This work was supported by the University of Basel, Department of Biomedicine, and the Swiss National Science Foundation (31003A-116624).

1. Setting up Organotypic Cerebellar Slice Cultures
Cerebellar slice cultures are prepared from postnatal day 8 P(8) mouse pups using the static incubation method 10. In our laboratory, we use B6CF1 mice. In some experiments also transgenic mice were used which express EGFP selectively in Purkinje cells. The preparation of slice cultures takes approximately 30 minutes per mouse pup, i.e. 3 hours for a litter of 6 mouse pups. All steps are carried out under sterile conditions in a lami...

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Animal experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were reviewed and permitted by Swiss authorities. The authors have nothing to disclose.

The methods presented here allow to study Purkinje cell dendritic development in organotypic cerebellar slice cultures and to quantitatively evaluate Purkinje cell dendritic expansion by measuring dendritic tree size and the number of dendritic branch points. Of course, a more extensive and sophisticated quantitative analysis of Purkinje cell dendrites is possible, e.g. by determining total dendritic length, performing a Sholl analysis or determining the fractal dimension of the dendritic tree. For this type of analysis ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>
			 Tissue culture Inserts </td>
			<td>
			 Millipore </td>
			<td>
			 PICM 03050 PICM ORG50 </td>
			<td>
			 The PICMORG50 inserts have a low rim and allow viewing of live cultures at a microscope </td>
		</tr>
		<tr>
			<td>
			 MEM </td>
			<td>
			 Gibco, Invitrogen </td>
			<td>
			 11012044 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Glutamax 1 </td>
			<td>
			 Gibco, Invitrogen </td>
			<td>
			 35050038 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Basal Medum Eagle </td>
			<td>
			 Gibco, Invitrogen </td>
			<td>
			 41010026 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Horse serum </td>
			<td>
			 Gibco, Invitrogen </td>
			<td>
			 26050070 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Phorbol 12-myristate 13-acetate (PMA) </td>
			<td>
			 Tocris </td>
			<td>
			 1201 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 (RS)-3,5-Dihydroxy-phenylglycine (DHPG) </td>
			<td>
			 Tocris </td>
			<td>
			 0342 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Rabbit anti-calbindin D-28K </td>
			<td>
			 Swant </td>
			<td>
			 CB38 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Anti NeuN, clone A60 </td>
			<td>
			 Chemicon, Millipore </td>
			<td>
			 MAB377 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Rabbit anti-GFP </td>
			<td>
			 Abcam </td>
			<td>
			 Ab290 </td>
			<td>
			 </td>
		</tr>
	</tbody>
</table>

the analysis of purkinje cell dendritic morphology in organotypic slice cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

Watch this Scientific Journal Video about The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures at JoVE.com

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

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JoVE Journal

Neuroscience

18.4K visualizações.  University of Basel. Apresenta-se um protocolo que permite visualizar e para avaliar quantitativamente a morfologia da árvore dendrítico das células de Purkinje individuais crescidas em culturas organotípicas do cerebelo. Este protocolo destina-se a promover estudos sobre os mecanismos de Purkinje desenvolvimento de células dendríticas.