Journal: Nature Metabolism
Article Title: A subset of dopamine receptor-expressing neurons in the nucleus accumbens controls feeding and energy homeostasis
doi: 10.1038/s42255-024-01100-0
Figure Lengend Snippet: 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 .
Article Snippet: For immunofluorescence, cryostat sections were collected and incubated overnight with blocking solution (1× PBS containing 5% goat serum, 5% BSA and 0.1% Triton X-100) and then incubated with the following primary antibodies, diluted with blocking solution, for 1 day at 4 °C: rabbit anti-cFos (1:2,000; Synaptic systems, cat. no. 226003), chicken anti-GFP (1:2,000; Aves Labs, cat. no. GFP-1010), chicken anti-mCherry (1:2,000; Novus Biologicals, cat. no. NBP2-25158), mouse anti-orexin-A (KK09) (1:500; Santa Cruz Biotechnology, cat. no. sc-80263), rabbit anti-GABA (1:1,000; Sigma, cat. no. A2052), rabbit anti-MCH (1:20,000; Phoenix Pharmaceuticals, cat. no. H-070-47) and rabbit anti-leptin receptor (1:1,000; Abcam, cat. no. 104403).
Techniques: Labeling, Virus, Expressing, Immunofluorescence, Activation Assay
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