We crossed Olig2 knock-in mice (Olig2^KICreER) (Takebayashi et al., 2002) or GFAP^Cre mice (Garcia et al., 2004; Herrmann et al., 2008) with reporter mice Rosa-CAG-LSL-eNpHR3.0-EYFP (Ai39 mice, Jackson Laboratory, Stock No: 014539) to obtain Olig2^KICreER/WT; Ai39 mice (hereafter termed Olig2^CreER- Ai39 mice) and GFAP^KICre; Ai39 mice (hereafter termed GFAP^Cre-Ai39 mice). In Ai39 mice, cre-inducible halorhodopsin (eNpHR3.0) fused with YFP at its C-terminus is driven by a specially designed expression cassette in a modified Rosa26 locus, which showed efficient and strong YFP fluorescence at the membrane (Madisen et al., 2009, 2012). The YFP is fused to the C-terminus of the halorhodopsin and is localized in the cytoplasmic side. It clearly revealed cellular morphology without YFP immunostaining. In the present study, we used theses double transgenic mice for immunofluorescence study.

We also crossed Olig2 knock-in mice (Olig2^KICreER) and ROSA-GAP43-EGFP reporter line (Soriano, 1999) to obtain Olig2^KICreER/WT; GAP43-EGFP mice (hereafter termed Olig2^CreER-GFP mice). This reporter mice were particularly useful for visualizing the morphology of cells that underwent recombination, because GAP43-EGFP is a fusion protein between GFP and the N-terminus (amino acids 1–20) of GAP-43, which has been implicated in plasma membrane targeting through palmitoylation (Liang et al., 2002). Although the fluorescence of the GAP43-EGFP was generally weaker than that of the Ai39 reporter line, the moderate amount of the fusion protein was suitable for immunoelectron microscopic detection. Taking these experimental conditions into account, we employed Olig2^CreER-, and GFAP^Cre-Ai39 mice for fluorescence microscopy and Olig2^CreER-GFP mice were used for electron microscopy.

All the mice were maintained in a mixed genetic background; Olig2^CreER-Ai39 mice (C57BL/6 and 129 S6/Sv strains), GFAP^Cre-Ai39 mice (C57BL/6 and FVB/N strains) and Olig2^CreER-GFP mice (C57BL/6 and 129 X1/svJ strains). They were housed in standard cages under a 12 h light/dark cycle and temperature-controlled conditions. In the present study, we used 3–5 mice for each transgenic mouse strain and all the mice were 11 weeks old. All the protocols for the animal experiments were approved by the Animal Care Committee of Nara Medical University in accordance with the policies established in the NIH Guide for the Care and Use of Laboratory Animals.

In the present study, we employed oral administration of tamoxifen. Tamoxifen (TM, Sigma Aldrich, Japan) was mixed with powdered chow (0.5 mg/g normal chow). Olig2^CreER-Ai39 mice for immunofluorescence experiments were allowed to access the tamoxifen-containing chow ad libitum from 11 to 16 weeks old (see Figure 1A). This oral administration method is convenient for continuous administration and results in efficient induction of recombination while minimizing stress on the mice (Casanova et al., 2002; Kiermayer et al., 2007; Feil et al., 2009; Welle et al., 2009). Olig2^CreER-GFP mice for immunoelectron microscopy were also fed with tamoxifen-containing chow as described above (see Figure 2A). All the mice appeared in healthy condition, although some mice were lost up to 10% of their body weight during the tamoxifen-fed period.

Mice were perfused with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Brains were removed and post-fixed in 4% PFA, and cryopreserved in 30% sucrose until they sank, and cryosections were cut at 30-μm thickness on a cryostat (Leica CM1860, Leica Microsystems, Japan). Immunofluorescence experiment was performed as described previously (Tatsumi et al., 2016). The primary antibodies were listed in the Table 1. Alexa-594- or 647-conjugated antibodies (1:1,000, Jackson ImmunoResearch, USA) were used as secondary antibodies. Blue-fluorescent Nissl stain (1:200, Neurotrace 435/455, ThermoFisher Scientific, Japan) was used as a counterstaining.

All the fluorescence images were obtained with a confocal laser scanning microscope (Nikon C2-NiE, Tokyo, Japan) as single planes or as z-stack projections (x-axis by y-axis, 1,024 by 1,024 pixels). For wide-field fluorescence images (e.g., half coronal section or whole sagittal section of mouse brain), we captured 50~80 images for whole area with 10 × objective lens (NA 0.45, UpanApo, Nikon) at 1.5 optical zoom by motorized x/y stage, and images were tiled to construct a large image. We obtained the z-stack multi-channel images at 1 μm intervals with 20 × objective lens (NA 0.75, UpanApo, Nikon) for the globus pallidus and the images generated orthogonal views (x-z and y-z planes) and a 3D rendering view (see Figures 3B,C). For high magnification images, we used 60 × water-immersion objective lens (NA 1.45, UpanApo, Nikon) at 1.5 optical zoom. All the images were processed by Nikon Nis-Elements software (version 4.11).

