Fig 2: Oxt neuron input-output wiring diagram. Schematic summary of monosynaptic input and axonal output connectivity of Oxt neurons. Color of each ROI is related to nine functional circuits. ROI size is correlated with Oxt output degree as done in Figure 2. Areas providing the monosynaptic input are highlighted with asterisk (*). Number of * indicated input strength: * < 10 cells, 10 = ** < 100, 100 = ***. Monosynaptic input areas without receiving Oxt output in the SO-Oxt map were indicated with underlined fonts. All abbreviations for brain regions can be found in Table 2.

Fig 3: Treatment with an agonist for ß2 adrenergic receptor attenuates the anti-tumor effect of OxtPVN neuron activation.(A) Control and AAV-hSyn-DIO-hM3Dq-mCherry (hM3Dq) viruses were injected into the paraventricular nucleus (PVN) of male adult OxtCre mice. Colitis-associated cancer (CAC) was then induced using azoxymethane (AOM) and dextran sodium sulfate (DSS). These mice were i.p. administered with clozapine-N-oxide (CNO) every other day and i.p. injected with saline or isoprenaline, a ß2 adrenergic receptor agonist, on a daily basis. After 3 weeks of treatment, mice were perfused with 4% paraformaldehyde (PFA). For control, double immunofluorescence staining for c-Fos (gray) and oxytocin (Oxt) (red) was performed. For hM3Dq, immunofluorescent staining for c-Fos (green) was performed and Oxt neurons were identified using hM3Dq-mCherry (red). Cell nuclei were counterstained with DAPI (blue). Scale bars, 50 µm. (B) The percentage of Oxt neurons expressing c-Fos in the PVN. ISO, isoprenaline. n = 7 (control, saline) or 6 (all other groups) mice per group. (C) Adult OxtCre mice were injected with adeno-associated viruses (AAVs) into the PVN. CAC was then induced using AOM and DSS. Subsequently, these mice were i.p. administered with CNO every other day and i.p. injected with saline or isoprenaline on a daily basis. These treatments were continued for 3 weeks (see also Figure 6—figure supplement 1C). Representative images of colorectal tissue after the treatments are shown. White eclipse outlines individual tumor. (D and E) Tumor number (D) and diameter (E). n = 7 (control, saline) or 6 (all other groups) mice per group. (F) Colorectal length. n = 7 (control, saline) or 6 (all other groups) mice per group. (G and H) Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) of tumor tissue. Representative images (G) and the density of PCNA-positive cells (H) are shown. Scale bars, 50 µm. n = 4 (saline) or 5 (ISO) mice per group. (I and J) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of tumor tissue. Representative images (I) and the density of TUNEL-positive cells (J) are shown. TUNEL labeling is in red. Cell nuclei were counterstained with DAPI (blue). Scale bars, 20 µm. n = 4 mice per group. Data are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Bonferroni’s post hoc test (B, D, E, H, J). Figure 6—source data 1.Source data for Figure 6, panels B, D-F, H and J.

Fig 4: Brain oxytocin (Oxt) receptor is crucial for OxtPVN neuron activation to suppress colitis-associated cancer (CAC).(A) Adult male OxtCre mice (2 months of age) were injected with AAV-hSyn-GFP (control) or AAV-hSyn-DIO-hM3Dq-mCherry (hM3Dq) viruses into the paraventricular nucleus (PVN), and then colitis-associated cancer (CAC) was induced using azoxymethane (AOM) and dextran sodium sulfate (DSS). Subsequently, these mice were administered with clozapine-N-oxide (CNO) (i.p.), and artificial cerebrospinal fluid (aCSF) or L-368,899 (i.c.v.), an Oxt receptor antagonist (OTR anta), every other day for 3 weeks. Mice were then perfused with 4% paraformaldehyde (PFA). For control, double immunofluorescence staining for c-Fos (gray) and Oxt (red) was performed. For hM3Dq, immunofluorescent staining for c-Fos (green) was performed, and Oxt neurons were identified using hM3Dq-mCherry (red). Cell nuclei were counterstained with DAPI (blue). Scale bars, 50 µm. (B) The percentage of Oxt neurons expressing c-Fos in the PVN. n = 7 (hM3Dq, OTR anta) or 6 (all other groups) mice per group. (C) The OxtCre mice (2 months of age) were injected with indicated adeno-associated viruses (AAVs) into the PVN, and then CAC was induced using AOM and DSS. Subsequently, these mice were administered with CNO (i.p.), as well as aCSF or L-368,899 (i.c.v.), the OTR antagonist (OTR anta), every other day for 3 weeks (see also Figure 2—figure supplement 1A). Representative images of colorectal tissue after the treatments are shown. White eclipse outlines the individual tumor. (D and E) Tumor number (D) and diameter (E). ns, not significant. n = 7 (hM3Dq, OTR anta) or 6 (all other groups) mice per group. (F) Colorectal length. n = 7 (hM3Dq, OTR anta) or 6 (all other groups) mice per group. (G and H) Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) of tumor tissues. Representative images (G) and the density of PCNA-positive cells (H) are shown. Scale bars, 50 µm. ns, not significant. n = 4 mice per group. (I and J) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of tumor tissues. Representative images (I) and the density of TUNEL-positive cells (J) are shown. TUNEL labeling is in red. Cell nuclei were counterstained with DAPI (blue). Scale bars, 20 µm. ns, not significant. n = 4 mice per group. Data are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Bonferroni’s post hoc test. Figure 2—source data 1.Source data for Figure 2, panels B, D-F, H and J.

Fig 5: Comparison between the Oxt output and Oxtr expression. A, Composite images of representative Oxt neuronal projection outputs (red: combined from both the PVH and the SO) and Oxtr expression (green) across the mouse brain. See also Movie 3. B, Quantitative comparison of relative Oxt projection pattern and Oxtr expression. Note that the cerebral cortex has very small Oxt/Oxtr ratio while the hindbrain and the midbrain shows higher ratio. C, Correlation between Oxt projection and Oxtr density (Spearman nonparametric correlation, *p < 0.05). Note no significant correlation across the whole-brain areas despite the significant correlation in the thalamus and the medulla. D, Examples of Oxt long range projection touching the surface of all major ventricles.