Proteomics Applications in Neuroanatomy : a search for new regional markers in the mammalian brain

Abstract

Many studies have been undertaken to acquire full knowledge on the cortical parcellation of the mammalian brain in distinct areas. These functional and anatomical entities are interconnected in a highly-specific manner and enable complex behavior, adjusted to the environment and habitat of the species. Various types of techniques have attempted to reveal the areal parcellation. The different methods used failed however in reaching a consensus about cortical organization due to differences in measurement method, the characteristic they focused on, and the lack of transferability of results to other species. These drawbacks were assumed to be get round by mapping the mammalian cortex with molecular areal marker distribution. The present research study introduces protein expression profiling as a novel approach in neuroanatomical studies. Gene expression profiling has been applied extensively in the past, but we moved the focus to the actual active molecules in all cellular processes and pathways, the proteins. The proteomics technique of 2-D DIGE, combined with mass spectrometry, was applied as screening method for new areal markers, with subsequent validation of their applicability with immunocytochemistry at the protein level and in situ hybridization at the mRNA level. Comparison of the protein content of three visual areas (17, 18 and 19) with 2-D DIGE located fifteen differentially expressed spots. Subsequent mass spectrometry identified ten proteins in these spots, from which we selected the low molecular weight subunit of neurofilament or NFL as potential areal marker. Indeed, we were able to prove a correlation between differential expression levels of NFL to differential laminar distribution patterns in all three visual areas. Immunocytochemistry revealed area-specific NFL protein distribution in the cat visual cortex with differences in staining intensity, cell and fiber density, type and organization. The boundaries between areas 17, 18 and 19 were set, with the 17/18 border as a transitional border-zone rather than a clear or clear-cut border. To assess the value of NFL as areal marker in the visual cortex of other mammalian species we performed immunocytochemistry on rat, mouse and macaque monkey tissue sections. In all three mammalian species, NFL protein distribution allowed setting boundaries between primary and secondary areas and subareas. This first part of the study delivered ‘proof of principle’; 2-D DIGE is adequate to screen for neurochemical differences between cortical areas that are mirrored by area-specific distribution patterns as detectable by immunocytochemistry. Since NFL was proven a valuable, new marker in the mouse visual cortex, we performed immunocytochemistry on coronal sections along the rostro-caudal axis to visualize this protein’s distribution throughout the entire mouse brain. Indeed, NFL protein displayed area-specific expression patterns throughout the entire forebrain. Cortical boundaries were set based on differences in laminar distribution patterns of neuronal cells and fibers. These borders were classified in three types (clear-cut, clear and transitional). When grouping corresponding area-specific patterns, this classification was clearly related to the functionality of cortical systems. We identified characteristic NFL protein outlines in the paleocortex, including the anterior cingulate, the insular and the retrosplenial cortex, the motor cortex and the sensory cortex. Immunoreactivity for NFL grants complementary information on the areal parcellation of the mouse brain, for which until this day exists no consensus brain map. NFL protein distribution opens new perspectives to study position and size of different cortical systems in less understood mammalian species. Next, we investigated if the area-specific expression pattern of the NFL protein could be correlated to an area-specific distribution pattern of the NFL mRNA. Neurons which translate NFL were located by means of in situ hybridization histochemistry as a fast and reliable screening method. Indeed, NFL mRNA appeared in the mouse cortex in an area-specific manner, what allowed cortical delineation. Not all boundaries between abutting regions were perfectly clear, but this presents no drawback on the advantages of in situ hybridization. In the final chapter of the results sections, proteome profiling was performed on two primary sensory areas of the mouse brain. 23 spots were located as differential, and were mass spectrometrically identified as 26 different proteins. The potential areal markers identified differed quite from the earlier identified candidates in the cat visual cortex. CKB and CRMP2 were selected and validated by means of in situ hybridization. While CKB mRNA allowed clear delination of mouse cortical areas, CRMP2 mainly visualized subcortical regions. To conclude, protein expression profiling and thus new areal markers deliver additional information in a fast, reliable and reproducible manner in relation to remaining issues in neuroanatomical research on smaller mammals, but also on complex species, like primates and humans.status: publishe