Financiado pelo NINDS, American Heart Association, American Federation for Aging Research, March of Dimes

1. Preparação Antes de começar, montar os instrumentos necessários cirúrgica, dissecção e meios de cultura, e sete dias de ratos velhos ou filhotes de rato. O protocolo é o mesmo para mouse e preparações de ratos. (I) Os materiais e equipamentos <ul><li> Tesoura operacional (comprimento em linha reta 6 &quot;, cat # RS6818, Roboz, Gaithersburg, MD) </li><li> Micro dissecando tesoura, (comprimento 4 &#39;&quot;, cat # RS5910. Roboz, Gaithersburg, MD) </li><li> Aço Carbono Dumont Pinças (cat # RS-5045.Roboz, Gaithersburg, MD) </li><li> Vidro modificado &quot;transferência&quot; pipetas (parte inferior da pipeta Pasteur removido e suavizada pela borda) </li><li> Aclar filme plástico cortadas a 2 &quot;em x 2&quot; em praças (Honeywell, # 812071, Pottsville, PA) </li><li> Cobrir esponjas 4 em por 3 in (Kendall, cat # 3157, Mansfield, MA) </li><li> 30 milímetros Millicell insere membrana (0,4 mm; Millipore, Bedford, MA) </li><li> Mcllwain helicóptero Tecido com dois gumes lâmina de barbear (McIlwain helicóptero tecido, empresa Vibratome, St. Louis, MO) </li><li> Microscópio invertido com CCD da câmera e software de análise de imagem </li></ul> (Ii) Média Dissecação (DM; 1 litro em pH 7,3) <table border="0"><tbody><tr><td> Salina balanceada de Hank </td><td> 1 frasco (95,2 g/1L) (Sigma: H2387) </td></tr><tr><td> NHCO 3 (4,2 mM) </td><td> 350 mg </td></tr><tr><td> HEPES (10 mM) </td><td> 2,83 g </td></tr><tr><td> Glicose (33,3 mM) </td><td> 6,0 g </td></tr><tr><td> Penicilina estreptomicina / (100x) </td><td> 10 mL </td></tr><tr><td> BSA (0,3%) </td><td> 300 mg </td></tr><tr><td> MgSO 4 7H 2 O </td><td> 1,44 g </td></tr></tbody></table> (Iii) meio de cultura (400 mL) <table border="0"><tbody><tr><td> MEM (Invitrogen 11575-032) </td><td> 200 mL </td></tr><tr><td> *** Sal Balanço de Hank (Invitrogen 24020-117) </td><td> 100 mL </td></tr><tr><td> Soro de cavalo (Invitrogen 26050-070) </td><td> 100 mL </td></tr><tr><td> HEPES buffer (1 M; Invitrogen 15630-080) </td><td> 5 mL </td></tr><tr><td> Penicilina estreptomicina / (100x) </td><td> 4 mL </td></tr><tr><td colspan="2"> *** Adicionar 12,8 g de glicose para Salt 500 mL de Hank Balance antes de fazer meio de cultura </td></tr></tbody></table> (Iv) Animais 7 dias de idade ou filhotes de ratos rato pós-parto, geralmente uma ninhada por experimento. 2. No Dia da Cultura <ol><li> Todas as ferramentas de dissecação e Aclar filme deve ser embebido em álcool etílico 70% por 30 minutos antes de começar. A superfície do capô, a dissecção da lâmina, e helicóptero tecido deve ser desinfetado com etanol. </li><li> Prepare vários (3-4) 6 placas de cultura de tecidos bem, adicione um meio de cultura mL por poço, e inserir uma membrana Millipore permeáveis ​​em cada poço. Coloque 6-bem placas no 37 ° incubadora até que esteja pronto para transferir fatias. </li><li> Prepare vários (4-6) 60 mm com placas DM e colocar no gelo. </li><li> Rapidamente decapitar com uma tesoura e mergulhe em 70% o álcool etílico, e rapidamente expor o cérebro com uma incisão sagital do crânio. Remova cuidadosamente o cérebro e coloque imediatamente em DM frio. Separá-lo em dois hemisférios e coloque cada lado medial do hemisfério para baixo em uma esponja de cobertura para 2-3 segundos. </li><li> Levantar delicadamente e coloque o hemisfério para a praça Aclar filme e coloque no helicóptero tecidos; corte coronal do cérebro em 350 seções mM. Quando terminar, retire com cuidado a praça Alcar com o hemisfério seccionados e submergir em DM frio. </li><li> Usando um microscópio de dissecação, separar as seções do hipocampo. Para ratos, deve haver cinco seções que abrangem rostral hipocampo caudal, e para mouse deve haver seções 3-4. </li><li> Tirar as placas de 6 poços com inserções de membrana do 37 ° de cultura de tecidos incubadora. Transferência de fatias do hipocampo individualmente para a membranas permeáveis ​​usando a pipeta de transferência de vidro. Se demasiado DM é liberado para a membrana, remover o excesso de DM com uma pipeta Pasteur. Coloque cinco fatias do hipocampo em cada membrana. Permitir que pelo menos três poços por condição, ou 15 fatias. </li><li> Incubar as placas de cultura a 37 ° em 5% CO incubadora umidificada por 12-14 dias antes do início do experimento de toxicidade. O meio de cultura podem ser mudadas duas vezes por semana. </li></ol> 3. Induzir e quantificação lesão neuronal do hipocampo em Cultura Slice <ol><li> Depois de 12-14 dias em cultura, adicionar 5 mg / mL de iodeto de propídio (PI) para o meio de cultura. Esta etapa irá permitir a visualização e quantificação de morte celular basal. </li><li> No dia seguinte, quantificar PIfluorescência na sub-região CA1 (e / ou CA3 ou denteado se desejado) de cada fatia do hipocampo e obter média das medições de fluorescência PI representando a morte de células basais (referido como tempo = 0 horas, ou T 0). Para aquisição de imagem e quantificação, usamos OpenLab software (Improvisação, UK), mas a aquisição de imagens e outros softwares de análise podem ser usados ​​para quantificar a intensidade de fluorescência. </li><li> Imediatamente após a aquisição da imagem de T 0 PI fluorescência, induzir lesão neuronal pelo seu método de escolha (temos usado 10 mM NMDA, oxigênio glucose-privação, ou LPS para uma hora como indutores de toxicidade neuronal 2,5). Após o estímulo tóxicos, substituir média com meio fresco contendo 5 mg / mL PI e manter fatias durante a noite. </li><li> No dia 3, significa quantificar PI fluorescência da condição experimental em 24h, ou T 24. </li><li> Após esta aquisição de imagem, mudar imediatamente médio e adicionar 10 mM NMDA e 5 mg / mL PI e fatias de incubar durante a noite para atingir a morte neuronal máxima. </li><li> No dia 4, significa quantificar PI fluorescência da morte neuronal máxima, definida como T max. Calcular a morte celular por cento da seguinte forma: (T 24 - T 0) / (T max-T 0). </li></ol> 4. Resultados representante <img src="/files/ftp_upload/2106/2106fig1.jpg" alt="Figura 1" /> Figura 1. (A) da linha de tempo para a geração de hipocampo e subsequente lesão experimental e de aquisição de imagem. (B) Imagens representativas de fatias do hipocampo basally (T 0), 24 horas após uma exposição de uma hora a 10 mM NMDA (T 24), e após mais 24 horas ou 10 mM NMDA para obter lesão neuronal máxima (T max).

