Supplemental Information. A Labeled-Line Neural Circuit. for Pheromone-Mediated Sexual Behaviors in Mice

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1 Neuron, Volume Supplemental Information A Labeled-Line Neural Circuit for Pheromone-Mediated Sexual Behaviors in Mice Kentaro K. Ishii, Takuya Osakada, Hiromi Mori, Nobuhiko Miyasaka, Yoshihiro Yoshihara, Kazunari Miyamichi, and Kazushige Touhara

0 Figure S. Whole brain analysis of anterograde mapping from VRp-expresing Sensory Neurons, related to Figure (A) Table showing brain regions analyzed for tdtomato+ cells in day, and.

2 0 Figure S. Whole brain analysis of anterograde mapping from VRp-expresing Sensory Neurons, related to Figure (A) Table showing brain regions analyzed for tdtomato+ cells in day, and. The intensity of anterograde labeling was determined by the % of animals with tdtomato+ cells in the area among all animals; blank: < 0%, +: 0%, ++: 0%, +++: 0%. Day, n =, day, n = 0, day, n =. (B) Schematic and representative coronal section showing tdtomato+ (shown in red) cells in day -sacrificed animals. DAPI staining is shown in blue. Scale bar, 0 μm. Abbreviations are as following. LSV, lateral septum nucleus ventral part. BNST, bed nucleus of stria terminalis. SHy, septohypothalamic nucleus. DEn, dorsal endopiriform nucleus. Pir, piriform cortex. StHy, striohypothalamic nuclesus. MPA, medial preoptic area. EA, sublenticular extend amygdala. MCPO, magnocell preoptic nucleus. VEn, ventral endopiriform nucleus. CxA, cortex-amygdala

3 0 transition area. VCl, ventral part of claustrum. PaXi, paraxiphoid nucleus of thalamus. VMH, ventromedial hypothalamic nucleus. Arc, arcuate nucleus. MeA, medial amygdala. CeA, central amygdala. BMA, basomedial amygdala. ACo, anterior cortical amygdala. PLCo, posterolateral cortical amygdala. BLV, basolateral amygdala ventral part. LaDL, lateral amygdala dorsolateral part. RMM, retromammillary nucleus medial part. PM, premammillary nucleus. DG, dentate gyrus. Or, oriens layer hippocampus. AHi, amygdalohippocampus. VS, ventral subiculum. PMCo, posteromedial cortical amygdala. BMP, basomedial amygdala posterior part. BLP, basolateral amygdala posterior part. RAPir, rostral amygdalopiriform. MnR, median raphe. GrDG, granular dentate gyrus. LMol, lacunosum moleculare. STr, subiculum transition area. Ent, entorhinal cortex.

4 Figure S. Histochemical Characterization of ESP-responding Neurons and Projection Neurons in the MeA, related to Figure (A) Number of cfos+ cells per mm in the MeApd and MeApv of mice exposed to ESP (black) or the control (white) in female (left) and male (right) mice. n = for ESP and n = for control (females); n = each for ESP and control (males). Student s t-test with Bonferroni correction, *p < 0.0, **p < 0.0.

5 0 (B) Spatial organization of MeA sub-nuclei along the anterior-posterior axis. Eight typical coronal sections are shown that approximately correspond to the eight sections analyzed below. (C and D) Distribution of cfos+ cells in ESP-exposed female (B) or male (C) mice. Cells were counted from eight unilateral coronal sections from Bregma.0 mm to. mm. Female mice, control n = 0, ESP n =. Male mice, control n =, ESP n =. (E) Representative coronal sections showing cfos (green) and vglut+ or GAD+ (red) mrna expression in the MeApv following exposure to the ESP stimulus. Small panels represent higher magnification of each boxed area in merged and separated channels. (F) Proportion of cfos+ and marker+ (vglut+ or GAD+) cells among all cfos+ cells. n = animals each. (G) Representative coronal sections showing VMHd- or MPA-projectors labeled with red Retrobeads and vglut+ or GAD+ mrna-expressing cells. vglut+ and GAD+ were labeled with different RNA probes on the same section. Signals are pseudo-colored in green, and co-localization with Retrobeads is shown individually. (H) Number of vglut+, GAD+, or marker-negative cells among Retrobeads+ cells per MeApv section. Percentage of Retrobeads+ and vglut+ cells among total Retrobeads+ cells is shown in bar graphs. n = animal. Scale bar, 00 μm. Error bars, SEM.

6 0 Figure S. Additional Analysis on Axonal Arbor Distribution of MeApv vglut+ Neurons, related to Figure (A and B) Quantitative analysis for normalization methods used in the axon mapping data of vglut+ MeApv neurons (Figure ). We did not observe a significant correlation between mgfp+ area in CAV-injected brain regions (y-axis) and the number of source cells in the MeApv for VMHd- and MPA- projectors (A), suggesting that the axonal distributions are heterogeneous even among the same type of projectors. In contrast, we observed a significant correlation (p = ) between mgfp+ area in CAV-injected regions (y-axis) and total mgfp+ area in all counted regions (x-axis) (B). Thus, we used the proportion of mgfp+ area in a single brain region among the total mgfp+ area in all regions as a normalization strategy.

