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. 2022 Nov 10;66:101634. doi: 10.1016/j.molmet.2022.101634

Figure 6.

Figure 6

Fasting abolishes the inhibitory regulation of raphe-ZIR 5-HT signaling on ZIR GABA neurons and food motivation. A, Representative images showing ZIR GAD-GFP neurons from a GAD-GFP mouse used for recordings and testing 5-HT modulation on ZIR GABA neurons. B, Representative images shows GAD-GFP (B1), anti-GABA immunostaining (B2), and neurons co-expressed with both GFP and anti-GABA immunoreactivity (B3). C, Representative traces showing the effects of 5-HT (50 μM) on ZIR GAD-GFP neurons from slices of regularly fed mice. D, Representative traces showing the effects of 5-HT (50 μM) on ZIR GAD-GFP neurons from slices of 24-h fasted mice. E, Pie graphs showing percentages of recorded neurons that responded to 5-HT (50 μM) with inhibitory, inhibitory followed by excitatory, and excitatory effects in both regularly fed and 24-h fasted mice. F, 5-HT-induced changes of resting membrane potentials in neurons from both fed (n = 32 neurons) and fasted (n = 31 neurons) mice. ∗∗∗∗p < 0.0001, paired t test. Unpaired t test. G, 5-HT (50 μM) had no effect statistically on firing rate of ZIR GABA neurons (n = 32) from regular fed mice but increased firing rate of neurons (n = 31) from 24-h fasted mice. The firing rate of 5-HT treatment was measured for a period of 2 min including 1-min application followed by 1-min washout. 5-HT effect: F(1,61) = 11.72, p = 0.0011; fasting x 5-HT interaction: F(1,61) = 0.8424, p = 0.3623; ns, no significance, ∗∗p < 0.01, RM two-way ANOVA followed by Bonferroni's multiple comparisons test. H, 5-HT at 0.5 μM (n = 18 neurons), 1 μM (n = 17 neurons), and 5 μM (n = 14 neurons) decreased firing rate of ZIR GABA neurons from regular fed mice. The firing rate of 5-HT treatment was measured for a period of 2 min including 1-min application followed by 1-min washout. 5-HT effect: F(1,46) = 49.29, p < 0.0001; dose x 5-HT interaction: F(2,46) = 0.0133, p = 0.9868; ∗∗p < 0.01, ∗∗∗p < 0.001, RM two-way ANOVA followed by Bonferroni's multiple comparisons test. I, 5-HT at 0.5 μM (n = 23 neurons), 1 μM (n = 24 neurons), and 5 μM (n = 20 neurons) had no effect on firing rate of ZIR GABA neurons from 24-h fasted mice. 5-HT effect: F(1,64) = 3.70, p = 0.06; dose x 5-HT interaction: F(2,64) = 0.2388, p = 0.7883; ns, no significance, RM two-way ANOVA followed by Bonferroni's multiple comparisons test. The firing rate of 5-HT treatment was measured for a period of 2 min including 1-min application followed by 1-min washout. J, Bar graph with data plots showing active lever presses in 24-h fasted mice with and without photostimulation (20 Hz) of raphe-ZIR 5-HT projections. ns, no significance, n = 12 mice, paired t test. K, Breakpoints in 24-h fasted mice with and without photostimulation (20 Hz) of raphe-ZI 5-HT projections. ns, no significance, n = 12 mice, paired t test. L, Food reward earned by 24-h fasted mice with and without photostimulation (20 Hz) of raphe-ZI 5-HT projections. ns, no significance, n = 12 mice, paired t test.