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Development of cerebellar neurons and glias revealed by in utero electroporation: Golgi-like labeling of cerebellar neurons and glias.

ABSTRACT: Cerebellar cortical functions rely on precisely arranged cytoarchitectures composed of several distinct types of neurons and glias. Studies have indicated that cerebellar excitatory and inhibitory neurons have distinct spatial origins, the upper rhombic lip (uRL) and ventricular zone (VZ), respectively, and that different types of neurons have different birthdates. However, the spatiotemporal relationship between uRL/VZ progenitors and their final phenotype remains poorly understood due to technical limitations. To address this issue, we performed in utero electroporation (IUE) of fluorescent protein plasmids using mouse embryos to label uRL/VZ progenitors at specific developmental stages, and observed labeled cells at maturity. To overcome any potential dilution of the plasmids caused by progenitor division, we also utilized constructs that enable permanent labeling of cells. Cerebellar neurons and glias were labeled in a Golgi-like manner enabling ready identification of labeled cells. Five types of cerebellar neurons, namely Purkinje, Golgi, Lugaro and unipolar brush cells, large-diameter deep nuclei (DN) neurons, and DN astrocytes were labeled by conventional plasmids, whereas plasmids that enable permanent labeling additionally labeled stellate, basket, and granule cells as well as three types of glias. IUE allows us to label uRL/VZ progenitors at different developmental stages. We found that the five types of neurons and DN astrocytes were labeled in an IUE stage-dependent manner, while stellate, basket, granule cells and three types of glias were labeled regardless of the IUE stage. Thus, the results indicate the IUE is an efficient method to track the development of cerebellar cells from uRL/VZ progenitors facing the ventricular lumen. They also indicate that while the generation of the five types of neurons by uRL/VZ progenitors is regulated in a time-dependent manner, the progenitor pool retains multipotency throughout embryonic development.

SUBMITTER: Kita Y

PROVIDER: S-EPMC3720936 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators (GECIs), have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson's disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (<5 kb). In utero electroporation (IUE) offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s, or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat (ITR)-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5-14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal activity in the rat brain.

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Development of cerebellar neurons and glias revealed by in utero electroporation: Golgi-like labeling of cerebellar neurons and glias.

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