Identifying Parabrachial Neurons Selectively Regulating Satiety for Highly Palatable Food in Mice

PB_cl neurons are activated by condensed milk consumption and are molecularly distinct from CGRP⁺ neurons. A, Schematic illustration of Fos induction protocol. Ninety minutes after mouse consumed condensed milk or water ad libitum for 30 min, brainstem slices containing PB_L were stained for Fos expression. B, C, Representative images of Fos⁺ neurons in PB_L after (B) condensed milk consumption and (C) water consumption. For representative images of Fos⁺ neurons in PB_L after consumption of other palatable liquid/food diets, see Extended Data Figure 1-1A–E. Small white dashed circles indicate subregions of PB_L, including sl (superior lateral subnucleus), cl (caudal lateral subnucleus), dl (dorsal lateral subnucleus), el (external lateral subnucleus), vl (ventral lateral subnucleus), scp (superior cerebellar peduncles), KF (Koelliker–Fuse subnucleus). Blue, DAPI stain. Scale bars, 50 μm. D, Total number of Fos⁺ neurons in PB_L of mice that consumed condensed milk (beige, n = 10 mice) and water (blue, n = 7 mice; two-tailed unpaired Student’s t test; **p = 0.007; t₍₁₅₎ = 3.118). Data are mean ± SEM. E, Numbers of Fos⁺ neurons in PB_cl and PB_sl of mice that consumed condensed milk (beige, n = 10 mice) and water (blue, n = 7 mice; two-way ANOVA; PB_cl: ****p < 0.0001; PB_sl: ****p < 0.0001; F_(1,30) = 116.7). Data are mean ± SEM. F–I, Staining of neurons activated by condensed milk consumption (anti-Fos, green) with (F) FoxP2⁺ (immune), (G) CGRP⁺ (immune), (H) Th⁺ (immune), or (I) Pdyn⁺ (in situ) neurons in PB_cl (magenta) after condensed milk consumption. J, Quantification of co-expression of Fos⁺ neurons and FoxP2⁺ and Pdyn⁺ neurons. Data are mean ±  SEM. K, Schematic illustration of strategy to express GFP in PB_L neurons activated by condensed milk consumption (PANs) in Fos^TVA mice using CANE. For representative image and quantification of co-expression of Fos⁺ neurons and Pdyn⁺ neurons in PB_cl after chocolate Ensure consumption, see Extended Data Figure 1-1F–H. L, The CANE method was used to capture PB_cl neurons activated by condensed milk consumption (green), and 10 d later, Fos was re-induced in PB_cl by a second bout of condensed milk consumption (magenta). Blue, DAPI. Scale bar, 20 μm. M, The percentage of Fos⁺ neurons among CANE⁺ neurons in PB_cl. Data are mean ± SEM.

Optogenetic activation of PB_cl PANs induces place preference and decreases condensed milk consumption. A, Schematic illustration of strategy to selectively express ChR2 or GFP in PB_cl PANs in Fos^TVA mice using CANE. B, Schematic illustration of RTPP test. C, Quantification of time the PAN-ChR2 group spent in preferred chamber before, during, and after optogenetic stimulation (n = 6 mice; one-way repeated measures ANOVA; *p = 0.01; *p = 0.04; p > 0.99; F_(1.46,7.29) = 9.22). Data are mean ± SEM. D, Quantification of time the PAN-GFP group spent in preferred chamber before, during, and after optogenetic stimulation (n = 5 mice; one-way repeated measures ANOVA; p > 0.99; p > 0.99; p > 0.99; F_(1.42,5.69) = 0.05). Data are mean ± SEM. E, Schematic illustration of CPP test. F, Quantification of time the PAN-ChR2 group spent in preferred chamber before and after 2 d of optogenetic stimulation (n = 8 mice; two-tailed paired Student’s t test; *p = 0.02; t₍₇₎ = 3.05) Data are mean ± SEM. G, Quantification of time the PAN-GFP group spent in preferred chamber before and after 2 d of optogenetic stimulation (n = 5 mice; two-tailed paired Student’s t test; p = 0.19; t₍₄₎ = 1.57) Data are mean ± SEM. H, Schematic illustration of liquid/food intake assay. I, Measured amount of condensed milk consumed at 1 h (n = 6 mice), 2 h (n = 3 mice), and 3 h (n = 3 mice) after condensed milk presentation with and without optogenetic stimulation (p > 0.99; p = 0.37; *p = 0.01; two-way repeated measures ANOVA; F_(3,14) = 4.38). Data are mean ± SEM. J, Measured amount of regular chow consumed at 1 h (n = 6 mice), 2 h (n = 3 mice), and 3 h (n = 3 mice) after chow presentation with and without optogenetic stimulation (p > 0.99; p = 0.98; p = 0.92; two-way repeated measures ANOVA; F_(3,14) = 0.93). Data are mean ± SEM.