Generation of GABAergic and dopaminergic interneurons from endogenous embryonic olfactory bulb precursor cells

Eva Vergao-Vera 1 2 Mara J. Yusta-Boyo 1 Fernando de Castro 0 Antonio Bernad 3 Flora de Pablo 1 Carlos Vicario-Abejn 1 2 0 Instituto de Neurociencias de Castilla y Len, Universidad de Salamanca , Salamanca, Spain 1 Growth Factors in Vertebrate Development Group, Centro de Investigaciones Biolgicas , CSIC, Madrid, Spain 2 Instituto Cajal , Consejo Superior de Investigaciones Cientficas (CSIC), E-28002 Madrid, Spain 3 Department of Immunology and Oncology, Centro Nacional de Biotecnologa/CSIC , Madrid, Spain During the embryonic period, many olfactory bulb (OB) interneurons arise in the lateral ganglionic eminence (LGE) from precursor cells expressing Dlx2, Gsh2 and Er81 transcription factors. Whether GABAergic and dopaminergic interneurons are also generated within the embryonic OB has not been studied thoroughly. In contrast to abundant Dlx2 and Gsh2 expression in ganglionic eminences (GE), Dlx2 and Gsh2 proteins are not expressed in the E12.5-13.5 mouse OB, whereas the telencephalic pallial domain marker Pax6 is abundant. We found GABAergic and dopaminergic neurons originating from dividing precursor cells in E13.5 OB and in short-term dissociated cultures prepared from the rostral half of E13.5 OB. In OB cultures, 22% of neurons were GAD+, of which 53% were Dlx2+, whereas none expressed Gsh2. By contrast, 70% of GAD+ cells in GE cultures were Dlx2+ and 16% expressed Gsh2. In E13.5 OB slices transplanted with EGFP-labeled E13.5 OB precursor cells, 31.7% of EGFP+ cells differentiated to GABAergic neurons. OB and LGE precursors transplanted into early postnatal OB migrated and differentiated in distinct patterns. Transplanted OB precursors gave rise to interneurons with dendritic spines in close proximity to synaptophysin-positive boutons. Interneurons were also abundant in differentiating OB neural stem cell cultures; the neurons responded to the neurotrophin Bdnf and expressed presynaptic proteins. In vivo, the Bdnf receptor TrkB colocalized with synaptic proteins at the glomeruli. These findings suggest that, in addition to receiving interneurons from the LGE, the embryonic OB contains molecularly distinct local precursor cells that generate mature GABAergic and dopaminergic neurons. INTRODUCTION In contrast to the local origin of the excitatory pyramidal and mitral/tufted neurons, the brain region in which cortical and olfactory bulb (OB) interneurons originate during the embryonic period is species-dependent. In humans, the main source of cortical GABAergic neurons appears to be the cortical ventricle zone (VZ) (Letinic et al., 2002); it is nonetheless thought that many embryonic rodent OB interneurons (as well as cortical interneurons) arise in the ganglionic eminences (GE) (Marn and Rubenstein, 2003). Dlx2/Gsh2/Er81-expressing precursor cells in the lateral ganglionic eminence (LGE) are a source of mouse OB interneurons (Stenman et al., 2003). Supporting evidence includes the in vivo observation that migratory cells that leave the GE reach the cortex and OB (Anderson et al., 1997; Anderson et al., 2001; de Carlos et al., 1996; Lavdas et al., 1999; Pencea and Luskin, 2003), and that GE precursor cells grafted in the walls of lateral ventricles migrate to the cortex and the OB (Wichterle et al., 1999; Wichterle et al., 2001). In addition, mice lacking Dlx1/2, Gsh1/2, Arx and Sp8 transcription factors, expressed in the GE, show markedly decreased expression of GABAergic and dopaminergic cell markers in the embryonic granule and periglomerular layers of the OB (Anderson et al., 1997; Bulfone et al., 1998; Corbin et al., 2000; Qiu et al., 1995; Stenman et al., 2003; Toresson and Campbell, 2001; Waclaw et al., 2006; Yoshihara et al., 2005; Yun et al., 2003; Yun et al., 2001). Other data suggest that, in addition to this exogenous neuron source, interneurons can be generated intrinsically within the embryonic OB. These include observations that GABAergic markers are restored in the Gsh2 knockout mouse OB at late embryonic stages (Corbin et al., 2000; Toresson and Campbell, 2001; Yun et al., 2003), that the Gsh1/2 double null mutation reduces, but does not abolish, glutamic acid decarboxylase 67 (GAD67; Gad1 Mouse Genome Informatics) and Er81 expression in the OB (Stenman et al., 2003; Yun et al., 2003), and that a considerable number of GABAergic neurons remains in the OB of Arx mice (Yoshihara et al., 2005). Finally, the Gsh2 null mutation does not affect OB neuron generation and differentiation from cultured LGE cells (Jensen et al., 2004). Overall, these findings indicate that the LGE may be the primary source of OB interneurons during the embryonic period (Stenman et al., 2003; Wichterle et al., 1999) but do not rule out dopaminergic and GABAergic neuron generation from local OB precursor cells, particularly before migratory LGE cells reach the OB. According to previous studies (Pencea and Luskin, 2003), the rostral migratory stream (RMS) reaches the rat OB by embryonic day (E) 16.5; this embryonic age would correspond approximately to mouse E14.5. A recent study using Arx to label migratory cells in the mouse (Yoshihara et al., 2005) corroborates the idea that the first GEderived cells are detected in the OB at E14.5. Here we show efficient differentiation of local E13.5 OB precursor cells into GABAergic and dopaminergic interneurons, when transplanted into E13.5 OB slices or into P5-P7 OB in vivo, or when plated in short-term dissociated cultures. GABAergic neurons isolated from the rostral half of E13.5 OB and from E13.5 GE were distinguished by their distinct migration patterns following transplant into the OB, and by their different gene expression in short-term dissociated cultures. This suggests that the OB contains a distinct, endogenous pool of interneuron precursor cells. Our results also show that these precursors differentiate in vivo into morphologically mature neurons, suggesting that they could integrate into the OB circuit. MATERIALS AND METHODS OB and GE short-term dissociated cultures Tissue culture reagents were purchased from Gibco-Life Technologies and Sigma. Mouse care was in accordance with European Union guidelines. The day on which a vaginal plug was found was considered E0.5. E13.5 OB cells were prepared from the rostral half of CD1 mouse OB to avoid contamination of LGE extending into the caudal part of the OB and of the accessory olfactory bulb (Jimnez et al., 2002). OB cells and GE (both medial and lateral eminence) cells were mechanically dissociated, then cultured for 6 or 17 days essentially as described (Vicario-Abejn et al., 1998). Cells were also isolated and cultured from the OB of E13.5 embryos from pregnant mice that were injected intraperitoneally (i.p.) with 5 -bromo2-deoxy-uridine (BrdU; 100 g/g) 4 or 14 hours before embryos were removed. Neural stem cell cultures Olfactory bulb stem cells (OBSC) were prepared from E13.5-14.5 mice. In some experiments, only the rostral half of the E13.5 OB was dissected. Cells were (...truncated)