Extended Data Fig. 9: Optogenetic activation of hindlimb PROKR2ADV neurons induced Fos in NTS-projecting spinal neurons and in adrenal medulla-projecting DMV efferent neurons.
From: A neuroanatomical basis for electroacupuncture to drive the vagal–adrenal axis
a, Schematics showing the experimental design for testing if optical stimulation of PROKR2ADV nerve fibers of the hindlimb ST36 region can activate spinal ascending projection neurons retrogradely labeled with Fluoro-gold from the nucleus tractus solitarius (“NTS”) in hindbrain. b, 473 nm blue light stimulation of the hindlimb ST36 region was sufficient to evoke Fos induction in NTS-projecting neurons located in the lamina I of the spinal cord in Prokr2Adv-CatCh (“CatCh”) mice (b, arrows), but not in control mice (n = 3 mice per group, two-side student’s unpaired t-test, t4 = 6.807, ***P < 0.001). This stimulation virtually did not induce any Fos in NTS-projecting neurons located in deep laminae (IV and V) of the spinal cord, in both control (1.77 ± 0.24%) and CatCh (2.27 ± 0.43%) mice (data not shown; n = 3 mice per group, two-side student’s unpaired t-test, t4 = 1.023, P = 0.364). c, Schematics showing the experimental design for testing if optical stimulation of PROKR2ADV nerve fibers of the hindlimb ST36 region can activate a subset of vagal efferent neurons located in the dorsal motor nucleus of the vagus (DMV) that were retrogradely labeled from the adrenal gland with Fluoro-gold. d, Optical stimulation of PROKR2ADV neurons of ST36 regions caused an increase in Fos induction in adrenal medulla-projecting DMV neurons compared with control mice (n = 3 mice per group, two-side student’s unpaired t-test, t4 = 8.159, ***P = 0.001). Arrows indicate retrogradely labeled DMV neurons with Fos induction. Arrowhead indicates the baseline Fos expression in ChAT-negative cells. e, No significant (NS) reduction of LPS-induced TNF-α and IL-6 production following 10 mW or 30 mW optical stimulation of PROKR2ADV fibers at the ST36 site of CatCh mice compared with control mice (two-way ANOVA, n = 5 mice per group; for TNF-α: F1, 20 = 0.124; NS, P = 0.728; post-hoc Tukey test: **P = 0.003, ***P < 0.001; for IL-6: F1, 20 = 0.714; NS, P = 0.408; post-hoc Tukey test: ***P < 0.001). Data are shown as mean ± SEM. Scale bars: 100 μm.