7 0 (C) Representative coronal section of the VMH showing mgfp (green in the left panel, black in the middle panel) and SypRuby (red in the left panel, black in the right panel) signals of VMHdprojectors in the MeApv. Scale bar, 00 μm. (D) Correlation between the number of SypRuby+ dots (y-axis) and the area of mgfp+ axons (xaxis). In this analysis, we pooled VMHd and MPA data from all VMHd-projectors, MPAprojectors, and pan-vglut labeling in male and female mice. We observed a positive correlation with R = 0.. (E) Correlation between the proportion of mgfp+ area ([VMHd]/[VMHd + MPA], x-axis) and the proportion of the number of SypRuby+ dots ([VMHd]/[VMHd + MPA], y-axis). We observed a strong positive correlation with R = 0..

0 0 Figure S. The VMHvl Is Necessary for Basal Lordosis in Female Mice, related to Figure (A) Schematic of the experimental setup and time line of procedures.

8 0 0 Figure S. The VMHvl Is Necessary for Basal Lordosis in Female Mice, related to Figure (A) Schematic of the experimental setup and time line of procedures. AAV-FLEx-DTA or AAV- FLEx-GFP as a control was injected into the VMHvl of female Esr-Cre mice. (B) Representative coronal sections showing Esr mrna expression (red) in the VMHvl and in the arcuate nucleus (Arc) of AAV-FLEx-DTA-injected mice (Esr::DTA) and AAV-FLEx-GFPinjected mice (Esr::GFP). (C) Representative coronal sections showing SF mrna expression (red) in the VMHd of Esr::GFP and Esr::DTA mice. (D) Number of Esr+ cells in the VMHvl and Arc per unilateral section. Esr+ neurons were drastically reduced in the VMHvl, but not in the Arc. Esr::GFP, n = unilateral region. Esr::DTA, n =. (E) Number of SF+ cells in the VMHd per unilateral section. The number of SF+ neurons was also reduced by expressing DTA in Esr+ neurons, likely due to weak Cre activity in SF+ neurons in Esr-Cre mice. Esr::GFP, n =. Esr::DTA, n =. (F) Raster plots of behavior episodes during the mating assay. (G) Lordosis percentage of Esr::GFP and Esr::DTA mice exposed to ESP or the control stimulus. Note that addition of ESP to Esr::DTA mice did not rescue the basal lordosis defects, although we cannot exclude the possibility that this may be caused by non-specific ablation of a subset of SF+ neurons in the VMHd. Grey lines, trials from individual animals. Esr::GFP, n = animals. Esr::DTA, n =.

9 Error bars, SEM. Scale bar, 00 µm. (D) and (E) Unpaired Student s t-test, **p < 0.0. (G) Paired Student s t-test, **p < 0.0.

10 0 Figure S. Additional Analysis of the Representation of Snake Signals and their Effect on Female Sexual Behaviors, related to Figures and (A) Number of cfos-expressing VMHd-projectors in the MeApv per section in female or male mice (left) and number of cfos-expressing MPA-projectors in the MeApv per section in female or male mice (right). Data for the control stimulus are the same as those used in Figure G. The number of animals used for each condition is shown below each bar. (B) Lordosis percentage of wild-type female mice pre-exposed to snakeskin or a control stimulus. Control, n = ; snakeskin n =. (C) Representative coronal sections from catfish analysis by using cfos coding (green) and intron (red) probes in the AOB. The stimulation conditions are shown in each image as in Figure A. Insets show high magnification images of the two boxed areas in each panel. Cells were counted from the glomerulus layer (GL) and the mitral cell layer (MCL).

11 0 (D) Proportion of dual-positive (nuclear and cytoplasmic cfos+) cells among total nuclear cfos+ expressing cells (A-index) in the GL and MCL. The number of animals used for each condition is shown below each bar. Control, Same, and Different conditions were collected as described in Figure B. (E) Representative coronal sections from catfish analysis in the MeApv by using NRa coding (green) and intron (red) probes. The stimulation conditions are shown in each image. Insets show high magnification images of the two boxed areas in each panel. (F) A-index in each condition of MeApv catfish in male and female mice as in panel D. D, dorsal, A, anterior, M, medial. Error bars, SEM. (A) and (B), Students t-test. (D) and (F), Oneway ANOVA on conditions with Bonferroni correction for female data set (D) and for data set of each sex (F). a vs. b, a vs. c, b vs. c, p < 0.0. Scale bars, 00 μm. 0

