Agradecemos a Dan McWhorter para narrar o protocolo no vídeo. Este trabalho é suportado pelo NIH Grant 5R01NS062182, uma doação da Federação Americana de Investigação em Envelhecimento e fundos institucionais concedido a HTG.

<ol>
	<li>Ghashghaei, H. T., Lai, C., Anton, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+migration+in+the+adult+brain:+are+we+there+yet.">Neuronal migration in the adult brain: are we there yet.</a> Nat. Rev. Neurosci. 8, 141-151 (2007).</li><li>Valiente, M., Marin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+migration+mechanisms+in+development+and+disease.">Neuronal migration mechanisms in development and disease.</a> Curr. Opin. Neurobiol. 20, 68-78 (2010).</li><li>Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+neocortex:+a+perspective+from+developmental+biology.">Evolution of the neocortex: a perspective from developmental biology.</a> Nat. Rev. Neurosci. 10, 724-735 (2009).</li><li>Jaglin, X. H., Chelly, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubulin-related+cortical+dysgeneses:+microtubule+dysfunction+underlying+neuronal+migration+defects.">Tubulin-related cortical dysgeneses: microtubule dysfunction underlying neuronal migration defects.</a> Trends Genet. 25, 555-566 (2009).</li><li>Carro, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptional+network+for+mesenchymal+transformation+of+brain+tumours.">The transcriptional network for mesenchymal transformation of brain tumours.</a> Nature. 463, 318-325 (2010).</li><li>Wu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+guidance+of+neuronal+migration+in+the+olfactory+system+by+the+protein+Slit.">Directional guidance of neuronal migration in the olfactory system by the protein Slit.</a> Nature. 400, 331-336 (1999).</li><li>Hu, H., Tomasiewicz, H., Magnuson, T., Rutishauser, U., U, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+polysialic+acid+in+migration+of+olfactory+bulb+interneuron+precursors+in+the+subventricular+zone.">The role of polysialic acid in migration of olfactory bulb interneuron precursors in the subventricular zone.</a> Neuron. 16, 735-743 (1996).</li><li>Shapiro, E. M., Gonzalez-Perez, O., Garcia-Verdugo, M. a. n. u. e. l., Alvarez-Buylla, J., &amp;amp, A., Koretsky, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+the+migration+of+neuronal+precursors+generated+in+the+adult+rodent+brain.">Magnetic resonance imaging of the migration of neuronal precursors generated in the adult rodent brain.</a> Neuroimage. , (2006).</li><li>Vreys, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+visualization+of+endogenous+neural+progenitor+cell+migration+along+the+RMS+in+the+adult+mouse+brain:+validation+of+various+MPIO+labeling+strategies.">MRI visualization of endogenous neural progenitor cell migration along the RMS in the adult mouse brain: validation of various MPIO labeling strategies.</a> Neuroimage. 49, 2094-2103 (2010).</li><li>Davenne, M., Custody, C., Charneau, P., Lledo, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+migrating+neurons+in+the+mammalian+forebrain.">In vivo imaging of migrating neurons in the mammalian forebrain.</a> Chem. Senses. 30, 115-116 (2005).</li><li>Mirzadeh, Z., Doetsch, F., Sawamoto, K., Wichterle, H., Alvarez-Buylla, 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+subventricular+zone+en-face:+wholemount+staining+and+ependymal.">The subventricular zone en-face: wholemount staining and ependymal.</a> J. Vis. Exp. , (2010).</li><li>Shen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+SVZ+stem+cells+lie+in+a+vascular+niche:+a+quantitative+analysis+of+niche+cell-cell+interactions.">Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions.</a> Cell Stem Cell. 3, 289-300 (2008).</li><li>Tavazoie, 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+specialized+vascular+niche+for+adult+neural+stem+cells.">A specialized vascular niche for adult neural stem cells.</a> Cell Stem Cell. 3, 279-288 (2008).</li><li>Mirzadeh, Z., Merkle, F. T., Soriano-Navarro, M., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Stem+Cells+Confer+Unique+Pinwheel+Architecture+to+the+Ventricular+Surface+in+Neurogenic+Regions+of+the+Adult+Brain.">Neural Stem Cells Confer Unique Pinwheel Architecture to the Ventricular Surface in Neurogenic Regions of the Adult Brain.</a> Cell Stem Cell. 3, 265-278 (2008).</li><li>Polleux, F. &. a. m. p. ;. a. m. p., Ghosh, 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+slice+overlay+assay:+a+versatile+tool+to+study+the+influence+of+extracellular+signals+on+neuronal.">The slice overlay assay: a versatile tool to study the influence of extracellular signals on neuronal.</a> Sci. STKE. , L9-L9 (2002).</li><li>Murase, S. &. a. m. p. ;. a. m. p., Horwitz, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deleted+in+colorectal+carcinoma+and+differentially+expressed+integrins+mediate+the+directional+migration+of+neural+precursors+in+the+rostral+migratory+stream.">Deleted in colorectal carcinoma and differentially expressed integrins mediate the directional migration of neural precursors in the rostral migratory stream.</a> J. Neurosci. 22, 3568-3579 (2002).</li><li>Suzuki, S. O. &. a. m. p. ;. a. m. p., Goldman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+cell+populations+in+the+early+postnatal+subventricular+zone+take+distinct+migratory+pathways:+a+dynamic+study+of+glial+and+neuronal+progenitor+migration.">Multiple cell populations in the early postnatal subventricular zone take distinct migratory pathways: a dynamic study of glial and neuronal progenitor migration.</a> J. Neurosci. 23, 4240-4250 (2003).</li><li>Ghashghaei, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+neuregulin-ErbB4+interactions+on+the+proliferation+and+organization+of+cells+in+the+subventricular+zone.">The role of neuregulin-ErbB4 interactions on the proliferation and organization of cells in the subventricular zone.</a> Proc. Natl. Acad. Sci. U. S. A. 103, 1930-1935 (2006).</li><li>Khodosevich, K., Seeburg, P. H., Monyer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+signaling+pathways+in+migrating+neuroblasts.">Major signaling pathways in migrating neuroblasts.</a> Front Mol. Neurosci. 2, 7-7 (2009).</li><li>Jacquet, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+neuronal+proliferation,+migration+and+differentiation+in+the+postnatal+brain+using+equine+infectious+anemia+virus-based+lentiviral+vectors.">Analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors.</a> Gene Ther. 16, 1021-1033 (2009).</li></ol>

Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pela North Carolina State University e da sua Faculdade de Medicina Veterinária. Não há conflitos de interesse declarados.

Migração neuronal na RMS é um componente essencial da neurogênese pós-natal no bulbo olfatório 1. Migração através do RMS ocorre em um plano tangente à superfície do cérebro. Neuroblastos tangencialmente migração são distintas das células migram radialmente com base na localização de sua fonte progenitor, bem como o destino divergentes de seus produtos finais neuronal 1, 2, 3. A população relativamente pura de células tangencialmente migrando na RMS pós-natal torna esta região anatômica definível uma ótima plataforma para estudar os mecanismos de migração tangencial. Decifrar os mecanismos celulares e moleculares da migração neuronal é fundamental para a compreensão de muitas doenças do neurodesenvolvimento (por exemplo, Ref. 4). Esse conhecimento pode facilitar ainda mais a identificação de mecanismos que sustentam a formação e propagação de tumores cerebrais (por exemplo, Ref. 5), e é essencial para direcionar neuroblastos reprogramado para locais de lesão ou neurodegeneração em futuras estratégias de substituição neuronal. Apesar dos progressos enormes no últimos anos, o atual entendimento dos mecanismos genéticos e moleculares subjacentes à migração neuronal, ea sua relação com eventos específicos subcelulares permanece fragmentado. Grande parte da dificuldade em fazer headways nessa busca é devido à dependência da maioria dos estudos sobre migração de fixação do tecido e imuno-histoquímica, que têm sido usados ​​para fazer inferências sobre os mecanismos indiretos da migração na RMS e outras correntes migratórias no cérebro embrionário. A abordagem &quot;fixo&quot; é sub-ótima, uma vez que só fornece um instantâneo de uma célula, enquanto a resolução temporal é um requisito fundamental para a compreensão de um processo altamente dinâmico como a migração celular. Alguns estudos criteriosos têm abordado os mecanismos de migração celular in vitro usando um método pelo qual explantes, semelhantes aos utilizados em nosso protocolo, são semeadas em substratos de plástico ou de vidro revestido com componentes da matriz extracelular (por exemplo, Refs. 6, 7). Embora os estudos utilizando este teste ter identificado uma série de mecanismos com funções claras em matéria de migração, a relevância de seus resultados para as condições in vivo não é clara. Mais recentemente, direta in vivo de imagem de neuroblastos através do uso de microscopia confocal desfibrado e RM foram realizados 8-10. No entanto, tais técnicas rendimento imagens de baixa resolução e, assim, são insuficientes na avaliação subcelular da migração neuronal. Outra abordagem, a preparação toda montagem criada pelo grupo Alvarez-Buylla 11, tem sido utilizada para gerar idéias fisiológicas importantes na regulação do nicho subependimal de células-tronco 12-14. No entanto, esta abordagem elegante, não pode ser utilizada para estudar a migração neuroblastos na RMS. Relatórios anteriores, utilizando slices organotípicas para a análise da migração nos 15 cérebro embrionário e pós-natal da RMS foram publicados 16-18. Temos aperfeiçoado esta abordagem e fornecer aqui o protocolo que permite a gravação de alto rendimento de orientação, velocidade e dinâmica intra-e intercelular da migração na RMS em alta resolução. O protocolo apresentado coloca alguns desafios que requerem prática, paciência e tempo dedicado tanto a preparação ea análise dos resultados. As sugestões a seguir irão ajudar com a realização deste protocolo: <ul><li> Em nossa experiência, os camundongos na faixa etária de P1 a P10 oferecem os melhores resultados. Correspondência de idade entre o doador eo receptor cérebros também melhora a reprodutibilidade dos experimentos. </li><li> Como a maioria dos reagentes e placas de cultura devem ser preparados na hora, nossa fatia de ensaio requer várias horas de preparação ininterrupta e de imagem em um único dia. Uma vez que os cérebros são colhidas passos conseqüente deve ser realizada o mais rápido possível. </li><li> Entre decapitação e preparação de fatias, todas as etapas devem ser realizadas com reagentes gelada, e manipulação de tecidos devem ser mantidas a um mínimo, a fim de evitar interrupções anatômica. </li><li> As ferramentas utilizadas para estas dissecações deve ser estéril e reservado para manipulações tecido vivo só. Nunca utilizar ferramentas que já foram em contato com fixadores tais como formaldeído. </li><li> As fatias são geralmente viáveis ​​por até 36 horas, com uma queda visível na velocidade de migração e orientação entre 24 e 36 horas. Cuidado com essa limitação no planejamento de suas experiências e análises. </li></ul> Ao nosso conhecimento, o ensaio apresentado fatia organotípicas oferece a melhor abordagem atual para decifrar os mecanismos genéticos e moleculares da migração neuroblastos do ZEE para o OB. Dada a informação emergente em relação à expressão de sinalização redes na RMS 19, futuros experimentos para vincular os identificadoscascatas de sinalização para a migração neuronal será maciçamente beneficiar do nosso ensaio. Nosso protocolo facilitará a identificação de moléculas de sinalização candidato que pode regular a migração neuroblasto autonomamente, ao mesmo tempo permitindo que os pesquisadores para avaliar o papel de quimiocinas e fatores secretados na regulação da migração de uma forma não-autônoma, como temos demonstrado recentemente 18, 20 . Nossa técnica apresentada pode ser facilmente modificado para o estudo do transplante de células-tronco, conseqüência dendríticas / axonal, e toda uma série de outros tópicos no desenvolvimento de neurobiologia e neurogênese adulta.

