Name: genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration
Uploaded: 2014-03-17T17:45:00
Description: Watch this Scientific Journal Video about Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration at JoVE.com

We thank N. Schwedhelm-Domeyer for excellent technical assistance, C. Hammer&#160;and S. Papiol for help with statistical analyses. Our work is funded by the Max Planck Society, the Deutsche Forschungsgemeinschaft, the Center for Nanoscale Microscopy, and Molecular Physiology of the Brain (CNMPB), G&#246;ttingen, Germany and by the GGNB Junior Group Stipend of the University of G&#246;ttingen.

CGNs can be prepared either from postnatal day (P) 5 mouse pups or P6 rat pups. We follow a protocol, described by Bilimoria and colleagues, which uses a mitotic inhibitor to select for postmitotic CGNs13.
Ethics statement: 
All experiments involving live animals have been conducted according to the animal protocol approved by the &#34;Verbraucherschutz und Lebensmittelsicherheit&#34; of Lower Saxony, Germany.
In vitro assay:
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<ol>
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Advantages and limitations of the described in vitro and in vivo methods:
Cultured CGNs from mouse and rats are equally well suited for morphological analyses. Owing to the bigger size of a rat cerebellum, the yield of CNGs from rat pups exceeds that of mouse pups 3-4x. Aside from CGNs, cortical and hippocampal neurons can be used as culture system as well. The calcium phosphate method results in a low (0.01-5%) transfection efficiency, which is desired to analyze the morphol...

The cerebellum is an excellent system to study mechanisms of axon growth and migration. The cerebellum has been the subject of anatomical studies since the dawn of neuroscience1. Modern microscopy and immunohistochemical techniques have significantly expanded and refined the initial discoveries by Santiago, Ramon, and&#160;Cajal2-4. Mouse genetics and molecular studies uncovered essential growth and transcription factors in the control of cerebellar development, which led to greater understanding of crucial events required for proper wiring of different types of neurons including cerebellar granule neurons (CGNs)5-7.
The cerebellum is a derivative of rhombomere 1 of the developing hindbrain8.&#160; The rhombic lip, which is part of the roof of the 4th ventricle, gives rise to cerebellar granule neuron progenitors, which will constitute the most numerous neuronal population in the adult cerebellum9. Following rostral migration, they settle in the cerebellar anlage. Here, mitosis of granule neuron precursors leads to the dramatic expansion of the external granular layer (EGL), which takes place postnatally in rodents. From the EGL, neurons start migrating inward through the molecular layer (ML), past the Purkinje cell layer to ultimately take up residence in the internal granular layer (IGL2). During this migratory process, they acquire a bipolar shape with two axons extending into the ML. Upon further migration, the cell body migrates away from the axons and the two processes fuse to form one bifurcated, T-shaped axon10. Subsequently, these axons fasciculate and are referred to as parallel fibers. Having settled in the IGL, CGNs grow dendrites, which form dendritic claws to establish synapses with mossy fibers. To examine fundamental processes in the developing cerebellum, a combined in vitro and in vivo approach allows for reliable results and conclusions.
CGNs are not only the most numerous neurons of the cerebellum but of the entire brain and can be cultured to high purity11-13. In culture, this highly homogeneous neuronal population becomes rapidly postmitotic and acquires a polar morphology with easily identifiable axons and dendrites. Cultured CGNs have proven to be extremely useful to study various aspects of neuronal development including progenitor proliferation, differentiation, axonal and dendrite development, neuronal migration, apoptosis and electrophysiological properties (14-19 and many others). The use of genetic manipulation has expanded the versatility of cultured CGNs and allowed for further mechanistic insight into the aforementioned events. Transfection of cultured neurons using low-efficiency calcium phosphate or lipophilic methods followed by immunocytochemistry with polarity markers or software-supported analysis facilitates the assessment of e.g. the morphology of individual neurons in a dense neuronal culture. With this approach, the role of proteins of interest in axon or dendrite growth can be studied20-25,26-28. This culture system however is less useful to analyze neuronal migration as migration is very limited in high-density cultures and would require cocultures. The in vitro analysis of axon and dendrite growth also allows for the examination of interconnected proteins of a signaling pathway using combinations of RNA interference (i), over-expression or small molecules.
To establish the relevance of the protein of interest in axon and dendrite growth regulation or neuronal migration, the in vivo electroporation (IVE) technique allows for the analysis in the developing cerebellar cortex. Owing to the fact that cerebellar development in rodents extends way into the first two postnatal weeks, the cerebellum represents an accessible brain structure for genetic manipulations to examine developing axons and dendrites, neuronal migration, synaptogenesis and apoptosis20-24,29,30,26,27,31-34. In addition, this model system is also useful for other aspects of neuronal development that require the intact cerebellar cortex such as axon pathfinding, wiring and connectivity of neurons and neuron-glia interactions Taken together, this protocol provides in vitro and in vivo techniques to tackle a complementary approach regarding neuronal morphogenesis and migration.

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			<td style="width:168px;">Comments (optional)</td>
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			<td height="43" style="height:43px;width:160px;">ECM 830 and tweezertrodes</td>
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			<td height="64" style="height:64px;width:160px;">Epifluorescence microscope and camera</td>
			<td style="width:125px;">Nikon</td>
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			<td height="43" style="height:43px;width:160px;">SP2 confocal microscope</td>
			<td style="width:125px;">Leica</td>
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			<td height="22" style="height:22px;width:160px;">ImageJ</td>
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			<td height="22" style="height:22px;width:160px;">Imaris 7.4.2</td>
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			<td height="43" style="height:43px;width:160px;">GraphPad Prism</td>
			<td style="width:125px;">GraphPad Software, Inc.</td>
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			<td height="22" style="height:22px;width:160px;">MS Excel</td>
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			<td style="width:88px;">70.760.502</td>
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			<td height="43" style="height:43px;width:160px;">Microlance 3 needle, 30 gauge</td>
			<td style="width:125px;">BD</td>
			<td style="width:88px;">302200</td>
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			<td height="43" style="height:43px;width:160px;">50 &#181;l gastight Syringe 1705</td>
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			<td height="43" style="height:43px;width:160px;">Glass coverslips&#160;</td>
			<td style="width:125px;">Thermo Scientific Menzel Glaeser</td>
			<td>CB00120RA1</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

To analyze the morphology of CGNs in response to different culturing conditions, we transfected the neurons on DIV 0 as described above. After transfection, we placed one set of neurons into full medium (BME, 10% calf serum, 2 mM PSG, 25 mM KCl) and another set into minimal medium containing insulin (BME, 25 mM glucose, 2 mM PSG, 10 &#956;g/ml insulin). We subjected the neurons to immunocytochemistry using the GFP antibody at DIV 1, 2, and 3, followed by measuring axons and dendrites for set 1 and axons only for set 2. O...

Watch this Scientific Journal Video about Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration at JoVE.com

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.

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 ...

Research

JoVE Journal

Neuroscience

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

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The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Laser Nanosurgery of Cerebellar Axons In Vivo

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Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

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The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

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

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 ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...