Este trabalho foi financiado por doações do Instituto Nacional de Distúrbios Neurológicos e Derrame (NS-19108), o Instituto Nacional de Saúde Infantil e de Doenças (PO1-HD09402), a Fundação de Pesquisa da enxaqueca e da Fundação White. Ms. Marcia P. Kraig assistida na preparação e manutenção de sistemas de cultura.

<ol>
	<li>Kunkler, P. E., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+waves+precede+electrophysiological+changes+of+spreading+depression+in+hippocampal+organ+cultures.">Calcium waves precede electrophysiological changes of spreading depression in hippocampal organ cultures.</a> J Neurosci. 18 (9), 3416-3425 (1998).</li><li>Kunkler, P. E., Hulse, R. E., Schmitt, M. W., Nicholson, C., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+current+source+density+analysis+in+hippocampal+organotypic+culture+shows+that+spreading+depression+occurs+with+uniquely+reversing+currents.">Optical current source density analysis in hippocampal organotypic culture shows that spreading depression occurs with uniquely reversing currents.</a> J Neurosci. 25 (15), 3952-3961 (2005).</li><li>Hulse, R. E., Swenson, W. G., Kunkler, P. E., White, D. M., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monomeric+IgG+is+neuroprotective+via+enhancing+microglial+recycling+endocytosis+and+TNF-&#x03B1;lpha.">Monomeric IgG is neuroprotective via enhancing microglial recycling endocytosis and TNF-&#x03B1;lpha.</a> J Neurosci. 28 (47), 12199-12211 (2008).</li><li>Hailer, N. P., Jarhult, J. D., Nitsch, 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 (4), 319-331 (1996).</li><li>Ransohoff, R. M., Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+physiology:+unique+stimuli,+specialized+responses.">Microglial physiology: unique stimuli, specialized responses.</a> Annu Rev Immunol. 27, 119-145 (2009).</li><li>Mitchell, H. M., White, D. M., Domowicz, M. S., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+pre-conditioning+neuroprotection+depends+on+TNF-&#x03B1;lpha+and+is+enhanced+by+blockade+of+interleukin-11.">Cold pre-conditioning neuroprotection depends on TNF-&#x03B1;lpha and is enhanced by blockade of interleukin-11.</a> J Neurochem DOI. , (2010).</li><li>Mitchell, H. M., White, D. M., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+study+of+neuroprotection+from+cold-preconditioning.">Strategies for study of neuroprotection from cold-preconditioning.</a> J Vis Exp. (43), (2010).</li><li>Kunkler, P. E., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+astrocytosis+from+excitotoxic+injury+in+hippocampal+organ+culture+parallels+that+seen+in+vivo.">Reactive astrocytosis from excitotoxic injury in hippocampal organ culture parallels that seen in vivo.</a> J Cereb Blood Flow Metab. 17 (1), 26-43 (1997).</li><li>Grinberg, Y. Y., Milton, J. G., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spreading+depression+sends+microglia+on+Lévy+flights.">Spreading depression sends microglia on Lévy flights.</a> PLoS ONE. 6 (4), (2011).</li><li>Koroleva, V. I., Oitzl, M. S., Bures, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threshold+of+synaptically+elicited+cortical+spreading+depression:+drug-induced+changes+in+rats.">Threshold of synaptically elicited cortical spreading depression: drug-induced changes in rats.</a> Electroencephalogr Clin Neurophysiol. 60 (1), 55-64 (1985).</li><li>Gubern, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+housekeeping+genes+for+quantitative+real-time+PCR+in+in-vivo+and+in-vitro+models+of+cerebral+ischaemia.">Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia.</a> BMC Mol Biol. 10, 57-57 (2009).</li><li>Tian, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quantification+of+ADAMTS+expression+in+an+animal+model+of+cerebral+ischemia+using+real-time+PCR.">The quantification of ADAMTS expression in an animal model of cerebral ischemia using real-time PCR.</a> Acta Anaesthesiol Scand. 51 (2), 158-164 (2007).</li><li>Pfaffl, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mathematical+model+for+relative+quantification+in+real-time+RT-PCR.">A new mathematical model for relative quantification in real-time RT-PCR.</a> Nucleic Acids Res. 29 (9), e45-e45 (2001).</li><li>Bustin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MIQE+guidelines:+minimum+information+for+publication+of+quantitative+real-time+PCR+experiments.">The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.</a> Clin Chem. 55 (4), 611-622 (2009).</li><li>Pusic, A. D., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+gene+micro+array+profiling+demonstrates+"T-cell-like"+activation+after+recurrent+spreading+depression+-+implications+for+migraine+pathogenesis.">Inflammatory gene micro array profiling demonstrates "T-cell-like" activation after recurrent spreading depression - implications for migraine pathogenesis.</a> Soc Neurosci abst. , (2010).</li><li>Quinn, B., Graybiel, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+differentiated+silver+intensification+procedure+for+the+peroxidase-diaminobenzidine+reaction.">A differentiated silver intensification procedure for the peroxidase-diaminobenzidine reaction.</a> J Histochem Cytochem. 44 (1), 71-74 (1996).</li><li>Caggiano, A. O., Kraig, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eicosanoids+and+nitric+oxide+influence+induction+of+reactive+gliosis+from+spreading+depression+in+microglia+but+not+astrocytes.">Eicosanoids and nitric oxide influence induction of reactive gliosis from spreading depression in microglia but not astrocytes.</a> J Comp Neurol. 369 (1), 93-108 (1996).</li></ol>

