Extended Data Fig. 3: Quantification of cNST and nodose labeling.

a, Genetic TRAPing of cNST neurons with fat stimuli (see Methods) is efficient and reliable. Note that animals must not be food deprived to prevent labeling unrelated circuits (Methods). We labelled the fat-induced TRAP2 neurons by infection with an AAV carrying a Cre-dependent fluorescent reporter⁴ (shown in green), and then performed a second cycle of fat stimulation followed by Fos antibody labelling⁴ (shown in red; see Methods). b, By comparing the number of neurons expressing the fluorescent reporter to the number neurons labelled by Fos antibodies, we determined that 90.7 ± 0.6% of Fat-Fos neurons were also TRAPed with the fat stimuli (n = 6). Scale bar, 20 μm. c, For experiments targeting AAV- FLEX-TetTox, or AAV-DIO-mCherry (or GFP) to the cNST we used fat-stimulated TRAP2 animals (see Methods). By comparing the number of neurons expressing AAV after TRAPing and infection, to the number of cNST neurons labeled after crossing similarly TRAped animals to Ai9⁶³ reporter mice, we estimate the infection of TRAPed neurons to be >90%: TRAP-AAV: 68 ± 1 neurons; Trap-Ai9: 71 ± 1 neurons (n = 8). Scale bar, 100 μm. The equivalent area of the cNST (bregma − 7.5 mm) was processed and counted for the separate experiments. Values are mean ± s.e.m. d, Shown is a whole mount image of a nodose ganglia from Vip-Cre animals infected with AAV- FLEX-TetTox virus (see Methods). Average number of labeled neurons from Vip-TetTox was 48 ± 13 neurons (n = 4), and the average of nodose neurons labeled with AAV- FLEX-TetTox virus in the Trpa1-Cre animals was 62 ± 23 neurons (n = 6; not shown). These numbers compare favorably (~50%) to the total number of VIP and Trpa1 neurons detected by crossing the Cre animals to reporter Ai9⁶³ mice: VIP ~100 neurons; Trpa1 ~120 neurons (data not shown). Scale bar, 100 μm. e, Shown is a whole mount image of nodose ganglia from Vip-Cre animals infected with AAV- DIO-hM3Dq (activator DREADD^36,38). VIP-DREADD labeling efficiency: 43 ± 4% (43 ± 4/100.5), n = 9. Scale bar, 100 μm. f-i, cNST-activation in response to intestinal delivery of fat and sugar is mediated via vagal signaling. AAV carrying a Cre-dependent GCaMP6s was targeted to the cNST of Penk-Cre animals⁴. f, Fiber photometry was used to monitor cNST activity in response to intestinal delivery of sugar and fat stimuli (see also Fig. 2b–d); to minimize any labeling in the AP and ensure the signals originate in cNST neurons, we used AAV targeting of GCaMP6s to the cNST (see panel I below). g, Neural responses following intestinal delivery of fat (10% linoleic acid, LA) or sugar (500 mM glucose, Glu). The light traces denote normalized three-trial averages from individual animals, and the dark trace is the average of all trials. The responses after bilateral vagotomy are shown in green. Black bars below traces indicate the time and duration of stimuli; n = 4 mice. NR, normalized response. Note robust, time-locked responses of cNST neurons to intestinal delivery of fat and sugar. Importantly, responses are abolished after bilateral vagotomy. h, Quantification of neural responses before and after vagotomy. Two-tailed paired t-test, P = 3.8x10⁻⁵ (sugar), P = 5x10⁻⁵ (fat). Data are mean ± s.e.m. i, Sample brains of two different injected animals demonstrating expression of GCaMP6s restricted to the cNST, with minimal expression in the AP; the top brain also demarks the location of the recording fiber (dashed rectangle). Scale bars, 200 μm.