The activity of neural precursor cells in the adult hippocampus is regulated by various stimuli; however, whether these stimuli regulate the same or different precursor populations remains unknown. Here, we developed a novel cell-sorting protocol that allows the purification to homogeneity of neurosphere-forming neural precursors from the adult mouse hippocampus and examined the responsiveness of individual precursors to various stimuli using a clonal assay. We show that within the Hes5-GFP ϩ /Nestin-GFP ϩ /EGFR ϩ cell population, which comprises the majority of neurosphere-forming precursors, there are two distinct subpopulations of quiescent precursor cells, one directly activated by high-KCl depolarization, and the other activated by norepinephrine (NE). We then demonstrate that these two populations are differentially distributed along the septotemporal axis of the hippocampus, and show that the NE-responsive precursors are selectively regulated by GABA, whereas the KCl-responsive precursors are selectively modulated by corticosterone. Finally, based on RNAseq analysis by deep sequencing, we show that the progeny generated by activating NE-responsive versus KClresponsive quiescent precursors are molecularly different. These results demonstrate that the adult hippocampus contains phenotypically similar but stimulus-specific populations of quiescent precursors, which may give rise to neural progeny with different functional capacity.
Developmental vitamin D (DVD) deficiency has been proposed as an important risk factor for schizophrenia. Our previous study using Sprague Dawley rats found that DVD deficiency disrupted the ontogeny of mesencephalic dopamine neurons by decreasing the mRNA level of a crucial differentiation factor of dopamine cells, the nuclear receptor related 1 protein (Nurr1). However, it remains unknown whether this reflects a reduction in dopamine cell number or in Nurr1 expression. It is also unclear if any particular subset of developing dopamine neurons in the mesencephalon is selectively affected. In this study, we employed state-of-the-art spinning disk confocal microscopy optimized for the imaging of tissue sections and 3D segmentation to assess post-mitotic dopamine cells on a single-cell basis in the rat mesencephalon at embryonic day 15. Our results showed that DVD deficiency did not alter the number, morphology, or positioning of post-mitotic dopamine cells. However, the ratio of Nurr1+TH+ cells in the substantia nigra pars compacta (SNc) compared with the ventral tegmental area (VTA) was increased in DVD-deficient embryos. In addition, the expression of Nurr1 in immature dopamine cells and mature dopamine neurons in the VTA was decreased in DVD-deficient group. Tyrosine hydroxylase was selectively reduced in SNc of DVD-deficient mesencephalon. We conclude that DVD deficiency induced early alterations in mesencephalic dopamine development may in part explain the abnormal dopamine-related behaviors found in this model. Our findings may have broader implications for how certain environmental risk factors for schizophrenia may shape the ontogeny of dopaminergic systems and by inference increase the risk of schizophrenia.
Flow cytometry is an important drug discovery tool because it permits high-content multiparameter analysis of individual cells. A new method dramatically enhanced screening throughput by multiplexing many discrete fixed cell populations; however, this method is not suited to assays requiring functional cellular responses. HEK293 cells were transfected with unique mutant glycine receptors. Mutant receptor expression was confirmed by coexpression of yellow fluorescent protein (YFP). Commercially available cell-permeant dyes were used to label each glycine receptor expressing mutant with a unique optical code. All encoded cell lines were combined in a single tube and analyzed on a flow cytometer simultaneously before and after the addition of glycine receptor agonist. We decoded multiplexed cells that expressed functionally distinct glycine receptor chloride channels and analyzed responses to glycine in terms of chloride-sensitive YFP expression. Here, data provided by flow cytometry can be used to discriminate between functional and nonfunctional mutations in the glycine receptor, a process accelerated by the use of multiplexing. Further, this data correlates to data generated using a microscopy-based technique. The present study demonstrates multiplexed labeling of live cells, to enable cell populations to be subject to further cell culture and experimentation, and compares the results with those obtained using live cell microscopy. ' 2009 International Society for Advancement of Cytometry Key terms multiplex; chloride channels; glycine receptor FLOW cytometry permits multiple cell parameters to be analyzed simultaneously with high sensitivity and a high degree of statistical robustness. It is emerging as an important drug discovery tool because it can screen test compounds against a wide variety of cellular targets simultaneously. However, achieving this goal is dependant on two factors: (1) the ability to automate the screening of large numbers of compounds, and (2) the ability to monitor multiple parameters simultaneously in live cells. Recent developments (reviewed by Sklar et al. (1)), while useful, require dedicated hardware on an unmodified flow cytometer.Multiplexed or duplexed analysis allows the screening of multiple discrete samples in one tube or well at the same time (2-4). A recent method (5), which introduced the idea of labeling discrete cell populations with a unique combination of fluorochromes, demonstrated the feasibility of employing flow cytometry to discriminate between different multiplexed labeled cell populations. However, this method required cellular fixation, preventing further culture or experimentation.Glycine receptors (GlyRs) are pentameric Cys-loop anion-permeant channels that mediate inhibitory neurotransmission in the spinal cord, brainstem, and retina (6,7). These receptors have recently emerged as therapeutic targets for chronic inflammatory pain and movement disorders (8-10). Our interest lies in discovering novel bioactive compounds with therapeutic potential and i...
High-dose chemotherapy with autologous stem cell rescue has been proposed as an intensive therapy for severe rheumatoid arthritis (RA). In view of previous observations of abnormal haemopoiesis in RA patients, the composition and function of peripheral blood stem cell harvests (PBSCH) was investigated. Compared with PBSCH from healthy allogeneic donors mobilized with the same dose of G-CSF (filgrastim; 10 microg/kg/d, n = 14), RA PBSCH (n = 9) contained significantly fewer mononuclear cells (375 v 569 x 10(6)/kg, P = 0.03) and CD34+ cells (2.7 v 5.8 x 10(6)/kg, P = 0.003). However, there were increased proportions of CD14+ cells (P = 0.006) and CD14+ CD15+ cells (the phenotype of previously described 'abnormal' myeloid cells, P = 0.002) in the RA PBSCH which translated into 3.5- and 7-fold increases respectively on a per CD34+ cell basis. There were no differences in T-cell activation status as judged by proportions of CD4+ and CD8+ expressing CD45RA, CD45RO, HLA-DR and CD28 (RA PBSCH, n = 7, donor PBSCH, n = 5, P = 0.2-0.7). Phytohaemagglutinin responses determined fluorocytometrically with induction of CD69 expression were reduced in CD4+ and CD8+ cells following filgrastim administration in 3/3 RA patients tested. Compared with bone marrow as a potential source of CD34+ cells, PBSCH contained 11-fold more T cells (P < 0.0005), 8-fold more B cells (P < 0.0005) and 4-fold more monocytes (P = 0.02). In short-term methylcellulose culture there were no differences in colony counts (CFU-GM, CFU-GEMM, BFU-E) per CD34+ cell from PBSCH from RA patients (n = 11) and healthy donors (n = 10). Long-term culture initiator cells were cultured successfully from cryopreserved PBSCH from RA patients (n = 9). In conclusion, PBSCH from RA patients differed significantly in composition from normal individuals, but in vitro studies support normal stem and progenitor cell function. Changes in T-cell function occur during mobilization in RA patients. This work provides reassurance for the use of PBSCH as haematological rescue and baseline data for clinical trials of graft manipulation strategies in patients with RA.
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