Nanobody-based RFP-dependent Cre recombinase for selective anterograde tracing in RFP-expressing transgenic animals

Screening of efficient mCherry-binding protein (MBP) pairs to design Cre recombinase dependent on RFPs

First, we aimed to construct Cre recombinase dependent on RFPs based on the reported tool named Cre-DOG30. In this system, N-terminal and C-terminal split Cre recombinase fragments are fused with specific nanobodies for target proteins, and target proteins mediate reunion of the split Cre recombinase fragments (Fig. 1a). To identify functional pairs of binding proteins, we selected 6 nanobodies and 2 DARPins previously reported to have a highly specific binding property to a monomeric RFP, mCherry31,32, and we constructed every combination (8 + 8 = 16 constructs). The codon-optimized DNA sequences were synthesized and were inserted instead of GFP-specific nanobodies in pAAV-EF1α-N-CretrcintG (Addgene ID: 69570) or pAAV-EF1α-C-CreintG (Addgene ID: 69571) using NheI and EcoRI sites. We renamed them as MBPs (mCherry-binding proteins) 1 – 8 in this study. MBPs 1-6 are nanobodies and MBPs 7 and 8 are DARPins. As a consequence, we obtained pAAV-EF1α-N-Cre-MBP(1-8)-WPRE and pAAV-EF1α-C-Cre-MBP(1-8)-WPRE.

Fig. 1: Luciferase assay screening of efficient MBP pairs for Cre-DOR.
figure 1

a Schematic presentation of the construct of Cre-DOR. N- or C-terminal split Cre recombinase is fused with mCherry-binding nanobody or DARPin. Binding of RFPs induces reunion of N-Cre and C-Cre into functional Cre recombinase. While mCherry and mRFP1 are monomeric, tdTomato is dimeric. bg Luciferase assay screening of functional pairs of MBPs for mCherry (b), mRFP1 (c), tdTomato (d), DsRed2 (e), No RFPs (f) and mRuby (g). Every intensity value was normalized by the maximum value and the normalized value is indicated as brightness of 256 levels of gray. VMA: vacuolar membrane ATPase subunit, N-VMA: N-terminal portion of VMA, C-VMA: C-terminal portion of VMA, 10 G: 10 glycine linker. Cre-DORN6C1, Cre-DORN5C4, and Cre-DORN8C8 are marked using *, †, and ‡, respectively.

Then we performed in vitro luciferase reporter assays to find adequate MBP pairs that could induce reunion to reconstruct an active Cre recombinase. N-Cre-MBP, C-Cre-MBP, FLEX-NanoLuc, and target RFPs were co-transfected into HEK293 cells by the calcium phosphate method. The FLEX switch consists of paired loxP and lox2272 sequences and enables the expression of a gene of interest only when Cre recombinase is functional33. NanoLuc is a small and bright luciferase from the deep-sea shrimp Oplophorus gracilirostris34. Recombinase activities were measured as luminescence derived from the bioluminescent reaction catalyzed by NanoLuc luciferase. We tested mCherry, mRFP1, and tdTomato as target proteins. These red fluorescent proteins were all derived from the same wild-type DsRed protein4. While mCherry and mRFP1 are monomeric, tdTomato is a tandem dimer of two subunits. We also tested mRuby, which is a monomeric variant of the red fluorescent protein eqFP611 derived from Entacmaea quadricolor35, as a negative control. As shown in Fig. 1b, c, heat maps of mCherry and mRFP1 showed similar patterns. The pair of N-Cre-MBP6 and C-Cre-MBP1 induced high activity for both mCherry and mRFP1. Twin pairs of the same MBP such as N-Cre-MBP1 and C-Cre-MBP1 or N-Cre-MBP2 and C-Cre-MBP2 showed weak reporter activities, possibly indicating competition for the same binding site by both N-Cre-MBP and C-Cre-MBP. In contrast, the heat map for tdTomato greatly differed from those of mCherry and mRFP1 (Fig. 1d). Unfortunately, we did not find any strong signal when we targeted the tetrameric RFP, DsRed (Fig. 1e). The twin pair of N-Cre-MBP8 and C-Cre-MBP8 showed the highest activity for tdTomato. The heat maps for mRuby and No RFPs (Fig. 1f, g) showed only weak recombinase activities around the maps. According to these heat maps obtained from luciferase assays, we selected the pair of N-Cre-MBP6 and C-Cre-MBP1 as a candidate pair for Cre-dependent on monomeric RFP.