Não há conflitos de interesse declarados.

SD é uma perturbação paroxística do cérebro cujas características são altamente conservada em todas as regiões do cérebro examinado espécies e até à data. SD é comumente estudada em neocórtex. No entanto, a complexidade do circuito neural desta estrutura (em comparação com o hipocampo), bem como a incapacidade de manter viva neocórtex in vitro com funções imunológicas de repouso por períodos prolongados, torná-lo menos útil como uma preparação em vitro para a definição das consequências a longo prazo de susceptibilidade SD. Em contraste, o hipocampo não é apenas a estrutura do cérebro que é mais sensível a SD, é também uma estrutura archicortical com uma estrutura mais simplificada do circuito neural e função, que é bem adequado para definir os meios pelos quais neuroimunes atividade neural pode modular a suscetibilidade SD .

modeling neural immune signaling of episodic and chronic migraine using spreading depression in vitro

Enxaqueca e sua transformação para enxaqueca crônica são encargos de saúde que precisam de opções de tratamento melhoraram. Procuramos definir como sinalização imune neural modula a suscetibilidade à enxaqueca, como modelo in vitro utilizando a depressão alastrante (SD), como um meio para desenvolver novos alvos terapêuticos para enxaqueca episódica e crônica. SD é a causa provável de aura de enxaqueca e dor da enxaqueca. É uma perda da função neuronal paroxística desencadeada por aumento da atividade neuronal, inicialmente, que lentamente se propaga dentro de regiões cerebrais suscetíveis. Função normal do cérebro é extremamente sensível a, e se baseia em, coincidente baixo nível de sinalização imune. Assim, a sinalização neural imunológico provavelmente afeta a atividade elétrica do SD, e, portanto, enxaqueca. Estudos percepção de dor em animais inteiros SD são repletas de dificuldades, mas os animais inteiros são adequados para examinar os aspectos biologia de sistemas de enxaqueca desde SD ativa trigeminal vias nociceptivas. No entanto, estudos com animais inteiros por si só não pode ser usado para decifrar os mecanismos celulares e circuitos neurais da SD. Em vez disso, em preparações in vitro, onde as condições ambientais podem ser controlados são necessários. Aqui, é importante reconhecer as limitações de fatias aguda e vantagens distintas das culturas fatia do hipocampo. Fatias de cérebro agudo não pode revelar mudanças sutis na sinalização imune desde a preparar as fatias sozinho dispara: pró-inflamatórias mudanças que nos últimos dias, o comportamento epileptiforme devido aos altos níveis de tensão de oxigênio necessário para vitalizar as fatias, e lesões celulares irreversíveis nos centros fatia anóxica. Em contraste, nós examinamos sinalização imunológico maduro em culturas fatia hipocampal desde a culturas intimamente paralelo, a sua contrapartida na vivo com a função trisynaptic madura; mostrar astrócitos quiescente, microglia e os níveis de citocinas, e SD é facilmente induzida em uma preparação não anestesiados. Além disso, as fatias são de longa duração e SD pode ser induzida em dias consecutivos sem lesão, fazendo esta preparação o único meio de data-capazes de modelar as conseqüências de doenças crônicas neuroimunes SD, e enxaqueca crônica, assim, talvez. Usamos técnicas eletrofisiológicas e não-invasivo para medir a imagem de células neuronais e as funções de circuito coincidente com SD. Neural variáveis ​​expressão imune gene são medidos com qPCR triagem, arrays qPCR, e, mais importante, o uso de pré-amplificação de cDNA para detecção de alvos ultra-baixo nível, como interferon-gama usando todo, regionais ou específicos de células avançado (via laser microscopia de dissecção) amostragem. Sinalização em cascata de citocinas é avaliada com metas fosfoproteína multiplexados relacionadas com a expressão de genes e alterações fosfoproteína confirmada através de células específicas de imunocoloração. Estratégias farmacológicas e siRNA são utilizados para imitar e modular de sinalização imune SD.

