Name: an alternative approach to study primary events in neurodegeneration using ex vivo rat brain slices
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Este trabalho foi financiado pela Neuro-Bio Ltd. Gostaríamos de agradecer ao Dr. Giovanni Ferrati e Dr. Sergio Rotondo (Neuro-Bio) para seus comentários e conselhos sobre o manuscrito.

Todos os estudos em animais foram realizados sob protocolos aprovados.
Nota: Nesta seção, a sequência das principais fases executadas durante o procedimento experimental e o intervalo de tempo sugerido é fornecido (Figura 1). Além disso, uma descrição passo a passo do protocolo é complementada por um painel ilustrativo, mostrando ações críticas que variam de remoção do cérebro a homogeneização do tecido após o período de incubação (Figura 2). Os detalhes sobre os materiais e instruções para construir o aparelho e a fase subsequente para a análise de WB são descritas anteriormente22.
1. cirúrgicas ferramentas, configurações de Vibratome e preparação de tratamento
Nota: Execute as etapas a seguir antes de iniciar o procedimento experimental:
<ol><li>Prepare dois diferentes artificiais cerebrospinal fluidos (aCSFs), que usou para cortar o cérebro e o outro para a etapa de incubação como descrito anteriormente29. Brevemente, as concentrações de trabalho (em mmol) dos dois aCSFs são para o "fatiar" aCSF: 120 NaCl, 5 KCl, 20 NaHCO3, 2.4 CaCl2, 2 MgSO4, 1.2 KH2PO4e 10 glicose; 6.7 HEPES sal e ácido HEPES 3.3; pH: 7.1. Tempo para a "gravação" aCSF: 124 NaCl, 3.7 KCl, 26 NaHCO3, 2 CaCl2, 1.3 MgSO4, 1.3 KH2PO4e 10 glicose; pH: 7.1.</li>
	<li>Misture bem as soluções e colocar no gelo e oxigenar a aCSF corte a fim de tê-lo frio ao remover o cérebro e o seccionamento subsequentes. Manter a temperatura (RT) e fornecimento de oxigênio para a aCSF de gravação.</li>
	<li>Coloque os instrumentos sob a capa de dissecação para a remoção de decapitação e cérebro, ou seja, guilhotina, kit cirúrgico de dissecação (tesouras, pinças, lâminas) e espátula.</li>
	<li>Configure os parâmetros de vibratome, selecionando a espessura da fatia (300 µm nesta preparação) e a frequência às 7 e a velocidade em 6. Inserir a câmara de vibratome no seu espaço apropriado e cercá-lo com gelo.</li>
	<li>Abra a válvula de carbogênio (95% O2, 5% CO2) para oxigenar o "corte" (aCSF) durante o corte do cérebro. Selecione a taxa de vazão mínima, em torno de 2 mL/min.</li>
	<li>Pegue dois tubos de 15 mL para preparar as condições para testar. Adicione em um tubo de 10 mL da "gravação" aCSF sozinho (grupo controle) e no outro, 10 mL da "gravação" aCSF enriquecido com T30 em uma concentração de 2 µM (grupo tratado). Vórtice tanto tubos e mantê-los no gelo até sua utilização na fase de preparação de instalação (secção 3).</li>
</ol>2. anestesia, extração de cérebro e de corte
Nota: Neste estudo, foram usados 9 tipo selvagem P14 ratos machos Wistar. Extração do cérebro e subsequente corte constituem uma parte crítica do protocolo, desde que eles devem ser realizados rapidamente (dentro de 10 min) a fim de evitar ou pelo menos abrandar os processos de degeneração do tecido.
<ol><li>Adicione 1,5 mL de 100% w/w de isoflurano em uma camada de algodão para a câmara de indução, colocar o animal dentro e feche a tampa. Espere até que o animal é anestesiado profundamente, confirmando a ausência do pedal reflexo e então decapitar o animal usando a guilhotina.</li>
	<li>Mantenha a cabeça do animal dentro de um pote contendo gelada corte aCSF durante a etapa de remoção do cérebro (Figura 2A -C).</li>
	<li>Faça uma incisão na pele sobre o crânio com uma lâmina cirúrgica, em seguida, recorte usando a tesoura, seguindo a linha mediana, desde a parte posterior do crânio e proceda anteriormente até atingir o osso acima do OBs (Figura 2A).</li>
