Further characterization of the juxtaglomerular neurons in the mouse main olfactory bulb by transcription factors, Sp8 and Tbx21
Highlights
► Immunoreactivities of transcription factors Sp8 and Tbx21 were examined in the mouse MOB. ► The intensities of the Sp8 and Tbx21 immunoreactivities were so diverse. ► Sp8 and Tbx21 characterized subpopulations of juxtaglomerular neurons differently. ► Sp8 and Tbx21 further confirmed the heterogeneity of NOS positive cells.
Introduction
The main olfactory bulb (MOB), the first relay station for transmission of olfactory information, has a rather concentric arrangement of seven layers and richly varied in chemical substances. Previously we reported the chemically defined neuronal subpopulations in the juxtaglomerular region of the mouse MOB using the immunocytochemistry for transmitter- or neuroactive substance-related molecules and calcium binding proteins (Kosaka and Kosaka, 2007a). Other group also reported chemically defined subpopulations of juxtaglomerular neurons of the mouse MOB using the immunocytochemistry in combination with transgenic mice (Parrish-Aungst et al., 2007). In addition to those chemical properties recent studies have indicated that several transcription factors are important for the development and differentiation of those neuronal subpopulations and that the expression of some transcription factors persist toward adulthood, thus characterizing subpopulations (Yoshihara et al., 2005, Waclaw et al., 2006, Allen II et al., 2007, Li et al., 2011); those transcription factors characterizing OB interneurons include Sp8 (Waclaw et al., 2006, Allen II et al., 2007, Li et al., 2011), Arx (Yoshihara et al., 2005), ER81 (Stenman et al., 2003, Allen II et al., 2007, Saino-Saito et al., 2007), Pax6 (Dellovade et al., 1998, Kohwi et al., 2005, Hack et al., 2005, Allen II et al., 2007, Brill et al., 2008), Meis2 (Allen II et al., 2007) and those characterizing mitral/tufted cells include Tbr1, Tbr2 and Tbx21 (Faedo et al., 2002, Yoshihara et al., 2005, Winpenny et al., 2011). Furthermore Waclaw et al. (2006) and Li et al. (2011) showed that in the Sp8 conditional knock-out mice “the most severe reductions are seen in the populations which continue to express Sp8 as mature neurons (Waclaw et al., 2006)”, and thus suggested that Sp8 is required for their normal production.
In the present study we extended our analyses on the juxtaglomerular neurons of the mouse MOB by examining the immunoreactivities of two well-known transcription factors, Sp8 and Tbx21, of the chemically defined subpopulations we reported previously (Kosaka and Kosaka, 2007a, Kosaka and Kosaka, 2007b, Kosaka and Kosaka, 2009a), that is, calretinin (CR), calbindin (CB), tyrosine hydroxylase (TH) and nitric oxide synthase (NOS) positive groups, as well as a newly reported secretagogin positive group (Mulder et al., 2009). Special attention was paid to the expression of these transcription factors in the TH and NOS positive juxtaglomerular neurons, for, as we reported previously, both TH and NOS positive juxtaglomerular neurons in the mouse MOB are heterogeneous (Kosaka and Kosaka, 2007b, Kosaka and Kosaka, 2008, Kosaka and Kosaka, 2009b).
Section snippets
Tissue preparations
All experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, 1996) and the institutional guidance for animal welfare (the Guidelines for Animal Experiment in Graduate School of Medical Sciences, Kyushu University) and have been approved by the Committee of the Ethics on Animal Experiment in Graduate School of Medical Sciences, Kyushu University. All efforts were made to minimize the number of
Results
In the present study we used samples fixed with three different concentrations of paraformaldehyde, 1%, 2% and 4% (PFA1, PFA2 and PFA4). Generally speaking, samples fixed with PFA1 and PFA2 showed much stronger immunostaining for Sp8, Tbx21, NOS, TH, secretagogin and CR. Especially the immunostaining of Tbx21 positive nuclei appeared to be prominently dependent on the fixatives used. On the other hand the CB staining was somewhat diffuse in PFA1-fixed samples. The immunostainings were usually
Discussion
Waclaw et al. (2006) examined the Sp8 immunoreactivity in the periglomerular cells in the mouse MOB, and reported that almost all CR positive and about 76% of GAD65-GFP positive periglomerular cells were Sp8 positive, which represented about 47% and 57% of Sp8 positive cells in the GL, respectively. On the other hand they also reported that “Sp8 expression is only rarely observed within the TH population (13.6%). Thus the majority of TH-positive cells likely correspond to GAD-positive
Acknowledgements
The authors are grateful to Drs. Emson, Heizmann, Nagatsu, Wagner and Yoshihara for providing the primary antibodies. The authors would like to thank Ms. Chie Tanaka and Ms. Kazuyo Sawai for their technical and secretarial assistance.
Grant sponsor: Grants-in Aid for Scientific Research from the Japan Society for the Promotion of Science (23300124, 20300120, 23500412, 20500309).
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