma pequena espátula para manipular o partes cerebrais dissecados. </li><li> Além disso, prepare-se pequenos pedaços de papel de filtro para remover o excesso de líquido do cérebro, dois pequenos pratos de Petri para o cérebro para ser dissecado em dois copos de vidro de 500 ml para conter água aquecida purificada e SPS, e cola de cianoacrilato para colar do cérebro para o fase de corte. </li><li> Prepare uma escova pequena para lidar com as fatias durante o corte e uma pipeta de Pasteur de vidro de boca larga para mover as fatias do banho de corte para o cha de recuperaçãomber. Desinfetar as ferramentas para impedir o crescimento de bactérias nos banhos quentes fatia. Além disso, usar luvas de laboratório limpas. </li></ol></li><li> Prepare uma fatia de recuperação câmara submersa tal como descrito por Gibb e Edwards 15. Continuamente gás do SPS nele com 95% O2 / 5% de CO2 e manter a 36 ° C. Certifique-se de que o gaseamento não resulta em pequenas bolhas de ar no banho que pode danificar fatias. </li><li> Quente ~ 300 ml da SPS a 36 ° C sobre uma placa de aquecimento com agitador magnético ou em um banho de água. Se uma placa de aquecimento é usado, tome cuidado para evitar que a solução do superaquecimento, o que pode resultar na precipitação de íons. No caso de isso acontecer, SPS frescos quentes em vez de arrefecimento do antigo. </li><li> Traga ~ 500 ml de água purificada para ferver com uma chaleira eléctrica e, num copo, misture 300 ml de com água fria para obter água um pouco mais quente do que a temperatura de corte. Utilize este aqueceu água purificada durante a decapitação e usar o remaining da água fervida mais tarde para a manutenção da temperatura fisiológica do banho de corte. </li><li> Anestesiar o rato por injecção de 0,1 ml de pentobarbital (60 mg / ml) por via intraperitoneal. Depois de alguns minutos, verificar se o animal não responder à forte do dedo do pé ou da cauda pitadas de verificar a falta de sensação. </li></ol> 2. Dissecando o cérebro <ol><li> Decapitar o mouse rapidamente cortando o pescoço com as tesouras grandes, perto da parte de trás do crânio, e deixar a cabeça cair no copo com água purificada aquecida para lavar o excesso de sangue. </li><li> Exponha o forame magno no crânio sob os músculos do pescoço e da pele, possivelmente removendo mais do pescoço com as pequenas tesouras. </li><li> Puxar a pele na parte superior do crânio de ser capaz de ver claramente as suturas cranianas, a fim de orientar a abertura do crânio. </li><li> Corte o crânio aberto, inserindo a ponta inferior das pequenas tesouras através do forame magno e imediatamente transformá-los em direção ao lado lateral. Com cuidado, cortar ao longo do bordo lateral do osso parietal até uma localização atrás dos olhos e da sutura fronto-parietal; em seguida, virar-se para o centro do crânio e cortar através da linha média para o outro lado do crânio (ver linha tracejada verde na Figura 2A). </li><li> Utilizando uma pinça de ponta fina, levante o crânio do canto fronto-parietal e puxe-o na diagonal para cima e para os lados para expor o cérebro (ver amarelo seta tracejada na Figura 2A). Certifique-se de que o crânio não está ligado a qualquer osso ou pele da cabeça, de modo que o cérebro vai ficar no lugar quando o crânio é levantado. No caso em que o cérebro está em movimento, com o crânio, utilizar as pequenas tesouras para remover qualquer tecido conjuntivo entre o crânio e o cérebro. </li><li> Quando o cérebro é exposta, não permitem que o cérebro a ser seco. Assim, execute os passos seguintes, mantendo o crânio eo cérebro em uma placa de Petri cheia com aquecido, gaseados SPS. </li><li> Retire a parte do cérebro usada para o eXperiment a partir do resto do cérebro utilizando um bisturi e uma espátula. No caso de utilizar o cerebelo, separar da parte frontal do cérebro por um único corte através do mesencéfalo e ponte (ao longo das linhas vermelhas nas Figuras 2B-D) e movê-lo para uma pequena placa de Petri que contém frescos, gaseados SPS. </li><li> Remover o