	<li>Puxe lateralmente os dois lados do crânio usando um par de pinças para poder ter acesso ao cérebro (Figura 2B). Insira uma espátula ventralmente ao cérebro, colher-se suavemente para fora e mantê-lo hidratado na gelada aCSF "fatiar" por aproximadamente 5 min (Figura 2).</li>
	<li>Elimine o cérebro no filtro de papel e cortar o cerebelo usando uma lâmina (Figura 2D). Cola o cérebro verticalmente sobre o disco de vibratome e inseri-lo no interior da câmara de corte, preenchê-lo com aCSF "fatiar" gelada e fornecer oxigênio (Figura 2E).</li>
	<li>Ajustar o intervalo de corte e prosseguir com o seccionamento do cérebro (Figura 2F). Recolha as seções que contém a região de interesse, tais como o septo medial (MS), a banda diagonal de Broca (DBB) e substância innominata (SI), como descrito anteriormente,22. De cima para baixo, as três fatias contêm o rostral (R), intermediário (I) e caudal (C) parte do BF (Figura 2).</li>
	<li>Dividir ao longo da linha mediana de cada seção com uma pequena tesoura, obtendo dois hemislices especulares (Figura 2 H), usado individualmente como o controle e a condição tratada no mesmo plano anatômico.</li>
	<li>Manter o hemisections na câmara de vibratome por 5-10 min, em seguida, transferi-los com uma pipeta de vidro em um subida pote contendo gravação aCSF. Mantenha a RT para cerca de 30 min para recuperar.</li>
	<li>Preencha, entretanto, três frascos de vidro com 3 ml de solução de gravação aCSF sozinho (anteriormente na etapa 1.6 para o grupo de controle) e três tubos de ensaio com 3 mL de aCSF enriquecido com T30 (previamente preparado no passo 1.6) para o grupo tratado.</li>
</ol>3. preparação da instalação
<ol><li>Transferir o hemisections para seus frascos de vidro correspondente (Figura 2I) a fim de ter três hemislices consecutivos (incluindo a região BF rostral, intermediária e caudal) para o grupo de controle (quadro verde, Figura 2J) e suas correspondência de contrapartes para o grupo tratado (quadro vermelho, Figura 2J).</li>
	<li>Transferi o tecido cerebral com duas escovas diferentes, uma para o controle hemislices e outro para as contrapartes tratadas, para evitar qualquer contaminação entre as condições.</li>
	<li>Sele cada frasco de vidro com a tampa correspondente, apresentando a agulha oxigenação (21G) (Figura 2J). Ligar o tubo principal do aparelho para a fonte de carbogênio (Figura 2 K). Entrega oxigênio para a hemislices durante todo o período de incubação com um caudal mínimo de 2 mL/min para evitar qualquer movimento do tecido cerebral e possíveis danos mecânicos durante o experimento. Inicie a incubação de 5h. Execute as etapas de 3.1-3.3 dentro de 5 min. Nota: Verifique frequentemente (cada 15-20 min) se o oxigênio é fornecido em todos os frascos e que seu fluxo não está se movendo o tecido do fundo.</li>
</ol>4. homogeneização da amostra
<ol><li>Parar o fluxo de oxigênio após a incubação e remover todas as tampas dos frascos. Transferi o hemislices, usando os pincéis adequados, para tubos de 1,5 mL, contendo 250 µ l de tampão de Lise, manter os tubos em gelo (Figura 2 L). Para fazer a lise de tampão, misturar PBS 1 x com cocktails de inibidores da fosfatase e protease tanto diluído a 1: 100 e homogeneizar o tecido com macacos diferentes para evitar a contaminação entre as amostras. Conclua essas ações dentro de 10-15 min.</li>
	<li>Centrifugue o cérebro lisado a 1.000 x g por 5 min a 4 ° C. Transferir o sobrenadante para tubos novos e manter as amostras a-80 ° C.</li>
</ol>5. leitura da concentração de proteínas e análise ocidental do borrão
<ol><li>Determinar a concentração de proteína de cada amostra e posteriormente, preparar as alíquotas para análise ocidental do borrão como descrito anteriormente,22.</li>
	<li>Realizar análise estatística para avaliar os efeitos do tratamento na expressão da proteína.</li>
</ol>

