Fig. 5: Serpinb2⁺ neurons project to LH LepR⁺ GABA⁺ neurons and their activation can counteract the effect of leptin.

a, Diagram indicating the MCH⁺, orexin-A⁺, GABA⁺ and LepR⁺ neurons in the LH. b,c, Example images of non-connected MCH⁺ (b) and orexin-A⁺ neurons (gray) (c) and Serpinb2⁺ neuron terminals (green fibers with red puncta) in the LH. Scale bars, 50 μm (left), 20 μm (right). Arrows indicate somas. d,e, Images showing the colocalization of Serpinb2⁺ neuron terminals (green fibers with red puncta) with GABA⁺ (d) and LepR⁺ (gray) (e) as indicated by arrows. Scale bars, 50 μm (left), 20 μm (right). One technical replicate of three biological replicates. f, Quantification of Serpinb2⁺ neuron terminal connected cells. Percentage represents eYFP⁺MCH⁺/eYFP⁺DAPI⁺, eYFP⁺orexin-A⁺/eYFP⁺DAPI⁺, eYFP⁺GABA⁺/eYFP⁺DAPI⁺ or eYFP⁺LepR⁺/eYFP⁺DAPI⁺. n = 3 sections from three mice. One-way ANOVA test, F_3,8 = 101. g, The timeline of monosynaptic retrograde rabies tracing of LH^LepR neurons. n = 4 biological replicates. h, Representative images showing the location of starter LH^LepR neurons in a LepR-Cre mouse; mCherry-TVA (red), rabies-GFP (green) and DAPI (blue). Scale bars, 100 µm (left), 50 µm (right). i, Representative histological images with cells retrogradely labeled from LH^LepR neurons (green) and Serpinb2⁺ neurons (red). Scale bars, 200 µm (left), 50 µm (right). j, cFos detection in the LH of DREAAD-treated Serpinb2-Cre mice. Virus expression in NAcSh (top); mCherry (red), cFos (green) and DAPI (blue). CNO-induced cFos immunofluorescence signals were observed in the LH; bottom panels are enlarged images of the squares above. Scale bars, 100 µm (top), 200 µm (middle) and 50 µm (bottom). k, The number of cFos⁺ cells (n = 4 sections from four mice). One-way ANOVA test, F_2,9 = 69.51. l, Diagram showing cannula implantation in LH for leptin delivery (left, middle). Results of total food consumption in 3 h by fasted mice with different doses of leptin administration (right). Scale bar, 500 μm. One-way ANOVA test, F_3,10 = 6.025. m, Same as k except food consumption is quantified under different conditions with or without Serpinb2⁺ neuron activation in the presence or absence of 1 μg (middle) or 300 ng (right) of leptin delivery. One-way ANOVA test. F_3,24 = 14.72 (left), F_3,24 = 5.285 (right). Data are presented as mean ± s.e.m. The graphics of the mouse and syringe in l and m were created with BioRender.com.

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