tronco cerebral para formar uma base recta e ampla para a colagem do cérebro a fase de corte ao cortar o cerebelo no plano horizontal. Resumidamente colocar o cerebelo em um pedaço húmido de papel de filtro de modo que o lado que foi cortado a partir do prosencéfalo está voltada para baixo. <ol><li> Cortar o tronco cerebral com um bisturi para formar a base (tal como indicado com a linha azul na Figura 2B), e devolver o cerebelo para o SPS. Quando são necessários outros planos de corte, cortar o bloco cerebelo em conformidade (linhas azuis nas Figuras 2C e D). </li></ol></li></ol> 3. Cortando o Cérebro <ol><li> Prepare o estágio de corte, verificando que é dry, antes da aplicação de uma pequena gota de supercola no meio do palco. </li><li> Levante o bloco cérebro do SPS com uma espátula com a correta voltada para cima, e retire todo o excesso de líquido do cérebro usando pequenos pedaços de papel de filtro. Então, com um único movimento, deslize o cérebro da espátula sobre a gota de cola no palco. </li><li> Para evitar que o cérebro de secagem e a cola de correr para cima sobre os lados do tecido, aplicar algumas gotas de SPS com uma pipeta de Pasteur e colocar a fase de corte na câmara de corte. Alinhar o bloco de cérebro de modo a que as regiões de interesse (por exemplo, do córtex cerebelar) estão virados para a lâmina e, portanto, vai ser submetido a menor quantidade de pressão mecânica antes de ser cortado. </li><li> Corte cada fatia de acordo com as instruções do fabricante, enquanto constantemente gaseificação do RPU na câmara de corte, bem como mantendo a temperatura a 34-37 ° C. Manter a temperatura da câmara de corte, neste intervalo, preenchendo o external câmara do cortador com água purificada aquecida e substituindo a água sempre que a temperatura cai muito baixa. Nota: Os parâmetros de corte devem ser experimentalmente encontrado para cada tipo de tecido e dos neurônios que estão sendo examinados. No caso de o Campden SMZ 700 fatiador com lâminas de cerâmica e células cerebelares de Golgi, usar 0,75 milímetros amplitude de vibração a 65 Hz e velocidade de 0,05 mm / seg avançando; para os neurónios cerebelares nucleares maior amplitude e frequência (1 mm e 90 Hz, respectivamente) e menor velocidade de avanço (0,01-0,02 mm / seg) são recomendados. Para tanto Golgi e os neurônios nucleares do cerebelo, espessura do corte deve ser de 300 mm. </li><li> Durante o corte, não permita que as fatias de dobrar sobre si mesmos. Para evitar que isto delicadamente apoiando-os com uma escova macia, de preferência tocando a fatia apenas em regiões que não são de interesse para o experimento. Tome cuidado para evitar que a escova de tocar a lâmina vibratome; especialmente no caso das lâminas de cerâmica, até mesmo um ligeiro contactopode danificar a lâmina. </li><li> Mova cada fatia da câmara de corte para a recuperação / exploração câmara usando um de boca larga, fogo-polido Pasteur de vidro pipeta. </li><li> Deixe as fatias descansar na câmara durante pelo menos 1 hora antes do início da experiência. Isto permite a degradação de tecidos e células danificadas ou a morrer e, portanto, resulta numa superfície de corte mais limpo. Durante a primeira hora, manter a temperatura da RPU na câmara no intervalo de 34-36 ° C; garantir que as fatias não são tocados por bolhas de ar que podem danificar as fatias ou torná-los flutuar. </li></ol> 4. Experiência <ol><li> Após 1 hora, deixar a temperatura da câmara de recuperação de arrefecer até à temperatura ambiente (17-25 ° C). NOTA: Este pode prolongar o período de tempo durante o qual as fatias pode ser utilizado por abrandar o crescimento de bactérias, bem como o metabolismo celular. Este período depende da região do cérebro e do tipo de célula. Por exemplo, certos tipos de células de Golgi pode ser encontrado para ser saudável em fatias8 h após o corte ao passo que outros tipos durar apenas 6 horas. </li><li> Depois de 1 hora se passou desde o corte, use as fatias para vários experimentos eletrofisiológicos. </li></ol>

