Molecular characterization of CNS interneurons: subtype diversity and fate determinants

In this project two approaches have been used to gain insights into the identity and molecular regulation of interneurons in the brain. First, a general molecular characterization of GABAergic neuron subtypes has been performed and second, novel fate determinants specifically for olfactory bulb interneurons have been identified and investigated in detail. GABAergic neurons, the largest population of inhibitory neurons in the brain, play crucial roles in information processing. While most of these neurons are interneurons, some, for example in the striatum, represent projection neurons. So far, biochemical, morphological, and electrophysiological properties served as exclusive criteria for the classification of GABAergic neurons. Although these parameters allow for a partial description of subpopulations, a systematic dichotomy is not available. Therefore, the general molecular characteristics of GABAergic neurons were analyzed and differences among distinct brain regions were defined. Transgenic GAD67-GFP mice in concert with flow cytometric cell sorting were used to isolate GABAergic neurons from defined regions of the postnatal mouse brain, namely olfactory bulb, cortex, striatum and cerebellum. Subsequently, gene expression profiling as well as cell surface proteome analysis were carried out and identified genes were validated by in situ hybridization and qPCR. Potential new marker genes for GABAergic neurons and candidate factors necessary for their differentiation and general functionality were determined. Clustering of gene expression data revealed major differences between hind- and forebrain GABAergic neurons indicating a correlation between their development and localization. For example, while GABAergic neurons of the forebrain are characterized mainly by three groups of transcription factors, namely the Distal-less-family, the POU-family and the ETS/FOX-family; specific members of the ZIC- and LHX-family define hindbrain inhibitory neurons. Olfactory bulb interneurons are generated throughout live by adult neuronal stem cells localized in the subventricular zone. While considerable information is available concerning the generation and migration of these cells, the molecular mechanisms regulating their terminal differentiation are barely understood. Therefore, mature interneurons from the periglomerular layer and their specific precursors were isolated by microdissection and magnetic cell sorting. Gene expression analysis was performed by microarray analysis. Several candidate factors to be involved in the differentiation of olfactory bulb interneurons were identified. The bHLH transcription factors NeuroD1 and NeuroD2 were analyzed for their functional relevance in vivo. Using in vivo electroporation, overexpression of these transcription factors was induced in postnatal forebrain stem cell populations as well as their progeny, namely neuronal precursors and mature neurons of the olfactory system. It was shown that high expression of NeuroD2 delayed the differentiation of Type A neuronal precursor cells into granule- and periglomerular neurons. In contrast, overexpression of NeuroD1 induced the premature and ectopic differentiation of precursor cells. Furthermore, NeuroD1 induced specifically a dopaminergic phenotype, indicating that it represents a novel key fate determinant for the specification of periglomerular interneurons, possibly with a GABA/dopamine bifunctional neurotransmitter phenotype. In conclusion, this study represents a comprehensive molecular basis for the understanding of spatially as well as temporally defined GABAergic neuron subpopulations and led to the identification of novel fate determinants for the differentiation of inhibitory interneuron subpopulations