Gostaríamos de agradecer a Britta Kaltwasser por sua excelente assistência técnica e Mahesh Kumar Teli para organizar a preparação filmagens de vídeo.

1. Animais <ol><li> Os experimentos foram realizados de acordo com as diretrizes do NIH para o cuidado e uso de animais de laboratório e aprovado pelas autoridades locais. Para todas as experiências, C57BL6 / J ratinhos do tipo selvagem, ApolipoproteinE - / - (ApoE KO) e murganhos SV129 (12-16 semanas de idade, 26-30 g de peso corporal, 5-6 animais por grupo experimental) foram utilizados. </li></ol> 2. A coloração dos vasos cerebrais com látex colorido <ol><li> Prepara-se uma mistura de 25 ul de tinta de carbono preto (Herlitz, Germany) com 0,5 ml de composto de látex (Pebeo, França) numa razão de 1:20 em um tubo de PE e aquecer a mistura a 37 ° C num banho de água. Recolher a mistura em uma seringa de 2 ml com uma agulha de 28-30G antes anestesiar o animal. </li><li> Dissolver 50 mg de pó de hidrocloreto de papavarine em 1 ml de solução salina normal estéril. Recolher a solução de uma seringa de insulina. Parta a agulha de outra seringa de insulina estéril. Coloque esta agulha no final de um c 20m tubo PE10 de comprimento. Agora anexar este tubo PE10 sobre a agulha da seringa de insulina contendo uma solução de cloridrato de papavarine a ser injectado através da veia femoral para assegurar o enchimento correcto e vasodilatação dos vasos. </li><li> Preparar outras duas seringas de insulina de 1 ml cada de solução salina contendo. Seringas de insulina permitir a entrega suave do fluido injectado a uma localização precisa. No entanto, devido à alta viscosidade do látex, alternativa seringas de 1 ml com agulhas removíveis mais largas devem ser usados. </li><li> Tomar todas as seringas e instrumentos esterilizados dissecando próximas à fase operacional. Espalhe uma folha draper cirúrgica em torno da fase de operação. A fase de operação devem ser suficientemente iluminada e um microscópio de operação pode ser utilizado, se necessário. </li><li> Agora medir o peso do corpo de um rato C57BL6 / J e, em seguida, induzir a anestesia com isoflurano a 5% vaporizado em 70% de N 2 O e 30% de O 2. Continuar a anestesia com 1% de isoflurano vaporizado. Após POSICIONAM adequadag do mouse em sua parte traseira e fixação dos membros, avaliar a profundidade da anestesia. Em seguida, corte a pele sobre a veia femoral esquerda e localizar a veia. Introduzir a ponta afiada da agulha colocado no tubo PE10 e injecte lentamente uma solução de cloridrato papavarine a uma taxa de 100 ul por minuto (50 peso corporal mg / kg). </li><li> Após a injecção, a cavidade abdominal cortar e perfurar o diafragma. Corte as costelas para expor o coração, sem danificar ou nódoas negras-lo. Clipe da aorta torácica descendente com uma pinça de artérias. Faça um corte afiado sobre o átrio direito para permitir que o sangue venoso drenado. Injectar 2 ml de solução salina no ventrículo esquerdo, seguida de injecção do látex colorido manualmente com uma ligeira pressão sobre 18-20 seg. Usar as seringas pré-cheias, uma após a outra. Evite injetar bolhas. </li><li> Após a injecção de látex, deixam o animal, durante 5 min. Decapitar o animal e retire cuidadosamente todo o cérebro. Cuide bem para não ferir a arquitetura do vaso e cortex, removendo o osso do crânio e meninges. Coloque o cérebro em um prato de 60 milímetros de cultura de células contendo 6-8 ml de PFA 4% para tirar uma foto. Coloque uma régua de medição debaixo do prato transparente. Tire fotos com uma ampliação de 25x da dorsal e da superfície ventral do cérebro com uma câmera acoplada ao microscópio cirúrgico. Fotografias devem ser tomadas em ângulo de 90 ° entre a placa de Petri e a objetiva do microscópio a fim de quantificar a distância original. </li><li> Repetir o processo em outros animais 4-5. </li></ol> 3. A coloração dos vasos cerebrais com Mistura de Tintas Carbon Black <ol><li> Para comparar os resultados da coloração de látex baseado vascular cerebral com nosso protocolo, preparar uma mistura de 100 ul de Herlitz Stempelfarbe tinta (CB1) com 900 ul de tinta Pelican Scribtol Schwarz (CB2), em um tubo de 1,5 ml EP. Preparar dois tubos de PE para fazer um volume total de 2 ml de mistura de CB1 e CB2 na proporção 1:9. Recolher a mistura de duas seringas de insulina separadas (1ml de cada). Tapar as seringas e coloque-os num banho de água a aquecer até 37 ° C. Recolha de 2 ml de soro fisiológico em mais duas seringas de insulina, e coloque-os num banho