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The authors declare that they have no competing financial interests.

Nós demonstramos um método para a preparação de fatias cerebrais agudas a partir de ratos em vez de temperatura fisiológica gelada. Demonstrou-se 11 que a qualidade das fatias obtidas em condições de calor é superior quando em comparação com as preparadas com condições de frio, desde que a lâmina tem fatiador de vibração vertical mínima. Cortando na temperatura fisiológica pode impedir artefatos fisiológicos causados ​​pela baixa temperatura, como as relacion...

O objectivo do método apresentado é obter de alta qualidade fatias cerebrais agudas para experiências in vitro electrofisiológicas, especialmente quando se utiliza adulto ou mesmo animais mais velhos. O método de fatiamento cerebral aguda, como descrito por Skrede e Westgaard 1 em duas frases elegantes, tornou-se um dos fundamentos da pesquisa em neurociência moderna e é empregado em inúmeras variações em todo o mundo. A qualidade das fatias é reflectido no número de neurónios por fatia vivo, o período de tempo durante o qual as células de manter as suas propriedades electrofisiológicas e morfológicas, bem como na integridade do tecido. Além disso, a duração máxima para gravações estáveis ​​depende da qualidade das fatias. Assim, ao longo das décadas, o método de corte original foi desenvolvido por grupos de pesquisa individuais para melhorar a recuperação após o corte fatia 2-10, muitas vezes por meio de modificações complexas de composição de cortar osoluções de recuperação r (como a adição de ácido ascórbico, tioureia ou mesmo de H 2 O 2), bem como intra-cardíaca de pré-perfusão do animal com soluções fisiológicas arrefecidos. Tal como foi recentemente mostrado 11, temperatura fisiológica durante o corte parece ser mais benéfico do que o arrefecimento para a saúde neuronal; a melhora é mais marcante quando se trabalha com adultos (2-8 meses) roedores. Evitar mudanças dramáticas de temperatura impede artefatos devido a processos dependentes da temperatura nas células, como plasticidade e 13 canais iónicos cinética 13,14. Tais mudanças podem influenciar a tensão de membrana e sinalização intracelular de cálcio, limiar pico e pico de forma. O método &quot;quente&quot; fatia aguda preparação aqui apresentado é um processo geral para a obtenção de alta qualidade fatias cerebrais agudas a partir de qualquer região do cérebro, incluindo no cerebelo, córtex e no hipocampo, núcleos do tronco cerebral 16 &lt;/ Sup&gt;, bem como o bolbo olfactivo, tanto em ratos e ratinhos. Notavelmente, o procedimento temperatura fatiamento fisiológico requer que a lâmina de corte vibra quase perfeitamente na horizontal e é, sem quaisquer defeitos estruturais. Tal precisão pode não ser possível com modelos de cortador de idosos; em tais casos, é recomendável a realização da preparação fatia em condições de frio como a temperatura baixa parece tornar o tecido mais resistente a danos mecânicos, mesmo que à custa de aberrações metabólicas.

<table><tbody><tr><td>Pentobarbital</td><td>CTS</td><td>170066</td><td>Concentration: 60 mg/ml in physiological saline.</td></tr><tr><td>Big scissors&amp;nbsp;</td><td>FST</td><td>14001-16</td><td>Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation&amp;nbsp;</td></tr><tr><td>Iris scissors&amp;nbsp;</td><td>Prestige medical</td><td>48,148</td><td>Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5&amp;#8211;2 cm</td></tr><tr><td>Fine tip forceps&amp;nbsp;</td><td>FST</td><td>11254-20</td><td></td></tr><tr><td>Scalpel&amp;nbsp;</td><td>FST</td><td>91003-12</td><td></td></tr><tr><td>Scalpel blade #11</td><td>FST</td><td>10011-00</td><td></td></tr><tr><td>Small spatula&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>2350</td><td></td></tr><tr><td>Filter paper</td><td></td><td></td><td>Any laboratory brand can be used.</td></tr><tr><td>Petri dishes</td><td>Duroplan</td><td>Z231509-1</td><td></td></tr><tr><td>Glass beakers&amp;nbsp;</td><td>SCHOT</td><td>10022846</td><td></td></tr><tr><td>Pasteur pipette&amp;nbsp;</td><td>Maple Leaf Brand</td><td>14672-029</td><td></td></tr><tr><td>Super glue&amp;nbsp;</td><td>LOCTITE</td><td>4091361/1</td><td></td></tr><tr><td>Slicer</td><td>Campden</td><td>7000-smz</td><td></td></tr><tr><td>Ceramic slicing blade</td><td>Campden</td><td>7550-1-C</td><td></td></tr><tr><td>Magnetic heater/stirrer</td><td></td><td></td><td>For heating up the SPS for the procedure</td></tr><tr><td>Electric kettle</td><td></td><td></td><td>For heating up water for temperature control</td></tr><tr><td>Slice recovery chamber + heating unit</td><td>Warner instruments</td><td>&amp;nbsp;BSC-HT +&amp;nbsp; BSC-BUW</td><td>Home-built models may also be used.</td></tr><tr><td>Thermometer</td><td></td><td></td><td>For monitoring SPS temperature during dissection and slicing</td></tr></tbody></table>