Characterization of recombinase activity of Cre-DORs in vitro

Next, we investigated the recombination efficiency of the pair of N-Cre-MBP6 and C-Cre-MBP1 (Cre-DORN6C1) using a fluorescent protein reporter. Four kinds of plasmids including N-Cre-MBP6, C-Cre-MBP1, target RFPs, and FLEX-H2B-GFP were co-transfected into HEK293 cells by the calcium phosphate method (Fig. 2a). H2B-GFP shows nuclear localization because H2B (histone 2B) protein binds to the DNA in the nucleus. Recombinase activities were measured as H2B-GFP expression induced by FLEX switching (Fig. 2b). The fluorescent signal of GFP was enhanced by immunostaining using a GFP antibody. Quantitative cell counting of fluorescent images showed that 81.8 ± 1.5% of mCherry-positive cells were GFP-positive and that 74.1 ± 1.6% of mRFP1-positive cells were GFP-positive, while 5.6 ± 0.6% of mRuby-positive cells were GFP-positive (n = 8 each) (Fig. 2c, d). The recombination efficiency of Cre-DORN6C1 + mCherry was 14.6-times higher than that of Cre-DORN6C1 + mRuby. The Cre-DORN6C1 system was found to be dependent on all three components of target RFPs, N-Cre-MBP6, and C-Cre-MBP1 in the system. Removal of N-Cre-MBP6 or C-Cre-MBP1 resulted in total loss of reporter activity. Cell counting showed that 0.9 ± 0.1% of mRFP1-positive cells were GFP-positive without Ccre-MBP1 and that 0.8 ± 0.1% of mRFP1-positive cells were GFP-positive without Ncre-MBP8 (n = 8 each). In all cases, the percentages of RFP-positive cells in GFP-positive cells were higher than 90% possibly because of the transfection method (mCherry: 98.9 ± 0.3%, mRFP1: 98.3 ± 0.3%, mRuby: 91.0 ± 1.9%, mRFP1ΔCCre: 94.4 ± 3.7%, mRFP1ΔNCre: 88.5 ± 5.6%). The heatmaps in Fig. 1 are reliable to predict the efficiency of recombination in HEK293 cells to some extent. For example, we found that Cre-DORN5C4 recognizes mCherry, but much less effectively mRFP1 in Fig. 2f, and this finding is predictable by heat maps shown in Fig. 1.

Fig. 2: Functional assay of Cre-DOR in HEK293 cells.
figure 2

a Schematic illustration of Cre-DOR transfection in HEK293 cells. Four kinds of plasmids (NCre-MBP, CCre-MBP, FLEX-H2B-GFP, and target RFPs) were transfected in HEK293 cells to assess Cre-DOR recombination efficiency. b Illustration of specific expression of H2B-GFP induced by Cre-DOR activated by mRFP1. c Fluorescent images of reporter H2B-GFP expression in transfected HEK293 cells to assess Cre-DOR (N-Cre-MBP6 and C-Cre-MBP1) efficiency for target RFPs. d Quantification of cell counts in transfected HEK293 cells to assess Cre-DOR (N-Cre-MBP6 and C-Cre-MBP1) efficiency for all components of the system. e Fluorescent images of reporter H2B-GFP expression in transfected HEK293 cells to assess Cre-DOR (N-Cre-MBP5 and C-Cre-MBP4) efficiency for target RFPs. f, g Quantification of cell counts in transfected HEK293 cells to assess Cre-DOR (N-Cre-MBP5 and C-Cre-MBP4) efficiency for all components of the system. Data are means ± SEM (n = 8 or n = 4 in g). Scale bar = 50 μm.

