Quantitative analysis of microglial cells in the degenerating cerebellum of the staggerer (RORA(sg/sg)) mutant mouse.

Elevated levels of pro-inflammatory cytokines, such as IL-1ss and IL-6, have been detected in the cerebellum of Rora(sg/sg) mice during the first postnatal month of neurodegenerative process. This suggests the existence of a microglial reaction in the context of an inflammatory process that would be triggered by the massive neuronal loss. To test this hypothesis, we qualitatively and quantitatively studied the microglial cell population using lectin and nucleosidic diphosphatase labeling of the cerebellum of 30-day-old mice. The massive neuronal loss induces a 11.7-fold smaller size of the Rora(sg/sg) cerebellum compared to wild-types. We showed that the Rora(sg/sg) microglia population is exclusively composed of cells displaying the characteristic morphology of activated cells, with enlarged, heavily stained cell bodies and few thick processes, in contrast to microglial cells in the wild-type. The density of microglia is 2.7-fold higher in Rora(sg/sg) than wild-type mice (22444+/-5011 cells/mm(3) versus 8158+/-1584 cells/mm(3)), although the absolute number is 4-fold smaller. These results show that neurodegeneration in the Rora(sg/sg) cerebellum leads to persistance of microglial activation while in wild-type it disappears around P10.

Quantification of transcriptional activities of reporter gene constructs in primary cultures of sympathetic neurons.

Primary cultures of sympathetic neurons provide an attractive cellular model for investigating the mechanisms of neurotransmitter phenotypic plasticity. However, it has not been possible to transfect these neurons by conventional techniques, and this has been a major impediment to molecular investigations of neuronal gene expression in this system. Here, reporter plasmids were transferred into the nuclei of cultured sympathetic neurons by microinjection. We developed and improved this procedure and were able to measure the transcriptional activities of two coinjected promoters in small groups of neurons, and even from a single neuron. Promoter activities can thus be quantified and normalized relative to that of a constitutively expressed promoter, allowing correction for variability in the injection and assay procedures. High and low promoter activities can be reliably quantified. Importantly, this method can be used not only for reporter plasmids but also for DNA fragments containing only a promoter and reporter gene without any vector sequence that might interfere with promoter. Using this approach, we measured neuronal promoter activities and found that one promoter region of the gene encoding choline acetyltransferase was up-regulated by more than sevenfold by leukemia inhibitory factor. This method thus provides the means to investigate the function of neuronal genes and the mechanisms that regulate their transcription in cultured sympathetic neurons.