Fig. 3: Au1GABA neurons mediate visceral analgesia through enhanced GABAergic output to ACCGlu in synergy with attenuated glutamatergic transmission via Au1Glu.
A Schematic illustration of RV monosynaptic retrograde tracing to identify presynaptic inputs to ACCGlu. B Representative images showing the injection site in the ACC and input cells in the Au1, n = 3, scale bar = 100 µm. C–E Colocalization analysis of RV+ neurons in the Au1 revealed more glutamatergic than GABAergic neurons (C, ***p = 0.0002, unpaired t-test, n = 22 sections for Glu and 21 sections for GABA, both from 3 mice), scale bar = 50 µm. Yellow arrows indicate co-localization. F Experimental setup of in vivo extracellular recording using optical electrode arrays in NCI mice. G Representative raster diagrams and firing rate of an ACC neuron responsive to blue light. H Blue light attenuated firing rate in a proportion of ACC neurons, which was abolished by a GABAA receptor antagonist bicuculline (1 mg/kg, IP injection, **p = 0.001, one-way ANOVA with Bonferroni test, n = 10 neurons from 3 NCI mice). I–K Optogenetic activation of Au1GABA axonal terminals in the ACC alleviated visceral pain responses in NCI mice (K, F1,5 = 36.710, **p = 0.002, two-way ANOVA with Bonferroni test, n = 6) at 40 (*p = 0.032), 60 (*p = 0.023), and 80 mmHg (***p = 0.0002), scale bar = 100 µm. L–N Optogenetic inhibition of Au1Glu axonal terminals in the ACC significantly alleviated visceral pain responses in NCI mice (N, F1,5 = 8.941, *p = 0.030, two-way ANOVA with Bonferroni test, n = 6) at 60 (*p = 0.030) and 80 mmHg (***p < 0.0001), scale bar = 100 µm. O–S With optogenetic inhibition of Au1GABA projections in the ACC, administration of GABAA receptor agonist muscimol (1 mM, 1 μL) into the Au1 alleviated visceral pain responses in NCI mice (S, pink symbols: **p = 0.004, **p = 0.003,***p = 0.0001, two-way ANOVA with Bonferroni test, n = 7), while chemogenetic activation of Au1Glu reversed the analgesic effect of muscimol (blue symbols: *p = 0.033, **p = 0.005). Scale bar = 100 µm. All data are presented as means ± SEM.
