Extended Data Fig. 8: NPYergic inputs to the lPBN activated by competing survival threats.

(a) Monosynaptic rabies tracing was performed in Npy-GFP mice to identify Npy-expressing inputs to the lPBN. (b) Three regions containing double-labeled Npy-positive (GFP) and rabies-positive (mCherry) neurons. Left, arcuate nucleus; middle, subparafascicular nucleus; right, ventrolateral periaqueductal grey. Scale bar, 1 mm (inset, 50 μm). (c) Labeling strategy to map NPY axon inputs to the lPBN. To visualize NPY inputs from the arcuate nucleus, post-hoc immunohistochemistry to AgRP⁷⁵ was used. (d-e) Representative image showing mCherry expression in SPF (d) and GFP expression in vlPAG (e) NPY neurons. Grey, DAPI. Scale bar, 1 mm (inset, 100 μm) (f-i) Representative image showing axons from the SPF (f), vlPAG (g) and arcuate nucleus (h) and a merged image showing the overlap (i). Scale bar, 100 μm. (j) Fiber photometry was used to record activity of SPF Npy-expressing neurons. Inset, representative image showing GCaMP6s expression in SPF NPY neurons below the fiber optic track. Scale bar, 200 μm. (k) Mean ΔF/F in 3 min bins of GCaMP6s signal in SPF NPY neurons after an injection of vehicle or ghrelin (600 μg/kg, n = 5 mice, two-way ANOVA, n.s.). (l) Mean ΔF/F in 5 min bins of GCaMP6s signal in SPF NPY neurons after an injection of vehicle or PEG (n = 5 mice, two-way ANOVA, n.s.). (m) Mean ΔF/F in 1 min bins of GCaMP6s signal in SPF NPY neurons after vehicle or TMT exposure (n = 5 mice, two-way ANOVA, main effect of group p = 0.05, group x time interaction p = 0.0288). (n) Fiber photometry was used to record activity of arcuate nucleus Agrp/Npy-expressing neurons. Inset, representative image showing GCaMP6s expression in arcuate NPY neurons below a fiber optic track. Scale bar, 200 μm. (o) Mean ΔF/F in 3 min bins of GCaMP6s signal in arcuate NPY neurons after an injection of vehicle (n = 6 mice) or 600 μg/kg ghrelin (n = 15 mice, two-way ANOVA, main effect of group p = 0.0044, group x time interaction p < 0.001). (p) Mean ΔF/F in 5 min bins of GCaMP6s signal in arcuate NPY neurons after an injection of vehicle or PEG (n = 4 mice, two-way ANOVA, n.s.). (q) Mean ΔF/F in 1 min bins of GCaMP6s signal in arcuate NPY neurons after vehicle (n = 5 mice) or TMT exposure (n = 4 mice, two-way ANOVA, n.s.). (r) Two possible NPY signaling mechanisms. (s) Channelrhodopsin-assisted circuit mapping – activation of arcuate NPY projections to the lPBN while monitoring activity in lPBN Y1R neurons. (t) Representative trace showing a lack of evoked current in an lPBN Y1R neuron following activation of NPY inputs. None of the 20 patched neurons showed inhibitory post synaptic currents (IPSC, n = 3 mice). (u) Representative trace of an evoked IPSC in a non-lPBN neuron following activation of NPY inputs (blue). 3/35 neurons patched showed inhibitory post synaptic potentials (n = 3 mice). (v) Monosynaptic rabies tracing was performed from lPBN Y1R neurons to determine whether arcuate NPY neurons form synaptic connections. (w) Representative image of arcuate NPY neurons and rabies-expressing presynaptic inputs. Scale bar, 200 μm. (x) Summary schematic. Data are expressed as mean ± SEM. ANOVA main effect of group: ^##p < 0.01. ANOVA interaction: ^†p < 0.05, ^†††p < 0.001.

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