Watch this Scientific Journal Video about Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro at JoVE.com

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Enxaqueca e sua transformação para enxaqueca crônica são um fardo imenso de saúde que precisam de opções de tratamento melhoraram. Procuramos definir como sinalização imune neural modula a suscetibilidade à enxaqueca, modelada<em&gt; In vitro</em&gt; Usando a depressão alastrante em culturas fatia do hipocampo, como um meio para desenvolver novos alvos terapêuticos.

Modelagem Neural Sinalização Imune de migrânea episódica e crônica Usando a depressão alastrante In Vitro</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

modeling-neural-immune-signaling-episodic-chronic-migraine-using

Research

JoVE Journal

Neuroscience

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

19.9K visualizações.  The University of Chicago Medical Center. Enxaqueca e sua transformação para enxaqueca crônica são um fardo imenso de saúde que precisam de opções de tratamento melhoraram. Procuramos definir como sinalização imune neural modula a suscetibilidade à enxaqueca, modelada In vitro Usando a depressão alastrante em culturas fatia do hipocampo, como um meio para desenvolver novos alvos terapêuticos.

Vídeo: Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

A Técnica de Janela crânio-fino para Crônica de dois fótons In vivo Imaging da Microglia murino em Modelos de Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Neurociência

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Combinando Behavioral Endocrinologia e Economia Experimental: Decisão da testosterona e Social Fazendo

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

Implante cirúrgico de crônicas Eletrodos Neurais para registrar a atividade única Unidade e Sinais eletrocorticográfica

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

Concepção e montagem de um Microdrive Ultra-leve motorizada para doenças crônicas Recordings Neurais em Pequenos Animais

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modelagem astrocitoma Patogênese<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Usando Cortical astrócitos ou células-tronco neurais de condicional, Geneticamente Modificadas Ratos

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Citometria de Fluxo protocolos para a superfície e intracelular Antigen análises de tipos de células neurais

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

In Vitro Modelagem de Cancerous Neural Invasion: o gânglio Modelo de raiz dorsal

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Cultivo In Vivo-gosto murino astrócitos usando o protocolo AWESAM rápida, simples e barato

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Crônica implantação de matriz Electrocorticographic todo-cortical no sagui comum

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

Combinando Comportamento e EEG para Estudar os Efeitos da Meditação Mindfulness na Memória Episódica

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...