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A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus.

ABSTRACT: In vitro assays are valuable tools to study the characteristics of adult neural precursor cells under controlled conditions with a defined set of parameters. We here present a detailed protocol based on our previous original publication (Babu et al., 2007) to isolate neural precursor cells from the hippocampus of adult mice and maintain and propagate them as adherent monolayer cultures. The strategy is based on the use of Percoll density gradient centrifugation to enrich precursor cells from the micro-dissected dentate gyrus. Based on the expression of Nestin and Sox2, a culture-purity of more than 98% can be achieved. The cultures are expanded under serum-free conditions in Neurobasal A medium with addition of the mitogens Epidermal growth factor and Fibroblast growth factor 2 as well as the supplements Glutamax-1 and B27. Under differentiation conditions, the precursor cells reliably generate approximately 30% neurons with appropriate morphological, molecular, and electrophysiological characteristics that might reflect granule cell properties as their in vivo counterpart. We also highlight potential modifications to the protocol.

SUBMITTER: Babu H

PROVIDER: S-EPMC3140691 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus.

Babu Harish H Claasen Jan-Hendrik JH Kannan Suresh S Rünker Annette E AE Palmer Theo T Kempermann Gerd G

Frontiers in neuroscience 20110714

In vitro assays are valuable tools to study the characteristics of adult neural precursor cells under controlled conditions with a defined set of parameters. We here present a detailed protocol based on our previous original publication (Babu et al., 2007) to isolate neural precursor cells from the hippocampus of adult mice and maintain and propagate them as adherent monolayer cultures. The strategy is based on the use of Percoll density gradient centrifugation to enrich precursor cells from the ...[more]

PMID: 21811434

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Project description: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.

Project description:After aversive stress, people either choose to return to their previously familiar social environment or tend to adopt temporary social withdrawal to buffer negative emotions. However, which behavior intervention is more appropriate and when remain elusive. Here, we unexpectedly found that stressed mice experiencing social isolation exhibited less anxiety than those experiencing social contact. Within the first 24 h after returning to their previous social environment, mice experienced acute restraint stress (ARS) displayed low social interest but simultaneously received excessive social disturbance from their cage mates, indicating a critical time window for social isolation to balance the conflict. To screen brain regions that were differentially activated between the poststress social isolation and poststress social contact groups, we performed ΔFosB immunostaining and found that ΔFosB + signals were remarkably increased in the vDG of poststress social isolation group compared with poststress social contact group. There were no significant differences between the two groups in the other anxiety- and social-related brain regions, such as prelimbic cortex, infralimbic cortex, nucleus accumbens, etc. These data indicate that vDG is closely related to the differential phenotypes between the poststress social isolation and poststress social contact groups. Electrophysiological recording, further, revealed a higher activity of vDG in the poststress social isolation group than the poststress social contact group. Chemogenetically inhibiting vDG excitatory neurons within the first 24 h after ARS completely abolished the anxiolytic effects of poststress social isolation, while stimulating vDG excitatory neurons remarkably reduced anxiety-like behaviors in the poststress social contact group. Together, these data suggest that the activity of vDG excitatory neurons is essential and sufficient to govern the anxiolytic effect of poststress social isolation. To the best of our knowledge, this is the first report to uncover a beneficial role of temporal social isolation in acute stress-induced anxiety. In addition to the critical 24-h time window, activation of vDG is crucial for ameliorating anxiety through poststress social isolation.

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A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus.

Publications

A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus.

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