de água também. </li><li> Anestesiar o animal e repita o mesmo procedimento como descrito acima, excepto por injecção de cloridrato de papavarine. Após a injecção de 2 ml salaine para o ventrículo esquerdo, injectar uma mistura de 2 ml de tinta de negro de fumo, em vez do látex colorido. Execute as injeções à mão com uma ligeira pressão sobre 18-20 seg por ml. Clipe da aorta torácica descendente antes das injeções. </li><li> Sacrificar o animal, remover o cérebro e tirar fotos. </li><li> Para preservação a longo prazo do cérebro, o cérebro colocar em PFA a 4% durante a noite, seguido de incubação com sacarose com uma concentração gradualmente crescente até que o cérebro flutuante submerge totalmente (5% seguido de 15% e, em seguida, 30%). Os cérebros dos animais foram perfundidas com o látex colorido pode também ser mantida na mesma forma. </li><li> Realize oprocedimento em outros animais 4-5. Para comparar a diferença na anatomia vascular cerebral devido às características genéticas ou cepas diferentes, repita o protocolo em igual número de ApoE KO e SV129 ratos, respectivamente. </li><li> Para estudar a anatomia vascular sob condições isquémicas, induzir isquemia focal cerebral transitória durante 45 min ou 90 min de acordo com um protocolo padrão de oclusão intraluminal da MCA, sob anestesia profunda (uma descrição detalhada da cirurgia está para além do âmbito deste artigo, o leitor é remetido para Doeppner et al., 2010). No final do período de reperfusão planeado (1 dia e 5 dias), realizar a coloração vascular com CB1 CB2 + tintas únicas e manchar todo o cérebro, com 2% de solução de TTC a 37 ° C durante 5-10 minutos para identificar o volume do enfarte . TTC é reduzido a formazano colorido vermelho pelas enzimas mitocondriais (desidrogenase particularmente succinato). Após a coloração TTC, as manchas de tecido metabolicamente ativos vermelho escuro, enquanto o tecido infartado permanece unstaiNed e aparece em branco devido a disfuncional e desnaturado enzima mitocondrial. Recolher imagens da mesma maneira como descrito acima. </li></ol> 4. Estudo da Cerebral territórios vasculares <ol><li> Para analisar a anatomia dos vasos cerebrais, imagens da superfície dorsal do cérebro ou manchado com látex colorido ou CB1 CB2 + tintas podem ser analisados ​​com software ImageJ (um software Java de domínio baseado público utilizado para o processamento e análise de imagens). Cérebros perfundidos com látex colorido pode mostrar graus variáveis ​​de coloração embarcação localizada na superfície dorsal, enquanto CB1 CB2 + perfusão irá manchar todos os vasos em ambas as superfícies ventrais e dorsais. </li><li> Abrir um arquivo de imagem com o software ImageJ. Identificar todos os pontos de anastomose entre o ACA e da MCA. Um ponto de anastomose é definido como o local onde o diâmetro do vaso é o mais estreito ou a meia distância entre os pontos mais próximos que ramificam da ACA e da MCA os ramos, respectivamente 2. <saté&gt; Desenhar uma linha imaginária (linha anastomótica), marcando os pontos anastomose com a ajuda da ferramenta de desenho linha segmentada eo plugin &quot;linha pontilhada&quot;. </li><li> Defina a escala em mm. Medir a distância entre a linha de anastomose e da linha média, 4 mm caudal ao poste frontal do cérebro, utilizando a ferramenta de desenho em linha recta e &quot;medida&quot; da ferramenta. Fazer o mesmo caudal de 6 mm a partir do pólo frontal. Analisar as imagens de 3-4 por animal e calcular o valor médio. Comparar os valores entre os grupos diferentes de animais para identificar a variação da anatomia cerebrovascular. Em C57BL6 / J animais, a linha de anastomose entre o ACA e da MCA pode ser rastreada mais perto da linha média, 4 mm de 6 mm de caudal do pólo frontal. Camundongos ApoE KO vai apresentar nenhuma diferença significativa a este respeito. Pelo contrário, em SV129 ratos da linha anastomótica mentirei mm mais e paralelamente à linha mediana ambos a 4 mm e 6 caudalmente do pólo frontal. </li><li> Para analisar a anatomia vascular in condições de isquemia, os mesmos cálculos acima mencionados. Identificar a fronteira infarto como a margem de tecido vermelho e branca representando tecido metabolicamente ativo e tecido sem manchas necróticas, respectivamente. Medir a distância da borda enfarte da linha média, 4 mm, 6 mm e caudal do pólo frontal do cérebro. Recolher o valor médio a partir de imagens 3-4 por animal. Comparar os resultados entre isquemia diferente e períodos de reperfusão. </li></ol>

