preparation of mouse brain slices for time-lapse imaging to determine cellular dynamics

Gelişmekte Olan Fare Beyninde Nöronal ve Glial Hücre Dinamiğinin Görselleştirilmesi

During the development of the cerebral cortex, (apical) radial glia in the pallial ventricular zone (VZ) lining the lateral ventricle produce first neurons and then glial progenitors with some overlapping period1. Neurons are also generated from intermediate progenitors or basal radial glia in the subventricular zone (SVZ) adjacent to the VZ, both of which originate from the (apical) radial glia2,3. In mice, radial glial cells produce only neurons on embryonic day (E) 12-14, both neurons and glial progenitors on E15-16, and glial progenitors from E17 onward4. The major population of glial progenitors generated during these embryonic stages preferentially differentiates into astrocytes, although some cells also differentiate into oligodendrocytes5. Neurons and astrocyte progenitors generated at these stages migrate toward the brain surface and enter the cortical plate (future cortical gray matter). Neuronal migration from the VZ to the cortical plate occurs in multiple phases. Neurons first adopt a multipolar morphology just above the multipolar cell accumulation zone (MAZ), overlapping the SVZ or intermediate zone, where they vigorously extend and retract multiple thin processes and slowly migrate (multipolar migration)6,7. After approximately 24 h, neurons transform into a bipolar morphology, extending a thick leading process toward the brain surface and a thin trailing process backward, and migrate linearly toward the brain surface using a radial process extending from the radial glia to the pial surface as a scaffold, which is called locomotion mode2,8. Because neurons in locomotion mode always reach the outermost surface of the cortical plate, passing through their predecessors just under the marginal zone, neurons are aligned in a birthdate-dependent inside-out manner in the cortical plate9,10,11.
In contrast, astrocyte progenitors migrate rapidly to the intermediate zone and cortical plate, with frequent directional changes. This migratory behavior is completely different from neuronal migration and is called erratic migration5. Astrocyte progenitors also migrate along blood vessels in a process called blood vessel-guided migration. Astrocyte progenitors switch between these migration modes and reach the cortical plate5,12. Although the positioning of astrocytes is not strictly determined by their date of production, a mild tendency for early-born astrocytes to settle in the superficial part of the cortical plate has been observed5. Interestingly, astrocytes that settle in the cortical plate are generated in embryonic stages and eventually differentiate into protoplasmic astrocytes, whereas postnatally generated astrocytes do not migrate actively, remain in the white matter, and differentiate into fibrous astrocytes5. How this stage-dependent specification of astrocytic subtypes occurs remains unclear.
A growing number of genes involved in neuronal migration have been identified, including those involved in neurodevelopmental and psychiatric disorders13,14. Therefore, it is crucial to elucidate the effects of mutations in these genes on the behavior of migrating neurons. As previously mentioned, neuronal migration occurs in multiple phases. Time-lapse observations can directly determine the phase that is mainly affected (cell cycle exit, multipolar-bipolar transition, migration speed of locomotion, etc.). However, the molecular mechanisms underlying the specification, migration, and positioning of astrocytes remain largely unknown. Given that astrocytes play crucial roles in synaptogenesis15 and blood-brain barrier formation during brain development16, developmental defects in astrocytes may result in neurodevelopmental disorders. Time-lapse studies on astrocyte progenitors may clarify these molecular mechanisms and their relationship with mental illness.
This protocol provides a method for time-lapse observation of cortical VZ-derived cells. A similar video protocol for the observation of neuronal migration has already been published17. Here, we describe the method for both migrating neurons and astrocyte progenitors. To label these cells with fluorescent proteins, such as green and red fluorescent proteins (GFP and RFP), plasmid mixtures containing appropriate components are introduced into the cortical VZ by in utero electroporation at appropriate stages18,19,20,21. The manipulated embryos are removed at the desired stages, and the brains are sliced and used for time-lapse observations using a laser scanning microscope. The migration speed, direction, and other behaviors, which are never addressed using fixed brain samples, can be examined using this method. Using in utero electroporation, expression, and knockdown/knockout vectors can be easily transferred concomitantly with fluorescent protein vectors, enabling us to conduct gain-of-function and loss-of-function studies of specific genes.