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Os autores declaram concorrentes interesses financeiros. Susan A. Greenfield é o fundador e presidente da Neuro-Bio Limited, uma empresa canadense e detém ações da empresa. Emanuele Brai e Antonella Cogoni são empregados da Neuro-Bio Ltd.

O principal aspecto do presente protocolo, baseado na técnica cérebro bem-estabelecida ex vivo , permite testar sincronicamente duas hemislices especulares, obtidas no mesmo plano anatômico, monitorando sua resposta após a aplicação de um determinado condição (controlar ou tratados); Este, portanto, oferece uma paradigma experimental tão rigidamente controlada quanto possível. A possibilidade de avaliar em um tempo - dose- e site-specific maneira diferente neuroquímicos relacionados com comprometiment...

AD é que uma patologia crônica caracterizada por imparidade neurodegenerativa progressiva, afetando áreas diferentes do cérebro, tais como o córtex entorhinal (CE), BF, hipocampo (HC) e bulbo olfatório (OB)1,2,3, 4,5. Estágios finais de desenvolvimento AD conduzem a um progressivo declínio cognitivo, tornando esta doença, a forma mais comum de demência, representando aproximadamente 70% de todos os casos6. Apesar das tentativas extensas para entender os estágios iniciais, causando AD, não há atualmente uma indicação experimental definida elucidá-los. Além disso, a teoria mais popular - a "hipótese amiloide" - é cada vez mais questionada desde que ele não fornece um perfil completo em explicar o Patobiológico AD, nem um alvo farmacêutico que se revelou eficaz7,8 ,9.
Uma teoria alternativa que está recebendo atenção crescente sugere que os mecanismos iniciais ocorrem durante neurodegeneração estão relacionados a um cluster neuronal principalmente sensível no AD3,10,11 , 12 , 13 , 14. este hub celular heterogênea englobada no âmbito dos projectos BF, mesencéfalo e tronco cerebral, para várias regiões, tais como o CE, HC e OB15,16. Apesar de sua diversidade na morfologia neuronal e síntese de neurotransmissor, esse núcleo de células compartilha uma característica comum em expressar a dor, que também pode ter uma função não-enzimática de17,18. Este papel não-clássica como um romance de sinalização molécula Medeia o fluxo de cálcio (Ca2 +) em neurônios que pode passar por eventos tróficos ou tóxicos em relação à dose de Ca2 + , disponibilidade e idade neuronal17,18 , 19.
Durante neurodegeneração, a perda celular observada pode ser, portanto, associada a esta função não enzimáticos17,18,20, que é atribuível a um peptídeo 30mer (T30) clivado de dor C-terminal 20. em consonância com os resultados anteriores, realizados na cultura de pilha e óptico de imagem18,21 preparações, temos demonstrado, através de uma nova abordagem baseada em ex vivo fatias cérebro rato contendo estruturas BF, que induziu T30 um perfil AD22. Especificamente, esta nova metodologia oferece um cenário mais fisiológico do que cultura celular desde que ele mantém muitas das características de um tecido intacto, variando de anatômica para preservação de circuitos, embora para uma janela de tempo de horas. Aplicamos este protocolo para explorar os eventos que ocorrem durante as primeiras fases de neurodegeneração, monitoramento da resposta aguda a T30 pedido.
Apesar da grande corpus de literatura sobre o uso do cérebro fatias para investigar as vias moleculares implícita no dano neuronal ou neurogênese23,24, este protocolo fornece pela primeira vez uma mais imediata e sensível à leitura comparada para o uso comum de organotypic fatias. No entanto, como é o caso de organotypic seções do cérebro, este procedimento de fatia aguda pode também ser adotado para fins diversos, tais como a avaliação de neuroprotective ou moléculas neurotóxicas, descoberta de mudanças moleculares primárias que ocorrem em um processo específico, a análise imuno-histoquímica e ensaios farmacológicos para o sistema nervoso central relacionadas com patologias.

