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Mechanisms and consequences of reduced dendritic growth of cerebellar Purkinje cells

Gugger, Olivia S.. Mechanisms and consequences of reduced dendritic growth of cerebellar Purkinje cells. 2012, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_9859

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Abstract

The cerebellum is a brain region crucial for coordination and motor learning. Being the principal output cell of the cerebellar cortex, Purkinje cell loss and degeneration play an important role in many cerebellar diseases. The most striking feature of cerebellar Purkinje cells is their large and extensively branched dendritic tree, which is almost flat and strictly arranged in the sagittal plane. The factors and molecules which control the growth and patterning of neuronal dendrites are still poorly understood. Previous research in our lab has shown that chronic activation of metabotropic glutamate receptor 1 (mGluR1) (Sirzen-Zelenskaya et al., 2006) or Protein Kinase C (PKC) (Metzger & Kapfhammer, 2000; Schrenk et al., 2002) in organotypic cerebellar slice cultures of postnatal mice severely inhibits the growth and development of the Purkinje cell dendritic tree.
Although we found that the similar effect induced by mGluR1 or PKC is mediated by independent mechanisms (Sirzen-Zelenskaya et al., 2006), the signaling events leading to inhibition of dendritic growth after both mGluR1 and PKC activation remain largely unknown. Another intriguing question is that of the physiological relevance of limiting dendritic size after chronic activation of a metabotropic glutamate receptor.
We addressed both aspects using organotypic cerebellar slice cultures. In this culture model, a 300 µm thick slice of cerebellar tissue is kept in culture, where the natural microenvironment of a cell with neighboring cell-cell interactions and local neuronal networks are preserved. In order to study Purkinje cell dendritic development, cerebella from mice were cultured shortly before Purkinje cells enter the developmental stage of rapid dendritic growth and expansion, and were maintained for 10-12 days.
Purkinje cells are especially affected in various diseases involving excitotoxicity. We have tested the hypothesis that it is the size of the dendritic tree which determines Purkinje cell sensitivity to excitatory overload. Therefore, we have grown Purkinje cells under conditions which result in a strong reduction of dendritic tree size. Then we have exposed the cultures to (RS)-a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), which is an appropriate model system to study excitotoxic neurodegeneration in the cerebellum as it selectively kills Purkinje cells but leaves cerebellar granule cells unharmed. Our results indicate that a reduction of the size of the dendritic tree in Purkinje cells does not offer any protection from glutamate-induced excitotoxicity. Staining for the vesicular glutamate transporter vGluT1 revealed a high density of glutamatergic synapses on the stunted dendritic trees of pre-treated Purkinje cells. This suggests that receptor density rather than total receptor load is important for determining the sensitivity of Purkinje cells to AMPA-mediated neurotoxicity.
In a second study we have searched for potential mechanisms limiting Purkinje cell dendritic growth and have concentrated on channels allowing the entry of Ca2+ ions, especially the TRPC3, P/Q-type and T-type Ca2+ channels.
To analyze the roles of these channels in mediating dendritic reduction induced by mGluR1 or PKC we have treated Purkinje cells in cerebellar slice cultures with the mGluR1 activator DHPG or the PKC activator PMA and simultaneously with different combinations of Ca2+ channel inhibitors. After the culture period, the size of the dendritic trees was evaluated.
Co-treatment with a combination of P/Q- and T-type inhibitors partially rescued the dendrite-reducing effects induced by DHPG or PMA treatment. In contrast, no improvement of dendritic growth was found in mice lacking TRPC3 function or using an acute pharmacological TRPC3 inhibitor. Furthermore, the partial rescue obtained by P/Q- and T-type inhibition was not further increased by additional co-treatments with inhibitors of GluRδ2 - or R-type Ca2+ channels, or of Ca2+ release from internal stores.
In conclusion, our results suggest that T-type and P/Q-type Ca2+ channels are part of the signaling pathways induced after chronic mGluR1 and PKC stimulation resulting in the inhibition of dendritic growth, while no involvement of TPRC3-, GluRδ2-, R-type Ca2+ channels or Ca2+ release from intracellular stores was found.
Advisors:Kapfhammer, Josef
Committee Members:Rüegg, Markus A. and Scheiffele, Peter
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Division of Anatomy > Developmental Neurobiology and Regeneration (Kapfhammer)
Item Type:Thesis
Thesis no:9859
Bibsysno:Link to catalogue
Number of Pages:146 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:48
Deposited On:08 May 2012 08:23

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