<ol>
	<li>Stoppin, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37 (2), 173-173 (1991).</li><li>McCullough, L., Wu, L., Haughey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+Function+of+the+PGE2+EP2+Receptor+in+Cerebral+Ischemia.">Neuroprotective Function of the PGE2 EP2 Receptor in Cerebral Ischemia.</a> J Neurosci. 24 (1), 257-257 (2004).</li><li>Czapiga, M., Colton, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+microglia+in+organotypic+slice+cultures.">Function of microglia in organotypic slice cultures.</a> J Neurosci Res. 56, 644-644 (1999).</li><li>Newell, D. W., Barth, A., Papermaster, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+and+non-glutamate+receptor+mediated+toxicity+caused+by+oxygen+and+glucose+deprivation+in+organotypic+hippocampal+cultures.">Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures.</a> J Neurosci. 15, 7702-7702 (1995).</li><li>Wu, L., Wang, Q., Liang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+Effects+of+prostaglandin+recetor+signaling+on+neuronal+survival.">Divergent Effects of prostaglandin recetor signaling on neuronal survival.</a> Neuroscience letter. 421, 253-253 (2007).</li><li>Hailer, N. P., Jarhult, J. D., Nitsch, R., R, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+microglial+cells+in+vitro:+Analysis+of+morphology+and+adhesion+molecule+expression+in+organotypic+hippocampal+slice+cultures.">Resting microglial cells in vitro: Analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures.</a> Glia. 18, 319-319 (1996).</li></ol>