<table><tbody><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich, Germany</td><td>S7653</td><td>Reagent for aCSF preparation</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich, Germany</td><td>P9333</td><td>Reagent for aCSF preparation</td></tr><tr><td>Sodium bicarbonate (NaHCO3)</td><td>Sigma-Aldrich, Germany</td><td>S5761</td><td>Reagent for aCSF preparation</td></tr><tr><td>Magnesium sulphate heptahydrate (MgSO4 (7H2O))</td><td>Sigma-Aldrich, Germany</td><td>63138</td><td>Reagent for aCSF preparation</td></tr><tr><td>Potassium phosphate monobasic (KH2PO4)</td><td>Sigma-Aldrich, Germany</td><td>P5655</td><td>Reagent for aCSF preparation</td></tr><tr><td>Hepes salt</td><td>Sigma-Aldrich, Germany</td><td>H7006</td><td>Reagent for aCSF preparation</td></tr><tr><td>Hepes acid</td><td>Sigma-Aldrich, Germany</td><td>H3375</td><td>Reagent for aCSF preparation</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich, Germany</td><td>G7528</td><td>Reagent for aCSF preparation</td></tr><tr><td>Calcium chloride dehydrate</td><td>Sigma-Aldrich, Germany</td><td>223506</td><td>Reagent for aCSF preparation</td></tr><tr><td>T30 peptide</td><td>Genosphere Biotechnologies, France</td><td></td><td>AChE-derived peptide tested</td></tr><tr><td>Surgical dissecting kit</td><td>World Precision Instruments, USA</td><td>Item #: MOUSEKIT</td><td>Brain removal step</td></tr><tr><td>Surgical blades</td><td>Swann-Morton, UK</td><td>BS 2982</td><td>Brain removal step</td></tr><tr><td>Filter paper</td><td>Fisher Scientific, USA</td><td>11566873</td><td>Brain preparation for slicing</td></tr><tr><td>Glue</td><td></td><td></td><td>Brain preparation for slicing</td></tr><tr><td>Vibratome</td><td>Leica, Germany</td><td>VT1000 S</td><td>Slicing</td></tr><tr><td>Brushes</td><td></td><td></td><td>Tissue handling</td></tr><tr><td>Oxygen canister</td><td></td><td></td><td>Sectioning and incubation phase</td></tr><tr><td>1x Phosphate buffer saline (PBS)</td><td>Fisher Scientific, USA</td><td>BP2438-4</td><td>Homogenization step</td></tr><tr><td>Phosphatase inhibitors</td><td>Fisher Scientific, USA</td><td>1284-1650</td><td>Homogenization step</td></tr><tr><td>Protease inhibitors</td><td>Roche complete PIC, USA</td><td>4693116001</td><td>Homogenization step</td></tr><tr><td>Pestles</td><td>Starlab, UK</td><td>I1415-5390</td><td>Homogenization step</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>Pierce 660 nm Protein Assay</td><td>Thermo Scientific, USA</td><td>22660</td><td>Protein concentration</td></tr></tbody></table>

an alternative approach to study primary events in neurodegeneration using ex vivo rat brain slices

