Untersuchungen der Funktion des Kleinhirns mit Hilfe von Mausmutanten

Abstract

Analysis of cerebellar function using mouse mutans- NDRF regulates differentiation and axon development of cerebellar basket cells -The cerebellar cortex is essentially comprised of three major neuronal subtypes, purkinje cells (PC), granule cells and several subtypes of interneurons including basket, stellate and golgi cells. Whereas PC and interneurons are GABAergic inhibitory neurons, granule cells use glutamate as their neurotransmitter.A crucial issue in neural development is the identification of mechanisms by which distinct types of neurons are specified, settled in their correct layers and differentiated. For granule cells the availability of specific markers and mouse mutants has led to a quite advanced understanding of their development. Among others, transcription factors like Math1 and NeuroD are necessary for the generation and survival of granule cells with little or no effects on PCs and interneurons. Development and function of cerebellar interneurons is less well understood. The few mouse mutants described with developmental alterations in those neurons rather identified transcription factors involved in determination of the GABAergic cell fate (like Heslike, MATH1, MASH1 and PTF1A) than in specific differentiation processes.The basic helix.loop-helix transcription factor NeuroD2 (NDRF) is expressed in postmitotic granule, stellate, and basket cells. To investigate its function in cerebellum development we inactivated the NeuroD2 gene in the mouse by homologous recombination in embryonic stem cells.The cerebellar granule cell layer of NeuroD2 null mutants was reduced in size. Nevertheless development and functional integration of those granule cells that accomplished the transition from the late precursor cell to a differentiated granule cell in the absence of NeuroD2 proceeded rather normal (most likely due to functional compensation by NeuroD). In contrast inhibitory interneurons were severely and constantly impaired. They were reduced in number to 1/3 and showed a severe delay in the expression of interneuronal marker antigens like GAD67 and Parvalbumin. This phenotypical alteration was accompanied by prolonged expression of the early interneuronal differentiation marker Pax2. Dendritic morphology was normal throughout all stages of development in both, stellate and basket cells. However, axo- and synaptogenesis showed pronounced differences between stellate and basket cells. Whereas stellate cells exhibited a normal distribution of GAD65 positive synapses at P25, axonal processes of basket cells and their pinceau synapses were nearly completely absent at this age. At P75 basket cell axonal processes had been developed further but still depicted a diffuse orientation, misshaped morphology and absence of synapse formation. Taken together the results suggest a important role for NeuroD2 in the differentiation of cerebellar cortical interneurons, especially basket cells, where the lack of NeuroD2 underlies severe deficits in axo- and synaptogenesis.