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Existem dois pontos importantes a seguir para garantir resultados reprodutíveis e consistentes ao replicar as experiências. Primeiro, é importante deixar que a idade fatias do hipocampo por 12-14 dias antes da experimentação. Com isolamento fatia, há uma resposta significativa da microglia e microglia permanecerá ativado por algum tempo, com um retorno a um estado de repouso ramificada ocorrem de dia 10 in vitro 3,6. Em segundo lugar, com média de PI intensidade de fluorescência é proporcional ao número de células lesadas 4. Fluorescência seqüencial é medido em três momentos: (i) em T 0, antes de NMDA ou estimulação OGD para medir os níveis basais de morte neuronal espontânea; (ii) a T 24, ou 24 horas após a estimulação com NMDA ou estímulo OGD ou outros, e (iii) a T max, após um tratamento final de fatias com um tratamento de 24 horas durante a noite letais com 10 mM NMDA, que produz a quantidade máxima de morte neuronal. A morte celular por cento é então calculada usando a fórmula (T 24 - T 0) / (T max-T 0). É fundamental para normalizar cada fatia para si mesmo usando este método porque o tamanho do hipocampo ea expressão dos receptores de glutamato e mudar significativamente a distribuição como um vai de rostral ao cérebro caudal, e um fracasso para normalizar cada fatia para si pode resultar em dados artifactual .

the organotypic hippocampal slice culture model for examining neuronal injury

Cultura fatia organotípicas hipocampo é um método in vitro para analisar os mecanismos de lesão neuronal em que a arquitetura básica e composição do hipocampo é relativamente preservado 1. O sistema de cultura organotípicas permite o exame dos efeitos neuronal, astrocíticos e microglia, mas como uma preparação ex vivo, não aborda os efeitos do fluxo de sangue, ou recrutamento de células inflamatórias periféricas. Para o efeito, este método de cultura é freqüentemente utilizado para examinar lesão hipóxico excitotoxic e aos neurônios piramidais do hipocampo, mas também tem sido usado para examinar a resposta inflamatória. Aqui nós descrevemos os métodos para a geração de culturas fatia do hipocampo do cérebro do roedor pós-parto, administrar estímulos tóxicos para induzir a lesão neuronal, e assaying e quantificar a morte neuronal no hipocampo.

Watch this Scientific Journal Video about The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury at JoVE.com

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

O modelo de cultura organoptypic hipocampo é uma fatia<em&gt; In vitro</em&gt; Modelo usado para examinar lesão neuronal em uma variedade de paradigmas. Neste artigo, vamos descrever os métodos de geração de culturas fatia e quantificar lesão neuronal.

O modelo de Cultura organotípicas Hippocampal Slice para o exame das lesões neuronais

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

the-organotypic-hippocampal-slice-culture-model-for-examining

Nanyang Technological University

Research

JoVE Journal

Neuroscience

19.0K visualizações.  Stanford University School of Medicine. O modelo de cultura organoptypic hipocampo é uma fatia In vitro Modelo usado para examinar lesão neuronal em uma variedade de paradigmas. Neste artigo, vamos descrever os métodos de geração de culturas fatia e quantificar lesão neuronal.

Vídeo: The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

Cultura Slice organotípicas de GFP-expressando embriões de camundongos para imagens em tempo real de crescimento dos nervos periféricos

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Neurociência

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotípicas Hippocampal Culturas Slice

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Um Ensaio Slice organotípicas para High-Resolution Imaging Time Lapse de migração neuronal no cérebro pós-natal

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Organotípicas culturas de mesencéfalo ventral Slice Embrionárias: Um Sistema para o Estudo de Desenvolvimento Neuronal dopaminérgicos In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

A Análise de Purkinje Morfologia de células dendríticas em Culturas fatia organotípicas

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Emparelhados Recordings células inteiras em organotï¿½icas fatias do hipocampo

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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 ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

Low-Density hipocampo Cultura Neuron primária

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic fatias Hippocampal culturas como um modelo para estudo neuroproteção e invasão de células tumorais

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic de arrastamento de Theta Hippocampal oscilações em comportar-se ratos

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...