We also tested the recombination efficiency of the pair of N-Cre-MBP5 and C-Cre-MBP4 (Cre-DORN5C4). Quantitative cell counting of fluorescent images showed that 57.9 ± 1.6% of mCherry-positive cells were GFP-positive and that 9.3 ± 1.1% of mRFP1-positive cells were GFP-positive, while only 0.9 ± 0.1% of mRuby-positive cells were GFP-positive (n = 8 each) (Fig. 2e, f). The recombination efficiency of Cre-DORN5C4 + mCherry was 61.0 times-higher than that of Cre-DORN5C4 + mRuby. The Cre-DORN5C4 system was also found to be dependent on all three components of target RFPs, N-Cre-MBP5, and C-Cre-MBP4 in the system. Removal of N-Cre-MBP5 or C-Cre-MBP4 resulted in total loss of reporter activity. Cell counting showed that 0.7 ± 0.1% of mRFP1-positive cells were GFP-positive without Ccre-MBP4 and that 0.8 ± 0.2% of mRFP1-positive cells were GFP-positive without Ncre-MBP5 (n = 8 each). Although Cre-DORN5C4 can induce more specific recombination dependent on mCherry, we decided to use Cre-DORN6C1 in the later in vivo experiments because of its high efficiency when it was applied for mRFP1.

In the heatmap of the luciferase assay shown in Fig. 1d, we found that the pair of N-Cre-MBP8 and C-Cre-MBP8 shows the highest recombination efficiency dependent on tdTomato. Therefore, we also tested the recombination efficiency of the pair of N-Cre-MBP8 and C-Cre-MBP8 (Cre-DORN8C8). Quantitative cell counting of fluorescent images showed that 46.9 ± 1.1% of tdTomato-positive cells were GFP-positive, while 12.8 ± 1.2% of mRuby-positive cells were GFP-positive (n = 4 each) (Fig. 2g). Although Cre-DORN8C8 shows mild specificity against tdTomato, we thought that the S/N ratio of Cre-DORN8C8 was not sufficiently high for use in vivo.

For benchmarking Cre-DOR efficiency, we performed a direct comparison of Cre-DORs with Cre-DOG (Supplementary Fig. 1). Quantitative cell counting of fluorescent images showed that 81.3 ± 0.8%, 72.3 ± 3.0%, and 60.4 ± 0.8% of the target fluorescent protein-positive cells were reporter fluorescent protein-positive in Cre-DORN6C1, Cre-DOG, and Cre-DORN5C4 experiments, respectively (n = 4 each). On the other hand, 6.5 ± 0.6%, 3.9 ± 0.6%, and 0.4 ± 0.1% of the non-target fluorescent protein-positive cells were reporter fluorescent protein-positive in Cre-DORN6C1, Cre-DOG, and Cre-DORN5C4 experiments, respectively (n = 4 each). These results show that Cre-DORN6C1 is more effective than Cre-DOG, while Cre-DORN6C1 has more background than Cre-DOG. These results also showed that Cre-DORN5C4 is more selective than Cre-DOG, while Cre-DORN5C4 is less effective than Cre-DOG.

To check the efficiency of Cre-DOR in the case of a moderate expression rate of target RFPs, we performed similar experiments using an mCherry-expressing HEK293 stable line (Supplementary Fig. 2). This stable line expresses mCherry in more than 90% of the cells (Supplementary Fig. 2a). Four kinds of plasmids including N-Cre-MBP6, C-Cre-MBP1, membrane-bound ALFA tag, and FLEX-nlsGFP were co-transfected into HEK293 cells (Supplementary Fig. 2b). Membrane-bound ALFA tag was transfected to confirm the transfected cells by immunocytochemistry using anti-ALFA tag antibody. Quantitative cell counting of fluorescent images showed that 79.8 ± 3.3% of the ALFA-positive cells were GFP-positive in the mCherry-expressing stable cell line and that 7.5 ± 2.1% of the ALFA-positive cells were GFP-positive in the control cell line (n = 4 each) (Supplementary Fig. 2c, d). These findings are very similar to the results shown in Fig. 2c, d.

