Dissection of Hippocampal Dentate Gyrus from Adult Mouse

Abstract

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

Exploring the potential relationship between Notch pathway genes expression and their promoter methylation in mice hippocampal neurogenesis

“…For qRT-PCR, Western blot and BSP assay, mice were anesthetized and the bilateral DG was rapidly microdissected under a stereomicroscope, strictly according to procedures as described by Hagihara et al (2009). The mouse was deeply anesthetized, the brain was carefully dissected out from the skull and placed into ice-cold phosphate-buffered saline (PBS).…”

Brain-Derived Neurotrophic Factor Val66Met Human Polymorphism Impairs the Beneficial Exercise-Induced Neurobiological Changes in Mice

“…To this aim, we dissected the DG from the remaining part of the hippocampus (collectively indicated as CA). The accuracy of our dissection was evaluated by measuring the mRNA levels of tryptophan 2,3-dioxygenase (TDO2) and TYRO3 protein tyrosine kinase 3 (Tyro3), two markers specifically expressed in the DG and in the CA regions, respectively (Hagihara et al, 2009), which revealed a clear separation of the two areas (Supplementary Figure S2). First, we assessed the total BDNF mRNA levels, finding that BDNF mRNA expression was higher in the DG compared with the remaining CA regions and that BDNF expression was modulated by exercise only in the DG area (area: F (1,44) = 106.8; Po0.0001; groups: F (3,44) = 14.38; Po0.0001; interaction: F (1,44) = 5.119; P = 0.004).…”

Redirecting N-acetylaspartate metabolism in the central nervous system normalizes myelination and rescues Canavan disease

“…Furthermore, the thalamus/hypothalamus was removed using a Wecker Micro Dissecting Spatula (Roboz Surgical Instruments Inc.). The hippocampus was removed as described elsewhere (53). Finally, part of the cortex was removed with a fresh razor blade.…”