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R., Nagel, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAT-Hsp70-mediated+neuroprotection+and+increased+survival+of+neuronal+precursor+cells+after+focal+cerebral+ischemia+in+mice.">TAT-Hsp70-mediated neuroprotection and increased survival of neuronal precursor cells after focal cerebral ischemia in mice.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 29, 1187-1196 (2009).</li><li>Todo, K., Kitagawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+enhances+leptomeningeal+collateral+growth+induced+by+common+carotid+artery+occlusion.">Granulocyte-macrophage colony-stimulating factor enhances leptomeningeal collateral growth induced by common carotid artery occlusion.</a> Stroke. 39 (6), 1875-1882 (2008).</li><li>Sugiyama, Y., Yagita, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte+colony-stimulating+factor+enhances+arteriogenesis+and+ameliorates+cerebral+damage+in+a+mouse+model+of+ischemic+stroke.">Granulocyte colony-stimulating factor enhances arteriogenesis and ameliorates cerebral damage in a mouse model of ischemic stroke.</a> Stroke. 42 (3), 770-775 (2011).</li><li>Busch, H. 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Perfusão de CB1 CB2 + por injecção manual pode ser realizada com sucesso por sem treino intensivo, uma vez que não envolve qualquer dispositivo específico para implicar uma certa pressão 2,3. A heterogeneidade dos resultados de perfusão em nosso protocolo também é insignificante. Só o animal um dos 16 animais não-isquêmicas e 3 de 20 animais isquêmicos mostraram perfusão incompleto. Nesses casos, a incorporação de bolhas durante a perfusão de solução salina levando a oclusão dos vasos foi ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Nome da empresa </td><td> Catalog No. </td></tr><tr><td> Scribtol Schwarz (CB2) </td><td> Pelicano, Alemanha </td><td> 221 135 </td></tr><tr><td> Stempelfarbe (CB1) </td><td> Herlitz PBS AG, Alemanha </td><td> 10417202 </td></tr><tr><td> Gedeo Látex </td><td> Pebeo, França </td><td> 13042B </td></tr></tbody></table>

intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels

A estrutura anatômica dos vasos cerebrais é um factor determinante para a hemodinâmica do cérebro, bem como a gravidade da lesão após lesões isquémicas. A vasculatura cerebral dinamicamente responde a diversos estados patofisiológicos e exibe diferenças consideráveis ​​entre estirpes e em condições de manipulações genéticas. Essencialmente, uma técnica adequada para a coloração de vasos intracranianos é essencial, a fim de estudar a patogénese da isquemia cerebral. Até recentemente, a um conjunto de diferentes técnicas tem sido utilizada para visualizar a vasculatura cerebral, incluindo a injecção de resina de baixa viscosidade, araldite F, gelatina misturada com diversos corantes 1 (isto é, vermelho carmim, India tinta) ou de látex, com 2 ou 3, sem carbono negro. Perfusão de composto de látex branco através da aorta ascendente foi primeiramente relatada por Coyle e Jokelainen 3. Maeda et al. 2 ter modificado o protocolo, acrescentando carbon tinta preta para o composto de látex para visualização melhor contraste dos vasos após a perfusão de solução salina do cérebro. No entanto, a perfusão ineficiente e enchimento insuficiente dos vasos são frequentemente experimentado devido à alta viscosidade do composto de látex 4. Assim, descrevemos uma técnica simples e de baixo custo, utilizando uma mistura de duas tintas de carbono disponíveis comercialmente pretas (CB1 e CB2) para visualizar o sistema vascular cerebral de um modo reprodutível 5. Nós mostrámos que a perfusão com CB1 CB2 + em ratos resulta na coloração de vasos cerebrais significativamente menores a uma densidade mais elevada em comparação com a perfusão de látex 5. Aqui, descrevemos nosso protocolo para identificar os pontos de anastomose entre o anterior (ACA) e artérias cerebrais médias (ACM) para estudar as variações dos vasos em ratos com diferentes origens genéticas. Finalmente, demonstrar a viabilidade da nossa técnica de um modelo de isquemia cerebral focal transiente em ratos através da combinação de CB1 +CB2 mediada por coloração com TTC vaso de coloração em vários graus de lesões isquémicas.