It is well known that Cre recombinase and Flp recombinase have orthogonality, and Flp recombinase dependent on GFP (Flp-DOG) has already been reported36. Therefore, we performed experiments to investigate the orthogonality of Cre-DOR and Flp-DOG using a mixture of RFP-positive and GFP-positive cells (Supplementary Fig. 3). We confirmed that Cre-DOR induced mRFP1-dependent expression of FLEX-H2B-BFP, while Flp-DOG induced GFP-dependent expression of dFRT-BFP. These results suggest that it can be useful to employ Cre-DOR with GFP-dependent Flp recombinase at the same time to manipulate specific cells expressing GFP and/or RFP in the same animal.

Cellular localization of target RFPs affects Cre-DOR activity

Cre recombinase exerts its activity within the nucleus. Therefore, it is possible that cellular localization of target RFPs affects Cre-DOR recombinase activity. If the Cre-DOR system is dependent on nuclear localization of target RFP proteins, it suggests a limitation of Cre-DOR application for selective gene expression in cells expressing membrane proteins fused with RFPs such as channelrhodopsin 2-mCherry or hM3Dq-mCherry in transgenic animals. On the other hand, it also suggests that the Cre-DOR system can be used for clarification of nuclear translocation of proteins such as nuclear receptors.

To test this hypothesis, we created mCherry and tdTomato fused with various localization signal peptides: CAAX motif for membrane localization, NES for cytosolic localization, and NLS for nuclear localization (Fig. 3a). We observed clear intracellular translocalization of mRFP1 and tdTomato by being fused with these motifs. mCherry/tdTomato fused with CAAX were localized in the plasma membrane, mCherry/tdTomato fused with NES were localized in the cytosol, mCherry/tdTomato without any motif were localized in both the cytosol and nucleus, and nls-mCherry/tdTomato were localized in the nucleus (Fig. 3b). Next, we performed in vitro luciferase reporter assays to investigate the effect of cellular translocation on recombinase activity. Plasmids for N-Cre-MBP, C-Cre-MBP, FLEX-NanoLuc, and target RFPs were co-transfected into HEK293 cells. When Cre-DORN6C1 or Cre-DORN8C8 targets RFPs with localization signals, the luciferase assay showed a clear difference between the cellular localizations (Fig. 3c). A gradual incremental tendency of recombination activity among membrane-bound, cytosolic, and nucleic localization of target RFPs was observed. Quantitative analyses of luciferase assay data showed that recombinase activity is significantly different between Cre-DORN6C1/Cre-DORN8C8 + mCherry/tdTomato-NES and Cre-DORN6C1/Cre-DORN8C8 + nls-mCherry/tdTomato (P < 0.0001, Tukey’s multiple comparison test, n = 11). Note that average fluorescence intensity of mCherry-NES or tdTomato-NES is higher than that of nls-mCherry or nls-tdTomato.

