Extended Data Figure 2. MPOA^Gal projections in males and downstream connectivity.

a, Synaptophysin-GFP (Syn-GFP) labelling of presynaptic sites in MPOA^Gal projections. b, Representative MPOA^Gal projections from a virgin male, identified by tdTomato fluorescence. c, Representative MPOA^Gal projections, identified by tdTomato fluorescence, after viral injection into the left MPOA. d, c-Fos⁺ fractions of virally labelled MPOA^Gal projections in fathers (n = 6, 3, 4, 3, 3, respectively, from top). Red line depicts the population average (see ref. 3). Data are mean ± s.e.m. e, Trans-synaptic retrograde rabies tracing from AVPe^TH neurons. f, MPOA^Gal neurons presynaptic to AVPe^TH neurons in females (left, arrowheads, 21.4% Gal⁺, 47/220 neurons, n = 3) and males (right, 16.7% Gal⁺, 4/24 neurons, n = 2). g, Direct and indirect MPOA^Gal→PVN^OXT connectivity. Asterisk, AVPe^TH neurons form excitatory synapses with PVN^OXT in females¹¹. h, Conditional monosynaptic retrograde tracing initiated from PAG. i, j, Injection sites with mCherry⁺ starter neurons in PAG of Vgat-ires-Cre (i, left) or Vglut2-ires-Cre (j, left) mice. Presynaptic, rabies⁺/Gal⁺ neurons are detected in MPOA when tracing is initiated from PAG^Vgat (i, right, arrowheads), but not PAG^Vglut2 (j, right), neurons. Scale bars, a, 50 μm; b, 250 μm; c, left, 500 and inset, 250 μm; f, 50 μm; i, j, left, 200 μm and right, 250 μm; i, insert, 50 μm.