The YFP intensity of each brain region in the low magnification images was measured using Nikon Nis-Elements software. To standardize the fluorescence intensities among different sections, the value of fluorescence intensity was divided by the value of background intensity, yielding a ratio. For objective evaluation of regional fluorescence, a researcher who was blind to the nature of an image performed measurements of regional fluorescence with the software and calculate the brain region/background ratios. We then classified these ratios into three groups (strong; larger than 20, moderate; 10–20, weak/absent; less than 10) and the groups were color-coded (strong; red, moderate; yellow, weak/absent; blue) for Table 2. For example, the panel B4 in the Figure 4 contained the regions of strong fluorescence such as the lateral septum (LS; 22.5) and the somatosensory cortex (S1BF; 28.6), the region of moderate fluorescence such as the motor cortex (M, 14.6) and the regions of weak or absent fluorescence such as the caudate putamen (CPu, 2.2). In the Table 2, it should be noted that the color-coded ratios were relative among the sections of each double transgenic line (i.e., Olig2^CreER-Ai39 or GFAP^Cre-Ai39 mice) and could not be directly compared.

Pre-embedding double immunoelectron microscopy was performed as described previously with a few modifications (Yamasaki et al., 2010). Vibratome sections were incubated in blocking solution (Aurion, Netherlands) for 30 min and then with a cocktail of primary antibodies (for GABAergic terminals; rabbit anti-GFP antibody and guinea pig anti-VGAT antibody, for glutamatergic terminals; rabbit anti-GFP antibody and guinea pig anti-VGluT1/2 antibodies) diluted with 1% BSA/ 0.002% saponin/PBS overnight. The sections were washed with 0.002% saponin/ PBS, incubated with 1.4 nm gold particles conjugated anti-guinea pig IgG (1:100, Nanoprobes, Stony Brook, NY) for 4 h. After washing, immunogold particles were intensified using a silver enhancement kit (R-Gent SE-EM; Aurion, Netherlands) for 60 min, washed, and incubated with peroxidase labeled anti-rabbit IgG (414341F, Nichirei Bioscience, Japan) for 4 h and visualized with chromogenic reaction of 3,3′-diaminozidine (DAB). Sections were further treated with 1% osmium tetroxide for 15 min on ice, stained by 2% uranyl acetate for 15 min.

In addition to the method described above, we performed another method of double immunoelectron microscopy. Briefly, vibratome sections were incubated a cocktail of primary antibodies (for GABAergic terminals; rat anti-GFP antibody and rabbit anti-VGAT antibody, for glutamatergic terminals; rat anti-GFP antibody and guinea pig anti-VGluT1/2 antibodies) diluted with 1% BSA/0.002% saponin/PBS overnight. The sections were washed, incubated with peroxidase-labeled anti-rat IgG (414311F, Nichirei Bioscience, Japan), and followed by DAB chromogenic reaction for GFP visualization. After washing, the sections were incubated either with biotinylated anti-rabbit antibody (BA-1000, Vector Laboratories, Japan, for GABA) or biotinylated anti-guinea pig antibody (BA-7000, Vector Laboratories, Japan, for glutamate), followed with peroxidase-labeled streptavidin (426061, Nichirei Bioscience, Japan) incubation, and then subjected to DAB chromogenic reaction again to visualize axon terminals with synaptic vesicles (VGAT or VGluT1/2 positive). Then, sections were treated with 1% osmium tetroxide for 60 min.

Although the latter DAB-DAB combination method yielded results comparable to those with immunogold-DAB method (see Figure 7), we found that the outlines of the DAB-labeled axon terminals were much easier to observe than those of the nanogold-labeled ones. We therefore employed the DAB combination method in semi-quantitative analyses of association between axon terminals and astrocytic fine processes (see below).

All the sections were dehydrated through a graded ethanol series, and flat-embedded in epoxy resin (Epon812 resin embedding kit, TAAB Laboratories Equipment, Japan). The resin was polymerized for 2 days at 60°C. We then carefully dissected out regions containing well-isolated GFP-positive bushy cells from a flat-embedded section under a light microscope (a representative image, inset in Figure 2B). The semi thin (1 μm) and ultrathin (70–80 nm) sections were cut on an ultramicrotome (Leica EM UC7, Leica Microsystems, Japan). The ultrathin sections were collected onto formvar-coated single whole grid, and observed under a JEOL transmission electron microscope (JEM-1400plus, JEOL, Japan).