Fig. 3: Cellular localization and recombinase activity of Cre-DOR.
figure 3

a Amino acid sequences of the CAAX motif (membrane localization signal), NES motif (nuclear export signal), and NLS motif (nuclear localization signal). b Fluorescent images of mCherry or tdTomato with or without (Control) each localization motif. Scale bar = 50 μm. c Luciferase assay of Cre-DORN6C1or Cre-DORN8C8 with RFPs having different cellular localizations. Data are means ± SEM. Statistical analyses were performed by one-way ANOVA followed by Tukey’s multiple comparison test (n = 11, ***P < 0.001). d Schematic representation of ligand-induced translocation of RFPs from the cytosol to the nucleus. Dexamethasone (Dex) induces translocation of GRs upon its binding. e Fluorescent images of mCherry-GR without or with Dex (1 μM, 1 h). Scale bar = 50 μm. f Luciferase assay of Cre-DORN6C1 activity targeting mCherry-GR without or with Dex (1 μM, 24 h). Data are means ± SEM. Statistical analyses were performed by one-way ANOVA followed by Tukey’s multiple comparison test (n = 8, **P < 0.01). g Schematic representation of light-induced translocation of RFPs from the nucleus to the cytosol. The LEXY domain consists of a modified AsLOV2 domain with NES. Blue light induces exposure of the NES and results in translocation of RFPs. h Fluorescent images of nls-mCherry-LEXY with or without blue light illumination (465 nm, 3.7 W/m2). Scale bar = 50 μm. i Luciferase assay of Cre-DORN6C1activity targeting nls-mCherry-LEXY without or with blue light illumination (n = 24, 12). Data are means ± SEM. Statistical analyses were performed by Student’s t-test (***P < 0.001). In Fig. 3e, h, nuclei are visualized by fluorescence from H2B-BFP that was co-transfected with mCherry-GR or nls-mCherry-LEXY.

Next, we aimed to control Cre-DOR activity by using chemical ligands. Glucocorticoid receptor (GR) is a nuclear receptor and it is translocated into the nucleus after binding its ligand, glucocorticoid37 (Fig. 3d). Ligand-induced translocation of GR has been detected by addition of a GFP to the N-terminus of GR38,39. Therefore, we attached RFPs to human glucocorticoid receptor alpha (hGRα) to control Cre-DOR activity. We found that mCherry-GR was localized in the cytosol without its ligand, dexamethasone (Dex) and that it translocated to the nucleus after incubation with Dex (1 μM, 1 h) (Fig. 3e). In Fig. 3e, nuclei are visualized by fluorescence from Histone 2B-BFP (pAAV-EF1α-H2B-BFP-WPRE) that was co-transfected with mCherry-GR. Cre recombinase activity of Cre-DORN6C1 targeting mCherry-GR was strongly increased by the presence of Dex (Fig. 3f).

An increase in Cre recombinase activity by nuclear localization of RFPs suggests that Cre-DOR activity can be manipulated by controlling the intracellular localization of target RFPs. To test this hypothesis, we used a light-inducible nuclear export domain called LEXY40. LEXY consists of an engineered LOV2 domain from Avena sativa phototropin-1 (AsLOV2), in which the C-terminal Jα helix was converted into an artificial NES. In the dark, the NES is tightly packed against the AsLOV2 core and is thus inactive. Exposure to blue light induces unfolding of the modified Jα helix, uncovering the NES (Fig. 3g). We confirmed that nls-mCherry-LEXY was localized mainly in the nucleus in the dark, and blue light illumination (465 nm, 7 W/m2) induced translocation of fused RFPs into the cytosol in 30 min (Fig. 3h). In Fig. 3h, nuclei are visualized by fluorescence from Histone 2B-BFP that was co-transfected with nls-mCherry-LEXY. We found that blue light illumination inhibited Cre recombination activity of Cre-DORN6C1 targeting nls-mCherry-LEXY as indexed by luciferase activity (Fig. 3i).