an organotypic slice assay for high-resolution time-lapse imaging of neuronal migration in the postnatal brain

Neurogênese no cérebro pós-natal depende da manutenção de três eventos biológicos: a proliferação de células progenitoras, a migração dos neuroblastos, bem como diferenciação e integração de novos neurônios em circuitos neurais existentes. Para a neurogênese pós-natal no bulbo olfatório, esses eventos são segregados dentro de três domínios anatomicamente distintas: a proliferação em grande parte ocorre na zona subependimal (ZEE) dos ventrículos laterais, migrando neuroblastos atravessar o fluxo migratório rostral (RMS), e novos neurônios diferenciar e integração dentro do bulbo olfatório (OB). Os três domínios servem como plataformas ideais para estudar os mecanismos celulares, moleculares e fisiológicos que regulam cada um dos eventos biológicos distintamente. Este artigo descreve um ensaio fatia organotípicas otimizado para o tecido cerebral pós-natal, em que as condições extracelular imitam de perto as em ambiente vivo para a migração de neuroblastos. Nós mostramos que o nosso ensaio prevê movimento uniforme, orientado, e rápida de neuroblastos dentro da RMS. Este ensaio será muito adequado para o estudo da regulação celular autónomos e não-autônoma da migração neuronal utilizando cruz-transplante abordagens de ratos em diferentes backgrounds genéticos.

Watch this Scientific Journal Video about An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain at JoVE.com

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

Este protocolo descreve um ensaio fatia organotípicas otimizado para o cérebro pós-natal e imagens de alta resolução de lapso de tempo de migração neuroblastos no fluxo migratório rostral.

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 ...

an-organotypic-slice-assay-for-high-resolution-time-lapse-imaging

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.9K visualizações.  North Carolina State University. Este protocolo descreve um ensaio fatia organotípicas otimizado para o cérebro pós-natal e imagens de alta resolução de lapso de tempo de migração neuroblastos no fluxo migratório rostral.

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

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

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 ...

Neurociência

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Lapso de tempo de imagens ao vivo de relação clonal células progenitoras neurais no desenvolvimento Zebrafish Encéfalo anterior

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Multieletrodos Recordings Array of Avalanches Neuronal em culturas organotípicas

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Responses imagem Neuronal em Preparações Slice of Organ Vomeronasal Expressando um sensor de cálcio geneticamente codificado

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

De alta resolução funcionais Métodos ressonância magnética para Midbrain Humanos

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

Lapso de tempo de imagem de migração neuroblastos em fatias agudas do cérebro anterior Rato Adulto

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

In vivo Eletroporação pós-natal e tempo-lapso de neuroblasto Migração na aguda fatias do cérebro do rato

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Manipulação genética de Cerebelar Granule Neurônios<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Estudar Neuronal Morfologia e Migração

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

Alta resolução<em&gt; In Vivo</em&gt; Manual Protocolo Segmentação para hipocampo humano subcampos Utilizando Ressonância Magnética 3T

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Ex vivo de imagens de pós-natal Cerebelar Granule migração celular Usando confocal Macroscopia

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...