13 0 0 Figure S. Additional Analysis of TRAP in the VMHd, related to Figure (A) Representative images of coronal sections from ESP-, snakeskin- or basal activity-trap mice exposed to ESP. cfos+ cells (green) were detected by anti-cfos staining. Bottom panels show merged and separated channels for mcherry and cfos, with yellow arrowheads representing dual-positive, and white arrowheads representing cfos+ only cells. (B) Representative coronal section showing cfos+ and mcherry+ in the dorsal hypothalamus (DH) and arcuate nucleus (Arc). (C E) Number of mcherry+ and double+ (cfos+ mcherry+) cells per unilateral section, % of double+ cells among all mcherry+ cells or all cfos+ cells in VMHd (C), Arc (D) and DH (E). Basal, n = unilateral regions, ESP, n =, snakeskin, n =. (F) Distribution of fiber tips in the animals analyzed in Figures F and G. Red x, ESP-TRAP mice, green x, snakeskin-trap mice. P, posterior. (G) A representative image showing the location of optic fibers above the VMHd, and eyfp expression in the VMHd. (H) Representative coronal sections showing eyfp (green) and cfos mrna (red)-expressing cells from the ESP- or snakeskin-trap animals exposed to ESP. Bottom panels show separated channels of the areas indicated by white dashed boxes in the top images. (I) Number of eyfp+ and double+ (cfos+ eyfp+) cells per unilateral section, % of double+ cells among all cfos+ cells or all eyfp+ cells. ESP, n = unilateral VMHd, snakeskin, n =. Scale bar, 00 µm (A, B and H) or mm (G). One-way ANOVA with Bonferroni correction (C E) or Student s t-test (I), *p < 0.0, **p < 0.0.

14 0 Figure S. SF+ VMHd Neurons Are Homogeneous with Respect to Their Axonal Projections, related to Figure (A) A representative coronal section showing mgfp and SypRuby expression in VMHd neurons following CAV-FLEx loxp -Flp injection into the PAGd and AAV-FLEx FRT -mgfp injection into the VMHd of female SF-Cre mice. Scale bar, 00 µm. (B) Fraction of source cells (defined by the expression of mgfp) in the VMHd for each injection condition. The number of animals for each condition is indicated at the bottom. Almost all source cells were restricted to the VMHd area. (C and D) Quantitative analysis for normalization methods. We observed a strong positive correlation (p = 0.00) between mgfp+ area in CAV-injected brain regions (y-axis) and the number of VMHd source cells (x-axis) for PAGd- or MPA-projectors (C). This was in contrast to the situation with MeApv source cells (Figure SA), suggesting that the axonal distribution of

15 0 SF+ VMHd neurons is highly homogeneous. We also observed a strong positive correlation (p = 0.00) between mgfp+ area in CAV-injected regions (y-axis) and the total mgfp+ area in all analyzed regions (x-axis) for PAGd- or MPA-projectors (D). Thus, for normalization, we divided the mgfp+ area in each region by the number of source cells and by the total mgfp+ area in all analyzed regions. (E) Representative images of binarized mgfp signals of a coronal section across eight brain regions collected from animals in which PAGd-projectors, MPA-projectors, or pan-vmhd projectors (CAV local injection) were labeled. (F and G) Proportion of mgfp+ area in each region normalized to the total mgfp+ area of brain regions (F), or mgfp+ area in each region normalized to the number of source cells in the VMHd (G). Error bars, SEM. Abbreviations, Sept, septum, Rapir, rostral amygdalopiriform area, lateral A, lateral amygdaloid nucleus, SN, substantia nigra, SubB, subbrachial nucleus, PAGd, dorsal periaqueductal gray. For other abbreviations, see Figures and.

16 0 Figure S. Histological Verification of Cannula Locations in the MPA and PAGd, related to Figure (A and B) Representative coronal images showing traces of the cannula for CNO infusion above the PAGd (A) or MPA (B). Scale bar, mm. (C F) Schematics showing the locations of cannula tips from the coronal (C and E) and sagittal (D and F) views, in the PAGd (C and D) or MPA (E and F). Grey circles indicate individual cannula tips; yellow circular shadows indicate the estimated areas where the injected CNO may spread based on the data described previously (Stachniak et al., 0). The position of each cannula was judged to be far enough from the VMHd, and therefore, drugs do not affect the cell bodies of SF+ neurons.

Nature Neuroscience: doi: /nn Supplementary Figure 1. Diverse anorexigenic signals induce c-fos expression in CEl PKC-δ + neurons

Nature Neuroscience: doi: /nn Supplementary Figure 1. Diverse anorexigenic signals induce c-fos expression in CEl PKC-δ + neurons Supplementary Figure 1 Diverse anorexigenic signals induce c-fos expression in CEl PKC-δ + neurons a-c. Quantification of CEl c-fos expression in mice intraperitoneal injected with anorexigenic drugs (a),