Functional assay of Cre-DOR in vivo using AAV vectors

To examine whether Cre-DORN6C1 functions in living animals, we generated AAV vectors encoding N-Cre-MBP6, C-Cre-MBP1, FLEX-nlsGFP, and target RFPs. GFP tagged with a nuclear localization signal (nlsGFP) is localized mainly in the nucleus. 600 nl of a mixture of virus vectors for Cre-DORN6C1, FLEX-nlsGFP (1 × 1012 vg/ml each), and target mRFP1 (5 × 1010 vg/ml) was unilaterally injected into the right side M1 cortex of wild-type 10-week-old male mice (Fig. 4a). The titer of the target RFP-expressing vector was lowered to induce scattered expression and make it easy to count fluorescent protein-expressing cells separately. Recombinase activities were measured as nlsGFP expression induced by FLEX switching (Fig. 4b). Four weeks after injection, mice were sacrificed for immunohistochemistry and brain slices were stained with anti-GFP. Clear expression of nlsGFP at the injected site in the M1 cortex was observed (Fig. 4c). Quantitative cell counting of fluorescent images showed that 50.2 ± 2.5% of mRFP1-positive cells were GFP-positive and that 93.5 ± 0.6% of GFP-positive cells were mRFP1-positive (n = 5 each) (Fig. 4d, e). The expression efficiency of GFP in the center area of injection was higher than that in the peripheral area. To check the specificity of the Cre-DORN6C1 system in vivo, we injected vectors for Cre-DORN6C1, FLEX-nlsGFP (1 × 1012 vg/ml each), and mRuby (5 × 1010 vg/ml) as a control (Fig. 4f). While we observed comparative amounts of mRuby-expressing neurons in the M1 cortex, we found only sparce nlsGFP-expressing neurons in the same area (Fig. 4g). Quantitative cell counting of nlsGFP and mRuby-positive cells showed a clear difference between Cre-DORN6C1 + mRFP1-injected mice and Cre-DORN6C1 + mRuby-injected mice. The cell counting showed that 1.5 ± 0.5% of mRuby-positive cells were GFP-positive and that 13.3 ± 4.1% of GFP-positive cells were mRuby-positive (n = 5 each) (Fig. 4d, e). The recombination efficiency of Cre-DORN6C1 + mRFP1 was 34.4-times higher than that of Cre-DORN6C1 + mRuby in these experiments. All these data suggest in vivo specificity of the Cre-DORN6C1 system using AAV vectors.

Fig. 4: Functional assay of Cre-DORN6C1 in vivo.
figure 4

a Injection schema of the Cre-DORN6C1 test with mRFP1 in wild-type mice. Four kinds of virus (NCre-MBP6, CCre-MBP1, FLEX-nlsGFP, and mRFP1) were injected in the M1 cortex at the same time. b Schematic representation of specific expression of nlsGFP induced by Cre-DOR activated by mRFP1. c Fluorescent images of the M1 cortex. Scale bar = 100 μm. d Quantification of cell counts to assess the efficiency of Cre-DORN6C1 (n = 5 each). e Quantification of cell counts to assess the fidelity of Cre-DORN6C1 (n = 5 each). f Injection schema of the Cre-DORN6C1 test with mRuby in wild-type mice. g Fluorescent images of the M1 cortex in which the four viruses were injected. Scale bar = 100 μm. Data are means ± SEM.

We also confirmed that Cre-DOR can be used in combination with intravenous injection of AAV vectors. AAV-EF1a-mRFP1-WPRE or AAV-EF1a-mRuby-WPRE (200 ul; 2 × 1011 vg/mouse) was injected systemically (Supplementary Fig. 4a). They are packaged by AAV PHPeb and can infect through the blood-brain barrier. Within the same day, 600 nl of a mixture of virus vectors for Cre-DORN6C1and FLEX-nlsGFP (1 × 1012 vg/ml each) was unilaterally injected into the right side M1 cortex of wild-type 10-week-old male mice (Supplementary Fig. 4a). Four weeks after injection, mice were sacrificed for immunohistochemistry and brain slices were stained with anti-GFP. Clear expression of nlsGFP at the injected site in the M1 cortex was observed (Supplementary Fig. 4b). While we observed comparative amounts of mRuby-expressing neurons in the M1 cortex, we found only sparce nlsGFP-expressing neurons in the same area. Quantitative cell counting of fluorescent images showed that 49.9 ± 3.9% of the mRFP1-positive cells were GFP-positive and that 93.3 ± 0.4% of the GFP-positive cells were mRFP1-positive (n = 4 each) (Supplementary Fig. 4c). Quantitative cell counting of nlsGFP and mRuby-positive cells showed a clear difference between Cre-DORN6C1 + mRFP1-injected mice and Cre-DORN6C1 + mRuby-injected mice. The cell counting showed that 1.7 ± 0.7% of the mRuby-positive cells were GFP-positive and that 10.5 ± 3.6% of the GFP-positive cells were mRuby-positive (n = 4 each) (Supplementary Fig. 4d). The recombination efficiency of Cre-DORN6C1 + mRFP1 was 29.9-times higher than that of Cre-DORN6C1 + mRuby in these experiments. All of these data confirmed in vivo specificity of the Cre-DORN6C1 system using AAV vectors.