In semi-quantitative analyses of association between axon terminals and astrocytic fine processes, a researcher took electron microscopic images that contained immunoreactive axon terminals (either VGAT-positive or VGluT1/2-positive) and astrocytic fine processes. Another researcher who was blind to the nature of vesicle transporter immunoreactions counted the numbers of immunoreactive terminals attached with astrocytic fine processes and the numbers of immunoreactive terminals without process attachments in the images. The counts were later summed and subjected to statistical analyses.

Figure 1A showed the schedule of oral tamoxifen administration for Olig2^CreER-Ai39 mice for immunofluorescence study. As shown in Figure 1B, continuous feeding with tamoxifen-containing chow markedly induced Cre-mediated recombination, yielding robust YFP labeling of Olig2-lineage cells. We previously reported that there were two types of Olig2-lineage cells in the adult brain (Tatsumi et al., 2008, 2016). The cell type with strong YFP fluorescence and bushy morphology were mature astrocytes (Tatsumi et al., 2016), and we confirmed that the bushy cells were positive for mature astrocyte markers in various brain regions, such as SOX9, S100β (Figures 1C1–C3, D1–D3, Figure S1) in the cerebral cortex, 3-PGDH (Figures 1E1–E3, Figure S1) in the facial nucleus (7N), GS (Figures 1F1–F3, Figure S1) in the subthalamic nucleus. The subcellular localization patterns varied among the mature markers; transcription factor SOX9 was found in the nucleus (Figure S1D), while the enzyme GS was primarily present in the cytoplasm, but was found little in the distal processes (Figure S1B). It should be noted that GS was distributed to the fine processes of Olig2-AS in addition to the cytoplasm in the cerebral cortex (Figure S1B; cx), but not in the other brain regions such as the globus pallidus or thalamus (Figure S1B; LGP, Th). The mechanisms underlying the differential subcellular distribution are unclear. The other type of YFP-positive cells was of oligodendrocyte lineage, including OPCs. We observed that mature oligodendrocyte marker CC-1 was positive in some of the YFP-expressing cells (Figures 1G1–G3, Figure S1), and immature oligodendrocyte marker NG2 was positive in other YFP-expressing cells (Figures 1H1–H3, Figure S1). The overall morphologies of these oligodendrocyte-lineage cells were clearly distinct from that of the bushy cells and these cells could not be detected in low magnification view, unless they were immunolabeled with anti-YFP antibody. This was most likely because polydendric processes were much simpler than bushy processes and the membrane-targeted YFP was less distributed in the oligodendrocyte-lineage cells. Taken together, the YFP-positive cells observed in low magnification view (Figure 1B) likely reflect the Olig2-lineage astrocytes.

As mentioned above, Olig2-AS expressed several astrocytic markers (arrows, Figures 1, 2), but we noticed that Olig2-AS expressed low level of GFAP, the cardinal astrocytic marker. To confirm this notion, we performed double immunofluorescence study with YFP and various astrocytic markers including GFAP in Olig2^CreER-Ai39 mice. Since GFAP immunoreactivity is not uniformly detected in the whole brain, we selected two regions, the lateral globus pallidus (LGP) and the substantia nigra (SN), where a number of GFAP-positive astrocytes exist. GFAP-positive cells co-expressed astrocytic markers such as SOX9 (arrowheads, Figures 2A2–A4), S100β (arrowheads, Figures 2B2–4,C2–C4) and GS (arrowheads, Figures 2D2–D4). On the other hand, Olig2-AS with bushy morphology also expressed astrocytic markers such as SOX9 (arrows, Figures 2A2,A4), S100β (arrows, Figures 2B2,B4,C2,C4) and GS (arrows, Figures 2D2,D4). We delineated territories of Olig2-AS with dotted line in the Figure 2 and even in the presence of GFAP-positive astrocytes, the Olig2-AS seldom overlapped with the GFAP-positive structures (Figures 2A3,B3,C3,D3).

In a low magnification view, Olig2-AS (YFP positive) were densely distributed in the LGP (Figure 3A1) and GFAP immunoreactivity was also strong (Figure 3A2). SOX9 positive cells were more widely observed in the LGP and the CPu (Figure 3A3). The orthogonal views (Figure 3B) and z-stack 3D images (Figure 3C) obtained with a confocal laser scanning microscope revealed that SOX9 positive Olig2-AS (arrows, Figures 3B,C) and SOX9 positive GFAP-positive astrocytes (arrowheads, Figures 3B,C) occupied adjacent but non-overlapping territories in the LGP. Semi-quantitative analyses revealed that 88% (204/232, n = 3) of SOX9-positive Olig2-AS were GFAP-negative and 12% (28/232, n = 3) of them co-expressed GFAP (Figure 3D). All of the SOX9-positive YFP-negative cells in the LGP co-expressed GFAP (281/281, n = 3) (Figure 3D). These findings illustrated that Olig2-AS constitute a sub-population of astrocytes distinct from GFAP-positive astrocytes.