O protocolo aqui descrito supera as limitações técnicas de visualização de látex convencional com base da vasculatura cerebral roedor. Figura 1A mostra que a perfusão seguinte do látex corado, apenas os vasos grandes na superfície ventral são corados, deixando toda a superfície dorsal imaculada. O resultado é também altamente variável. Apenas um animal de cada seis mostra coloração parcial da ACA e da MCA visível na superfície dorsal do cérebro (dados não apresentados). Por outro lad...

Watch this Scientific Journal Video about Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels at JoVE.com

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Análise da anatomia cerebrovascular roedor desempenha um papel importante na investigação do acidente vascular cerebral experimental. Neste contexto, a perfusão intravascular com látex corado tem sido considerada como um instrumento padrão para vários anos. No entanto, esta técnica implica limitações técnicas distintas, que minam a sua reprodutibilidade. Aqui, nós descrevemos um método simples para visualizar os vasos cerebrais, de uma forma reprodutível. A injecção de uma mistura de duas tintas pretas de carbono disponíveis comercialmente, através dos resultados do ventrículo esquerdo do miocárdio, em enchimento adequado de vasos cerebrais com visualização de alto contraste. Nós temos aplicado com sucesso esta técnica para identificar os pontos de anastomose entre territórios vasculares cerebrais de ratos com diferentes origens genéticas. Finalmente dar provas de que este método novo e simples para a coloração de navio podem ser combinados com cloreto de trifeniltetrazólio (TTC) de coloração - uma ferramenta amplamente utilizada para observar e analisar volumes do enfarte em ratos.

Perfusão intravenosa de tinta Carbon Black Permite visualização dos vasos cerebrais

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

intravascular-perfusion-carbon-black-ink-allows-reliable

Research

JoVE Journal

Neuroscience

15.3K visualizações.  University of Duisburg-Essen Medical School. Análise da anatomia cerebrovascular roedor desempenha um papel importante na investigação do acidente vascular cerebral experimental. Neste contexto, a perfusão intravascular com látex corado tem sido considerada como um instrumento padrão para vários anos. No entanto, esta técnica implica limitações técnicas distintas, que minam a sua reprodutibilidade. Aqui, nós descrevemos um método simples para visualizar os vasos cerebrais, de uma forma reprodutível. A ...

Vídeo: Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Modelo de isquemia focal cerebral por oclusão Suture Endovascular da artéria cerebral média no Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Neurociência

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

Reprodução de vídeo automatizada interativo para estudos da comunicação animal

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Labeling fluorescentes retrógrada Permite alvejado Extracelular Gravação Single-unidade dos neurônios identificados<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

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

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dois registros eletrofisiológicos de sinapticamente evocadas Respostas astroglial e neuronal em fatias hipocampais agudas

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Eletrofisiologia no isolado do tronco encefálico-espinal Preparações do cordão umbilical de recém-nascidos Roedores permite a gravação de saída Neural Rede Respiratory

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

Purificação de embarcações do cérebro do rato

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

Isolamento e Canulação da cerebral parenquimatosa Arteríolas

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

Intracerebroventricular e intravascular A injeção de Viral partículas e fluorescentes microesferas no cérebro Neonatal

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Um modelo do rato de leve hipoperfusão intra-uterino com estenose Microcoil

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...