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. 2017 Jan 26;2(2):e90565. doi: 10.1172/jci.insight.90565

Figure 4. CNO-induced changes in cardiac functions are due to hM3Dq activation in peripheral glial cells, likely ganglionic SGCs.

Figure 4

(A) AAV8-GFAP-GCaMP6–expressing astrocytes in visual cortex visualized through chronic polished window using 2-photon imaging before (left) and after (right) CNO i.p. injection in Gfap-hM3Dq mice (original magnification, 60×; scale bars: 40 μm). (B) Intracellular Ca2+ activity in cortical astrocytes in response to CNO i.p. administration, with or without i.p. trospium chloride injection (7 cells per condition, 2 repeats). (C) Trospium chloride, when administrated i.p., abolished CNO-induced increases in tachycardia (2-way ANOVA, n = 8–11 for each group; ***P < 0.0001, interaction between saline-treated Gfap-hM3Dq mice and littermate controls; ###P < 0.0001, interaction between saline- and trospium-treated Gfap-hM3Dq mice). (D) A cocktail of ganglionic blockers decreased baseline heart rates and blocked prazosin-induced tachycardia in both Gfap-hM3Dq and littermate control mice (n = 8–9 for each group). (E) The cocktail of ganglionic blockers did not block CNO-induced tachycardia in Gfap-hM3Dq mice (2-way ANOVA, ***P < 0.0001, interaction between blocker-treated Gfap-hM3Dq mice and littermate controls, n = 8–10 for each group). (F) CNO administration (0.5 mg/kg, s.c.) led to increases in heart rate in Gfap-hM3Dq+/–::Cx43–/–/Cx30–/– mice (n = 6), Gfap-hM3Dq+/–::d/nSNARE+/– mice (n = 3), Gfap-hM3Dq+/–::A1R–/– mice (n = 3), and Gfap-hM3Dq+/–::A2aR–/– mice (n = 3). Perturbing purinergic signaling with selective antagonists failed to block CNO-induced tachycardia in Gfap-hM3Dq mice; antagonists included P2X3 and P2X2/3 receptor antagonist (A-317491; n = 3) and adenosine A1 receptor antagonist (DPCPX; n = 3). Pre-block using nonselective P2 purinergic antagonist reduced but did not abolish CNO-induced tachycardia (PPADs; Mann-Whitney U test, *P < 0.01, n = 3). (G) Schematic model for CNO-induced, SNS-regulated cardiac changes in vivo in Gfap-hM3Dq mice.