Functional assay of Cre-DOR in mRFP1-expressing transgenic mice

Next, we examined selective expression by Cre-DORN6C1 in mRFP1-expressing transgenic animals. In Esr2-mRFP1 mice, neurons in the paraventricular nucleus (PVN) are visualized by mRFP1. 1 μl of a mixture of AAV9-EF1α-NCre-MBP6-WPRE (6 × 1012 vg/ml), AAV9-EF1α-CCre-MBP1-WPRE (6 × 1012 vg/ml) and AAV9-CAG-FLEX-palGFP-WPRE (6 × 1012 vg/ml) was injected in the PVN of Esr2-mRFP1 transgenic mice (Fig. 5a). Recombinase activities were measured as expression of GFP tagged with a palmitoylation signal (palGFP) induced by FLEX switching (Fig. 5b). palGFP is sorted to the plasma membrane and has been used to trace neuronal fibers anterogradely41,42. Four weeks after injection, mice were sacrificed for immunohistochemistry and brain slices were stained with anti-GFP and anti-mRFP1. Clear expression of palGFP at the injected site in the PVN was observed (Fig. 5c). It has been reported that mRFP1-expressing neurons in the PVN of Esr2-mRFP1 mice include oxytocin neurons43 and oxytocin neurons send their axons into the posterior pituitary. In accordance with these previous findings, we observed clear projection from the palGFP-expressing neurons in the PVN and dense axonal terminals in the posterior pituitary (Fig. 5d). Quantitative cell counting of palGFP-positive cells in the PVN showed a clear difference between mRFP(+) mice and mRFP(−) mice (Fig. 5e). The cell counting showed that 24.1% of mRFP1-positive neurons in the PVN express palGFP on average (Fig. 5f). These results showed the usability of Cre-DORN6C1 for detection of selective neural projection in mRFP1-expressing transgenic animals. We also confirmed that Cre-DORN6C1 can be functional in other parts of the brain such as the islands of Calleja (ICj) in Esr2-mRFP1 mice (Supplementary Fig. 5).

Fig. 5: Anterograde tracing using Cre-DOR in Esr2-mRFP1 transgenic mice.
figure 5

a Stereotaxic injection schema of Cre-DORN6C1 in Esr2-mRFP1 transgenic mice. Three kinds of virus (NCre-MBP6, CCre-MBP1, FLEX-palGFP) were injected in the paraventricular nucleus (PVN). b Schematic representation of specific expression of palGFP induced by Cre-DOR activated by mRFP1. c Immunofluorescent images of the PVN of injected mice. Scale bar = 100 μm. d Representative images of axonal projection from palGFP-expressing neurons in the PVN of Esr2-mRFP1 transgenic mice. AL anterior lobe, PL posterior lobe. Scale bar = 100 μm. e Quantification of mRFP1-induced expression of palGFP in WT and Tg Esr2-mRFP1 mice. f Cell counting results showing the average percentage of palGFP-positive cells in the PVN of the Cre-DOR virus-injected Esr2-mRFP1 transgenic mice.