Apesar de numerosos estudos que tentam desenvolver modelos animais confiáveis que refletir o primário processa neurodegeneração subjacente, muito poucos foram amplamente aceites. Aqui, propomos um novo procedimento adaptado de conhecidos ex vivo cérebro fatia técnica, que oferece um mais perto na vivo-como cenário do que em vitro preparações para investigar os eventos iniciais provocando degeneração celular, como observada na doença de Alzheimer (AD). Esta variação consiste em etapas simples e facilmente reproduzíveis, que permitem a preservação do cytoarchitecture anatômica da região selecionada do cérebro e sua funcionalidade local em um ambiente fisiológico. Diferentes áreas anatômicas podem ser obtidas o mesmo cérebro, proporcionando a oportunidade de realizar várias experiências com os tratamentos em questão em um local-, dose e tempo-dependente. Limitações potenciais que poderiam afetar os resultados relacionados com esta metodologia estão relacionadas com a conservação do tecido, ou seja, a manutenção da sua integridade anatômica durante as etapas de corte e a incubação e a espessura de corte, que pode influenciar a análise bioquímica e imuno-histoquímica. Esta abordagem pode ser utilizada para diferentes fins, tais como explorar mecanismos moleculares envolvidos em condições fisiológicas ou patológicas, triagem de drogas ou ensaios de dose-resposta. Finalmente, este protocolo também poderia reduzir o número de animais utilizados em estudos comportamentais. O aplicativo aqui relatado foi recentemente descrito e testado pela primeira vez na ex vivo fatias cérebro rato contendo o prosencéfalo basal (BF), que é uma das regiões cerebrais afetadas principalmente no AD. Especificamente, foi demonstrado que a administração de um peptídeo tóxico derivado do C-terminal da acetilcolinesterase (AChE) poderia solicitar um AD-como o perfil, provocando, ao longo do eixo antero-posterior do BF, uma expressão diferencial de proteínas alteradas na AD, como o receptor nicotínico alpha7 (α7-nAChR), fosforilada Tau (p-Tau) e beta amiloide (Aβ).

O protocolo aqui apresentado indica que a administração de um peptídeo tóxico, T30, modula de forma local-dependente da expressão do nAChR, α7-p-Tau e Aβ nas seções contendo BF (Figura 3A). O receptor nicotínico mostra um aumento significativo no hemislice rostral-tratados, comparado ao seu homólogo de controle (fatia 1, p = 0.0310) (Figura 3B), enquanto a fatia intermediária não revela qualquer alteração ...

Watch this Scientific Journal Video about An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices at JoVE.com

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

Apresentamos um método que pode fornecer mais insights sobre os primeiros eventos subjacentes a neurodegeneração e baseia a técnica de cérebro estabelecido ex vivo , combinando as vantagens dos experimentos in vivo e in vitro . Além disso, representa uma oportunidade única para comparação directa do grupo tratado e não tratado no mesmo plano anatômico.

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, ...

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Research

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Neuroscience

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

6.9K visualizações.  Culham Science Centre. Apresentamos um método que pode fornecer mais insights sobre os primeiros eventos subjacentes a neurodegeneração e baseia a técnica de cérebro estabelecido ex vivo , combinando as vantagens dos experimentos in vivo e in vitro . Além disso, representa uma oportunidade única para comparação directa do grupo tratado e não tratado no mesmo plano anatômico.

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

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Cultures Organotípicas fatia de Estudos de Pós-natal Neurogênese

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Biologia do Desenvolvimento

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

Preparação de fatias do hipocampo aguda de ratos e camundongos transgênicos para o Estudo das Alterações Synaptic durante o envelhecimento e amilóide Patologia

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Neurociência

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation de neurônios de fatias do cérebro de roedores para estudar a transmissão sináptica e Imagem Terminais pré-sináptica

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Medicina

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Cérebro Fatia Biotinilação: Uma<em&gt; Ex Vivo</em&gt; Abordagem medir Região específicas do tráfego de proteínas de membrana plasmática em adultos Neurônios

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

A Análise de Neurovascular Remodelação em Entorhino-hipocampal culturas Organotípicas Fatia

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

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 ...

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.

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.

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 ...

Neuronal Death Assay to Evaluate Cell Death in Ex Vivo Epilepsy Model

Source: Carvalho, M., et al., <a href="https://app.jove.com/v/61330">A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures.</a> J. Vis. Exp. (2021)
This video demonstrates the technique of assessing neuronal death in rhinal cortex-hippocampus organotypic slices. Neuronal death occurs via evolving epileptic seizures &#8212; resembling in vivo epilepsy &#8212; and is detected through dual-staining of the dead cells using propidium iodide and immunofluorescence.

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

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