Volume 27, Issue 6, Pages 1650-1656.e4 (May 2019) SatB2-Expressing Neurons in the Parabrachial Nucleus Encode Sweet Taste  Ou Fu, Yuu Iwai, Kunio Kondoh, Takumi Misaka, Yasuhiko Minokoshi, Ken-ichiro Nakajima  Cell Reports  Volume 27, Issue 6, Pages 1650-1656.e4 (May 2019) DOI: 10.1016/j.celrep.2019.04.040 Copyright © 2019 The Author(s) Terms and Conditions

Cell Reports 2019 27, 1650-1656.e4DOI: (10.1016/j.celrep.2019.04.040) Copyright © 2019 The Author(s) Terms and Conditions

Figure 1 SatB2PBN Neurons Are Required for Sweet Taste Sensing (A) Representative coronal section of the parabrachial nucleus (PBN) in wild-type (WT) mice showing SatB2 expression. Dashed area, superior cerebellar peduncle. (B) Ablation of SatB2PBN neurons in SatB2-Cre mice injected with adeno-associated virus (AAV) encoding Cre-dependent diphtheria toxin chain A (DTA). Representative coronal section of the PBN in mice injected with AAV encoding Cre-dependent DTA showing SatB2 expression. Dashed area, superior cerebellar peduncle. (C) The number of SatB2+ neurons was significantly lower in SatB2-DTA mice compared to those in SatB2-Cre mice injected with control AAV encoding Cre-dependent mCherry (see Figure S1). (D–H) Ablation of SatB2PBN neurons led to the loss of normal sweet taste sensing (D), but had little impact on sensitivities to umami (E), bitter (F), sour (G), and salty (H) taste. All of the experiments were carried out with 10- to 16-week-old male mice. Data are presented as means ± SEMs (3 sections from 3 mice per group were analyzed in C). n = 6–7 mice per group in (D)–(H). ∗∗p < 0.01, ∗∗∗p < 0.001, as compared with the corresponding control group. Unpaired Student’s t test in (C). Two-way ANOVA with Bonferroni post hoc test in (D)–(H). Cell Reports 2019 27, 1650-1656.e4DOI: (10.1016/j.celrep.2019.04.040) Copyright © 2019 The Author(s) Terms and Conditions

Figure 2 Vglut2 Neurons in the Waist Area Responded to Various Taste Stimuli In Vivo (A) Diagram of a coronal mouse brain showing the site of viral injection (left). Representative coronal section demonstrating the expression of GCaMP6s (green) and SatB2 (red), and the approximate placement of optic fiber (dashed lines) (right). (B–G) Bulk fluorescence of GCaMP6s-expressing Vglut2 neurons was not changed during water licking (B), but was increased by sweet (C, 500 mM sucrose), umami (D, 300 mM glutamate + 0.5 mM inosine monophosphate [IMP] + 0.1 mM amiloride), bitter (E, 1 mM denatonium), sour (F, 30 mM citric acid), and salty (G, 500 mM NaCl) taste stimuli. Each lick is shown as a sharp peak that starts from time 0. (H) The summary of average Ca2+ responses of Vglut2 neurons during licking. All of the experiments were carried out with 10- to 16-week-old male mice. Data are presented as means ± SEMs. n = 4–5 mice in (B)–(G). ∗p < 0.05, ∗∗p < 0.01, as compared with the water licking control group. One-way ANOVA with Dunnett’s post hoc test. Cell Reports 2019 27, 1650-1656.e4DOI: (10.1016/j.celrep.2019.04.040) Copyright © 2019 The Author(s) Terms and Conditions

Figure 3 SatB2PBN Neurons Responded to Sweet Taste In Vivo (A) Diagram of a coronal mouse brain showing the site of viral injection (left). Representative coronal section demonstrating the expression of GCaMP6s and the approximate placement of a microendoscope lens (dashed lines) (right). (B) Narrowly tuned response of SatB2PBN neurons to sweet taste. Mean Ca2+ responses of GCaMP6s-expressing SatB2PBN neurons before and during licking. Sweet substances (500 mM sucrose, 200 mM sucrose, and 10 mM sucralose) induced significant increases in Ca2+ responses compared to those observed before licking (−2 to 0 s). By contrast, umami (300 mM glutamate + 0.5 mM IMP + 0.1 mM amiloride), bitter (10 mM denatonium, 1 mM quinine), sour (30 mM citric acid), and salty (500 mM NaCl) taste did not induce significant change in Ca2+ responses. (C) Time course of mean Ca2+ responses of GCaMP6s-expressing SatB2PBN neurons before and during the licking of sweet (500 mM sucrose) or bitter (10 mM denatonium) solutions. The baseline was a 5-s period before licking. (D) Heatmap of Ca2+ responses from individual GCaMP6s-expressing SatB2PBN neurons before and during licking of taste solutions (n = 30 cells from 3 mice). All of the experiments were carried out with 12- to 14-week-old male mice. Data are presented as means ± SEMs for ΔF/F values. ∗∗p < 0.01, as compared with the “before licks” group in (B) or the denatonium taste group in (C). One-way ANOVA with Dunnett’s post hoc test in (B), 2-way ANOVA with Bonferroni post hoc test in (C). Cell Reports 2019 27, 1650-1656.e4DOI: (10.1016/j.celrep.2019.04.040) Copyright © 2019 The Author(s) Terms and Conditions

Figure 4 VPMpc-Projecting SatB2PBN Neurons Encode Positive Valence to Induce Appetitive Lick Behavior (A) Diagram of a sagittal mouse brain showing the site of viral injection and the placement of optic fibers for the photostimulation of axon terminals in VPMpc, CeA, or BNST. (B) Representative section showing the expression of ChR2-EYFP in the PBN. Dashed area, superior cerebellar peduncle. (C) Optogenetic activation of the SatB2PBN→VPMpc circuit led to increased numbers of licks for water in a brief access test, as in the case of optogenetic activation of the soma of SatB2PBN neurons. Similar effects were not observed when activating the SatB2PBN→CeA circuit or the SatB2PBN→BNST circuit. Optogenetic stimulation started immediately after the first lick of water. (D) Representative tracking traces in SatB2PBN→VPMpc mice and SatB2PBN→CeA mice in the optogenetic place preference test. (E) SatB2PBN→VPMpc mice showed a significant place preference for the photostimulation-paired side during the optogenetic place preference test, compared to the SatB2PBN→CeA control mice. Preference score was defined as (time spent in laser ON side)/(time spent in both laser ON and OFF sides). All of the experiments were carried out with 10- to 16-week-old male mice. Data are presented as means ± SEMs. n = 5–7 per group in (C) and n = 5 per group in (E). ∗p < 0.05, ∗∗p < 0.01 as compared with the laser OFF group in (C) (paired Student’s t test) or with SatB2PBN→CeA circuit in (E) (unpaired Student’s t test). Cell Reports 2019 27, 1650-1656.e4DOI: (10.1016/j.celrep.2019.04.040) Copyright © 2019 The Author(s) Terms and Conditions