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

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

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.

Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain

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

Подготовка срезов мозга мыши к покадровой визуализации для определения клеточной динамики

Для начала усыпьте усыпленную мышь с соответствующими плазмидами. Для приготовления чашки для культур добавьте в стеклянную чашку 1,9 миллилитра питательной среды и осторожно положите на нее вкладыш для клеточной культуры. Затем добавьте на фильтр 500 микролитров питательной среды и положите его на лед.Извлеките эмбрионы и поместите их в чашку Петри, содержащую ледяной PBS. Под микроскопом отбирают эмбрионы на основе экспрессии зеленого флуоресцентного белка или других флуоресцентных маркеров. После препарирования мозга поместите его в расплавленный агарозный гель, взятый в криоформу, и прокатайте его несколько раз.Как только агароза застынет при комнатной температуре, положите ее на лед. С помощью вибрирующего микротома надрежьте мозги коронально до толщины 350 микрометров и расположите их на фильтре. Затем удалите 600 микролитров питательной среды как изнутри, так и снаружи фильтрующего стакана.Через пять минут добавьте 100 микролитров свежей питательной среды внутрь вкладыша для клеточной культуры. Для цейтраферной съемки можно получить изображения с интервалом около 10Z с помощью 20-кратного объектива с большим рабочим расстоянием. Анализ покадровых изображений мозга мыши позволил определить траектории EGFP-положительных мигрирующих клеток и RFP-положительных мигрирующих клеток от 2 до 21 часа.

Research

JoVE Journal

Neuroscience

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

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

In Utero Electroporation for Time-Lapse Imaging of Neuronal Migration in Embryonic Mouse Brain

Внутриутробная электропорация для покадровой визуализации миграции нейронов в эмбриональном мозге мыши