Extended Data Figure 8: Glucose responders are not sensing osmolarity.

Recently, Williams et al. (2016)²⁸ identified vagal neurons that indiscriminately responded to high concentrations of several stimuli delivered in very large stimulus volume for hundreds of seconds. We believe these responses, largely independent of the quality of the stimulus, are intestinal osmolarity signals. a, Shown are heat maps summarizing responses to interleaved 60s stimuli of 500 mM glucose and 500 mM mannitol. Each row represents the average activity of a single cell during 3 interspersed exposures to the stimulus. Stimulus window is indicated by the dashed white lines. Of 134 neurons that responded to intestinal application of 500 mM glucose for 60 s, 101 did not exhibit statistically significant responses to mannitol (upper panel). However, 33 (~25%) showed responses to both 500 mM glucose and 500 mM mannitol (lower panel). N = 5 mice. When the intestinal stimulus consisted of a short pulse (i.e. 10 s; 33 μl volume) no responses were detected to 500 mM mannitol (data not shown). b, Sample traces (3 trials each) of a neuron responding to glucose (red) but not mannitol (blue). c, Sample traces (3 trials each) of a neuron responding to glucose and mannitol. Scale bars indicate % maximal response. d, Heat maps showing responses to a 60s stimuli of 1 M mannitol, 1 M fructose, 1 M mannose, and 1 M NaCl. Note that the same cells respond indiscriminately to the various stimulus (n = 4 mice). e, The graph shows preference plots for Fructose versus AceK (n = 8 mice, two-tailed paired t-test, p = 0.27). Note that Fructose, a caloric sugar, does not create preference, but activates osmolarity responses. f, Williams et al. (2016)²⁸ suggested GPR65-expressing vagal neurons function as the nutrient sensors. We generated mice where GCaMP6s expression was targeted to GPR65-expressing vagal neurons, and examined their responses to a 10s stimulus of 500 mM glucose or osmolarity signals (i.e. 1 M each of fructose, mannose, and NaCl for 60 s). Shown are normalized responses of from 3 different mice to the 4 stimuli; each trace represents a different responding neuron. Note that 500 mM glucose for 10 s does not activate GPR65 neurons. In contrast, they are activated by 60 s 1 M fructose, mannose and NaCl (see also Figure 4). g, Summary histogram of GPR65 tuning profile to 10s 500 mM glucose, and 60s 1 M fructose, 60s 1 M mannose, and 60s 1 M NaCl; n = 4 mice.