CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

Developmental neurobiology of the cerebellum

The series of developmental processes that drive the establishment of the mature pattern of connections, and hence the correct flow of information, in the adult central nervous system are regulated by complex interactions among different cell types involving an increasing variety of factors. We are studying the role of afferent fibers on the differentiation of their targets by using the olivocerebellar system (comprising of the inferior olivary neurons, their axons, i.e. the climbing fibers, and their target cells in the cerebellar cortex, i.e. the Purkinje cells) as a model. In particular, the study focuses on the role of the differentiative factors released by the afferent fibers. The studies on cell-cell interactions during neural differentiation by disclosing new players intervening in the development of a given region of the nervous system attempts to provide a better understanding of the process of the making of the brain.

Neuron-astrocyte interactions during cerebellar development

The development of the central nervous system is characterized by complex cell-cell interactions that drive the differentiative processes of individual cells. Among them, neuron-neuron interactions have been widely studied. In the embryonic cerebellum, postmitotic Purkinje cells migrate radially outwards in the cerebellar anlage (Altman, 1982), where they are contacted by climbing fibers that constitute their major early input (Morara et al., 2001), whereas granule cell precursors migrate dorsally, proliferate and later migrate inwards to settle in the internal granular layer (see Komuro and Yacubova, 2003 and ref. therein).

During this period waves of growth and regressive processes occur in several cell types at the level of, for example, Purkinje cell dendrites and climbing fiber axons and terminals on Purkinje cells. Whereas numerous developmental studies on neuron-neuron interactions have been performed, studies on neuron-astrocyte interactions have been mainly restricted to the early developmental phases of neuronal migration and axon elongation. In the cerebellar cortex, for example, Bergmann glial cells (astrocytes that closely enwraps Purkinje cells) have been shown to constitute a riding trail for radial granule cell migration, as extensively studied by several authors following the seminal paper by Rakic (1971). Only recently, in contrast, the relation between Bergmann glial cell and Purkinje cell differentiation received a specific attention (Yamada et al, 2000; Lordkipanidze and Dunaevsky, 2005). It has been hypothesized that radial processes of Bergmann glial cells provide a structural substrate for the directional growth of Purkinje cell dendrites, influencing the final shape of the dendritic tree. Moreover, selective ablation of Bergmann glia cells can lead to a severe disruption of Purkinje cell dendrites in adult rats (Cui et al, 2001), thus suggesting a role for these astrocytes also in the maintenance of Purkinje cell dendrites in the adult. However, very little is still known about a possible role of astrocytes at the final stages of neuronal development, the synaptogenetic process which is a crucial step in the establishment of the adult pattern of connections.

CGRP and other neuropeptides in the developing climbing fibers

 

In a search for putative anterograde factors expressed and released from afferent fibers during cerebellar development we found that the neuropeptides Neuropeptide Y (NPY), somatostatin and calcitonin Gene-Related Peptide (CGRP) are transiently expressed (or upregulated) in climbing fibers during the period of cerebellar process reshaping. The attention has been focused on CGRP which is selectively expressed in climbing fibers in the rat, and it has been shown to exert a differentiating and proliferating activity on glial cells in the peripheral and central nervous system (Lazar et al, 1991; Cheng et al, 1995). In addition, components of the receptor complexes of the CGRP family of peptides, Calcitonin-Like Receptor (CLR), Receptor Activity-Modifying Proteins (RAMP)1-3 and RCP, have been detected at the mRNA level in glial cells (Moreno et al, 1999; 2002), and CGRP was shown to increase cAMP levels and induce immediate early gene expression in cultured astrocytes and microglial cells (Haas et al, 1990; Priller et al, 1995; Moreno et al, 2002).

CGRP expression in olivocerebellar fibers starts at embryonic days at their entry in the cerebellar anlage where they represent the major input to Purkinje cells, and lasts up to the beginning of their climbing stage (Morara et al, 1989; 1995).

Moreover, in the time window of peptide expression, the corresponding receptor (revealed by a monoclonal antibody directed against a partially purified receptor) is present on the cell surface in glial cells (mainly Bergmann glial cells) while in the Purkinje cells the receptor shows a significant cell surface localization only at later stages, when climbing fiber synaptogenesys is almost complete (Morara et al., 2000). It has then been hypothesized that CGRP plays a role a neuropeptide that is anterogradely released by climbing fibers during development, and thus influencing not only astrocyte, but also, by a feedforward mechanism, neuron differentiation.

Effects of CGRP on cerebellar astrocytes

Since synaptic transmission between neurons (the key element of neuron-neuron interaction in the information process) has been recently reported to be profoundly influenced by neuron-glia interaction which is regulated by calcium signaling in astrocytes, we analyzed whether calcium signaling in astrocytes could also be affected by CGRP released from climbing fibers during development. In calcium imaging experiment it was found that CGRP affects astrocytic calcium signaling by way of transient increases in intracellular concentration. This effect was recorded not only in cultured astrocytes but also in Bergmann glial cells in acute cerebellar slices. Astrocytes responded to CGRP over a large range of peptide concentrations, from low nanomolar up to millimolar range. The release was inhibited by application of thapsigargin thus showing that the transient increase was caused by release from intracellular calcium stores.

More recently, the identification and cloning of the receptor allowed to raise new antibodies directed against identified CGRP receptor components. Receptor Component Protein (RCP), a cytoplasmic protein that links the activated peptide-receptor complex to the cAMP signaling cascade (Evans et al, 2000; Prado et al, 2001), was found to be expressed in cerebellar astrocytes in situ, at neonatal stages. In particular, a confocal analysis revelaed that when CGRP is still expressed by climbing fibers, RCP is expressed at the membrane level in Bergmann glial cells whereas in Purkinje cells it its expression is largely confined to the cytoplasm, in agreement with the previous confocal analysis carried out with a different antibody directed against a partially purified CGRP receptor (Morara et al, 2000).

The project is now aimed at analyzing the cerebellar distribution and intracellular localization of the different CGRP receptor components during development by using confocal and electron microscopy immunocytochemistry with recently developed antibodies. In parallel, any regulatory effect on cerebellar neuron differentiation will be analyzed in cerebellar primary cultures enriched in Purkinje cells, whereas the intracellular signaling cascade activated in astrocytes will be identified in cerebellar astrocytic cultures. Finally, on the basis of preliminary findings on the regulation of astrocytic cytokine production by this peptide, the involvement of this peptide in neurodegenerative, inflammatory-based disorders will be addressed by a quantitative densitometric analysis for immunmofluorescence and confocal microscope that has been recently developed.

Collaborations

  • F. Grohovaz, DIBIT, "Vita e Salute" University, HSR, Milano, Italy.
  • M. V. Catania, Institute of Neurological Sciences, Catania, Italy.
  • I. M. Dickerson, Department of Neurobiology, University of Rochester, Rochester, NY, USA.
  • H. Kettenmann, Max Delbrück Center, Berlin, Germany.
  • R. Quirion, Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada.

 

PI photo

Stefano Morara

Contact information

email  E-mail

email  +39 02 26144220

Participating staff
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