Given the clear contrast in distribution patterns of Olig2-AS and GFAP-positive astrocytes, we mapped distribution of GFAP-positive astrocytes in the brain of GFAP^Cre-Ai39 mice and compared it with that of Olig2-AS. The GFAP^Cre-Ai39 mice showed strong YFP fluorescence in recombinant cells and the strong labeling was suitable for comparison with the strong fluorescence of Olig2-AS visualized after continuous oral administration of tamoxifen (see above). There is, however, a caveat: some of the recombinant cells may not be astrocytes but neurons in specific brain regions such as the OB and hippocampus, where neurogenesis takes place from GFAP-positive neuronal stem cells (Kempermann et al., 1997; Eriksson et al., 1998; Seri et al., 2001). In these regions, we additionally performed GFAP immunofluorescence to distinguish neurons from astrocytes (Figures S2A,B), since neurons quickly lose GFAP protein expression as they mature while astrocytes retain GFAP expression. The genetically labeled astrocytes in the GFAP^Cre-Ai39 mice are hereafter referred to as GFAP-astrocytes (GFAP-AS). Figures 4, 5 show low-magnification images of coronal brain sections of the same rostro-caudal position of both Olig2^CreER−Ai39 mice and GFAP^Cre-Ai39 mice. As a reference for brain nuclei, we illustrated the corresponding section images (Figures 4A, 5A) and the borders of anatomical regions (Figures 4B,C, 5B,C) based on the atlas image according to the mouse brain map in stereotaxic coordinates (Franklin and Paxinos, 1997). In the following sections, we focus on the major brain regions where a significant difference was observed between Olig2-AS and GFAP-AS distribution. Regarding the densities of labeled cells in each brain region, we applied arbitrary criteria (strong, moderate and low/absent) (for classification details, see Materials and Methods) and summarize the distribution patterns of Olig2-AS and GFAP-AS in the Table 2.

We reported previously that Olig2-AS expressed GAT-3 in the globus pallidus and the cerebral cortex (Tatsumi et al., 2016). In this study, we noticed that the whole brain distribution map of Olig2-AS was very similar to that of GAT-3 (Figures 6A,B, respectively). On the other hand, the glutamate transporters, GLT-1/GLAST distributions were much less overlapped with those of Olig2-AS (Figures 6A,C).

The transporter expression patterns prompted us to examine the spatial relationships between fine processes of Olig2-AS and axon terminals of inhibitory or excitatory neurons. The double immunofluorescence experiments using anti-GFP antibody and anti-vesicular GABA transporter (VGAT) antibody or anti-vesicular glutamate transporters (VGluT1/2) antibodies were performed in the LGP of Olig2^CreER -GFP mice (Figures 7A,B). The reason we chose the LGP is that the nucleus receives glutamatergic inputs from the subthalamic nucleus in addition to dense GABAergic inputs from the striatum (Kita and Kitai, 1987). We observed the strong expression of VGAT (Figure 7A2) and the moderate expression of VGluT1/T2 in the LGP (Figure 7B2) and Olig2-AS overlapped with both VGAT and VGluT1/2 immunoreactivities in the low magnification views (Figures 7A3,B3). At this light microscopic level, Olig2-AS appeared to be associated with both excitatory and inhibitory terminals, but the actual associations should be verified at an electron microscopic level. We therefore performed double immunoelectron microscopy with GFP and VGAT or VGluT1/2 antibodies in the Olig2^CreER-GFP mice. We found that GFP-immunoreactive astrocytic processes (arrows, Figures 7C1,C2) tended to be associated with VGAT metal particle-labeled axon terminals (Figure 7C1, T_in), but not with VGluT1/2 metal particle-positive terminals (Figure 7C2, T_ex). When we changed the labeling methods to the opposite way (axon terminals were labeled with GFP and astrocytic processes with metal particles), similar tendency was observed (Supplementary Figures S3A,B). We further observed the preferential association of astrocytic processes with GABAergic terminals in the cases that both astrocytic and neuronal elements were double-labeled with DAB (Figures 7D1,D2, Supplementary Figures S3C,D). Semi-quantitative analyses revealed that 56.8% (54/95, n = 3) of VGAT-immunoreactive terminals made contact with fine processes of Olig2-AS, while 15.4% (18/117, n = 3) of VGluT1/2 immunoreactive terminals did so (Figure 7E).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.