Yazar Spotlight: Gelişmekte Olan Beyinde Hücre Göçünü ve Gen Rollerini Keşfetmek

Embriyonik Fare Beyin Dilimlerinde Göç Eden Nöronların ve Glial Progenitörlerin Hızlandırılmış Görüntülenmesi

During the development of the cerebral cortex, neurons and gray cells are derived from the ventricular zone and migrate to other part of the brain surface. Many genes are involved in this process, including those responsible for neurodevelopmental and psychiatric disorders. We are addressing their functions on the three behaviors in this process.Recently, we have reported that astrocyte progenitors assume two distinct migration modes:Erratic and blood-vessel guided migration. These observations were made using a combination of serotype-specific labeling and time observation methods introduced in this video. To level cells, we utilize in utero electroporation system, which we developed to visualize individual cells with a high signal to noise ratio.This in vivo gene transfer system also enable us to easily perform gain of function or loss of function experiments on the given genes by electroporation of their expression or neutron vectors. Using this experimental system, we aim to observe the cell behaviors of neurons, glial, and blood vessels, and elucidates the crosstalk between them. The findings from these studies will contribute to understanding the pathogenesis of neurodevelopmental disorders.

Hücresel dinamikleri belirlemek için hızlandırılmış görüntüleme için fare beyin dilimlerinin hazırlanması

Başlamak için, uygun plazmitlerle uygulanan ötenazi fareyi elde edin. Kültür kabı hazırlığı için, cam bazlı bir tabağa 1.9 mililitre kültür ortamı ekleyin ve üzerine dikkatlice bir hücre kültürü eki koyun. Daha sonra filtreye 500 mikrolitre kültür ortamı ekleyin ve buzun üzerine yerleştirin.Embriyoları çıkarın ve buz gibi soğuk PBS içeren bir Petri kabına yerleştirin. Mikroskop altında, yeşil floresan proteini veya diğer floresan belirteçlerin ifadesine dayalı olarak embriyoları seçin. Beyni inceledikten sonra, bir kriyo kalıbına alınan erimiş agaroz jele koyun ve birkaç kez yuvarlayın.Agaroz oda sıcaklığında katılaştığında buzun üzerine koyun. Titreşimli bir mikrotom kullanarak, beyinleri koronal olarak 350 mikrometre kalınlığa kadar dilimleyin ve filtrenin üzerine yerleştirin. Ardından filtre kabının hem içinden hem de dışından 600 mikrolitre kültür ortamını çıkarın.Beş dakika sonra, hücre kültürü ekinin içine 100 mikrolitre taze kültür ortamı ekleyin. Hızlandırılmış görüntüleme için, uzun çalışma mesafeli 20X lens kullanarak yaklaşık 10Z yığın görüntüleri elde edin. Fare beyninin hızlandırılmış görüntülerinin analizi, EGFP pozitif göç eden hücrelerin ve RFP pozitif göç eden hücrelerin yörüngelerini iki ila 21 saat arasında sağladı.

preparation-mouse-brain-slices-for-time-lapse-imaging-to-determine

Research

JoVE Journal

Neuroscience

Preparation of Mouse Brain Slices for Time-Lapse Imaging to Determine Cellular Dynamics

44 Görüntüleme.  Keio University School of Medicine. Başlamak için, uygun plazmitlerle uygulanan ötenazi fareyi elde edin. Kültür kabı hazırlığı için, cam bazlı bir tabağa 1.9 mililitre kültür ortamı ekleyin ve üzerine dikkatlice bir hücre kültürü eki koyun. Daha sonra filtreye 500 mikrolitre kültür ortamı ekleyin ve buzun üzerine yerleştirin.Embriyoları çıkarın ve buz gibi soğuk PBS içeren bir Petri kabına yerleştirin. Mikroskop altında, yeşil floresan proteini veya diğer floresan belirteçlerin ifad...

Video: Hücresel dinamikleri belirlemek için hızlandırılmış görüntüleme için fare beyin dilimlerinin hazırlanması

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...