Anterograde tracing of mRFP1-expressing neurons in Grpr-mRFP1 rats

Finally, we examined selective expression by Cre-DORN6C1 in mRFP1-expressing transgenic animals. In gastrin-releasing peptide receptor (Grpr)-mRFP1 transgenic rats, neurons in the posterior amygdala are visualized by mRFP1. 1 μl of a mixture of AAV9-EF1α-NCre-MBP6-WPRE (6 × 1012 vg/ml), AAV9-EF1α-CCre-MBP1-WPRE (6 × 1012 vg/ml) and AAV9-CAG-FLEX-palGFP-WPRE (6 × 1012 vg/ml) was injected in the medial amygdala area of male Grpr-mRFP1 transgenic rats (Fig. 6a). Four weeks after injection, rats were sacrificed for immunohistochemistry and brain slices were stained with anti-GFP and anti-mRFP1. Clear and selective expression of palGFP at the injected site in the posterodorsal medial amygdala (MePD) was observed (Fig. 6b). We observed clear projection from the palGFP-expressing neurons in the MePD. We found a bundle of smooth passing fibers in the stria terminalis (ste) and dense axonal terminals with varicosity in the posterior bed nucleus of the stria terminalis (BSTp or STMP) (Fig. 6c). These findings suggest that mRFP-expressing neurons in the MePD send their axons to the BSTp (Fig. 6e). Finally, we confirmed this neural projection using a retrograde tracer. We injected 300 nl of green retrobeads in the BSTp of Grpr-mRFP1 transgenic rats (Fig. 6d). One week after injection, rats were sacrificed. We detected some mRFP1 neurons that included green retrobeads in the MePD. These results support our idea that mRFP-expressing neurons in the MePD send their axons to the BSTp (Fig. 6f).

Fig. 6: Anterograde tracing using Cre-DOR in transgenic mRFP1-expressing rats.
figure 6

a Stereotaxic injection schema of Cre-DORN6C1 in Grpr-mRFP1 transgenic rats. Three kinds of virus (NCre-MBP6, CCre-MBP1, FLEX-palGFP) were injected in the medial amygdala. b Immunofluorescent images of the MePD of injected rats. Scale bar = 100 μm. c A representative image of axonal projection from palGFP-expressing neurons in the MePD of a Grpr-mRFP1 transgenic rat. Scale bar = 100 μm or 1 mm. d Injection schema of green retrobeads in a Grpr-mRFP1 rat. Scale bar = 100 μm or 1 mm. e Illustration of anterograde tracing using Cre-DOR in Grpr-mRFP1 rats. f Illustration of retrograde tracing using green retrobeads in Grpr-mRFP1 rats. All the rat brain atlas images were derived from Swanson, L.W. (2004) Brain maps: structure of the rat brain, 3rd edition (Creative Commons Attribution-NonCommercial 4.0 International License: creativecommons.org/licenses/by-nc/4.0/) with permission of Dr. Swanson.

To visualize each neuronal morphology, we also performed sparse labeling of mRFP-expressing neurons in the preoptic area of Grpr-mRFP1 transgenic rats. Three μl of a mixture of AAV9-EF1α-NCre-MBP6-WPRE (3 × 1011 vg/ml), AAV9-EF1α-CCre-MBP1-WPRE (3 × 1011 vg/ml) and AAV9-CAG-FLEX-hrGFP-WPRE (3 × 1011 vg/ml) was unilaterally injected into the preoptic area of 10–15–week-old male Grpr-mRFP1 transgenic rats (Supplementary Fig. 6a). Although the expression of hrGFP was sparse, 92.5% of the hrGFP-expressing neurons were confirmed to be mRFP1-expressing neurons. It is likely that Grpr-positive neurons in the preoptic area are peptidergic neurons from their neuroanatomical features. Numerous axonal varicosities and aspiny dendrites are features of many peptidergic neurons. Indeed, hrGFP-labeled neurons appear to be aspiny (Supplementary Fig. 6b), and we found numerous axonal varicosities labeled with hrGFP in the preoptic area (Supplementary Fig. 6b, arrowheads) or more caudal and ventrolateral region from the injection site of the preoptic area.

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