slice it hot: acute adult brain slicing in physiological temperature

Aqui apresentamos um protocolo para a preparação de fatias cerebrais agudas. Este procedimento é um elemento crítico para experiências de patch-clamp electrofisiológicas que determina em grande parte a qualidade dos resultados. Demonstrou-se que omitindo o passo de arrefecimento durante o processo de corte é benéfico na obtenção de cortes e células saudáveis, especialmente quando se lida com as estruturas cerebrais altamente mielinizadas de animais maduros. Embora o mecanismo preciso pelo qual temperatura elevada apoia a saúde neural só se pode especular em cima, é lógico que, sempre que possível, a temperatura em que o corte é realizado deve ser perto de condições fisiológicas para evitar artefatos de temperatura relacionados. Outra vantagem importante deste método é a simplicidade do processo e, por conseguinte, o tempo de preparação curto. No método demonstrado ratinhos adultos são usadas, mas o mesmo procedimento pode ser aplicado com ratos mais jovens, bem como os ratos. Além disso, os seguintes cl remendoamp experiência é realizada em fatias horizontais do cerebelo, mas o mesmo procedimento pode também ser utilizado em outros planos, bem como outras áreas do cérebro posterior.

Fatias preparados do modo descrito pode ser usado para várias experiências electrofisiológicas e Optogenetic. Na Figura 3A e 3C, que mostram um exemplo representativo de uma fatia horizontal cerebelar e uma fatia cerebral cortical coronal, respectivamente, vistas sob interferência diferencial (DIC) ópticas. Na fatia cerebelar, vários tipos de neurônios do cerebelo pode ser facilmente reconhecido por sua forma e localização das células do corpo, permitindo alvo registros eletro...

Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Slice It Hot: Agudo Adulto Cérebro Cortando na temperatura fisiológica

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

slice-it-hot-acute-adult-brain-slicing-in-physiological-temperature

Research

JoVE Journal

Neuroscience

26.4K visualizações.  Hebrew University of Jerusalem. In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Vídeo: Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

Preparação de fatias de cérebro aguda usando um otimizado N-metil-D-método de proteção de recuperação glucamine

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Neurociência

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Subcelulares Gravações patch-clamp do domínio somatodendrítico da substância negra dopamina Neurônios

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

Estereotáxica guiada por Atlas Laser Microdissection de captura de regiões do cérebro afetadas por lesão traumática

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Uma abordagem alternativa para estudar eventos primários na neurodegeneração usando fatias de cérebro de rato Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Imagem de dois fotões de atração Microglial processos para ATP ou de serotonina no cérebro aguda fatias

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Culturas de fatias flutuantes de curto prazo do cérebro humano adulto

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

Minimizando a hipóxia em fatias hipocampais de camundongos adultos e envelhecidos

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

<meta charset="utf8"></head><body>Preparando fatias cerebrais agudas do pólo dorsal do hipocampo de roedores adultos

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Preparando fatias cerebrais agudas do pólo dorsal do hipocampo de roedores adultos

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Corte cerebral aguda do rato para investigar atividade espontânea da rede hipocampal

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Slice It Hot: Agudo Adulto Cérebro Cortando na temperatura fisiológica

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