The N-methyl-D-aspartate (NMDA) receptor is a type of glutamate receptor, which is involved in neuronal function, plasticity and development in the mammalian brain. However, how the NMDA receptors contribute to adult neurogenesis and development of the dentate gyrus is unclear. In this study, we investigate this question by examining a region-specific knockout mouse line that lacks the NR1 gene, which encodes the essential subunit of the NMDA receptors, in granule cells of the dentate gyrus (DG-NR1 KO mice). We found that the survival of newly-generated granule cells, cell proliferation and the size of the granule cell layer are significantly reduced in the dorsal dentate gyrus of adult DG-NR1 KO mice. Our results also show a significant reduction in the number of immature neurons and in the volume of the granule cell layer, starting from 3 weeks of postnatal age. DG-NR1KO mice also showed impairment in the expression of an immediate early gene, Arc, and behavior during the novelty-suppressed feeding and open field test. These results suggest that the NMDA receptors in granule cells have a role in adult neurogenesis in the adult brain and contributes to the normal development of the dentate gyrus. Significance statementA type of glutamate receptors, the NMDA receptors, are known to be generally important in brain development. However, a previous study, which genetically ablated the NMDA receptors in the developing dentate gyrus, did not detect malformation of the dentate gyrus. Here, we performed more detailed analyses of the knockout mice and showed that the extent of neurogenesis and the size of the neuronal layer are reduced in the postnatal and adult dentate gyrus. We found that the knockout mice exhibit behavioural abnormality during adulthood. These results indicate that the NMDA receptor is essential for normal development of the dentate gyrus and that a malfunction of the NMDA receptors during the postnatal period could lead to life-long disturbance in brain functions.
Gene expression analysis in individual neuronal types helps in understanding brain function. Genetic methods expressing fluorescent proteins are widely used to label specific neuronal populations. However, because cell type specificity of genetic labeling is often limited, it is advantageous to combine genetic labeling with additional methods to select specific cell/neuronal types. Laser capture microdissection is one of such techniques with which one can select a specific cell/neuronal population based on morphological observation. However, a major issue is the disappearance of fluorescence signals during the tissue processing that is required for high-quality sample preparation. Here, we developed a simple, novel method in which fluorescence signals are preserved. We use genetic labeling with fluorescence proteins fused to transmembrane proteins, which shows highly stable fluorescence retention and allows for the selection of fluorescent neurons/cells based on morphology. Using this method in mice, we laser-captured neuronal somata and successfully isolated RNA. We determined that ∼100 cells are sufficient to obtain a sample required for downstream applications such as quantitative PCR. Capability to specifically microdissect targeted neurons was demonstrated by an ∼10-fold increase in mRNA for fluorescent proteins in visually identified neurons expressing the fluorescent proteins compared with neighboring cells not expressing it. We applied this method to validate virus-mediated single-cell knockout, which showed up to 92% reduction in knocked-out gene RNA compared with wild-type neurons. This method using fluorescent proteins fused to transmembrane proteins provides a new, simple solution to perform gene expression analysis in sparsely labeled neuronal/cellular populations, which is especially advantageous when genetic labeling has limited specificity.
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