Figure 4.
BNST–PVN-RVLM neural signaling mediates IL-1β–induced changes in heart rate and IL-6. (A) Schematic for anterograde tracing of axonal projections and terminals from PBS-TRAPed or IL-1β–TRAPed BNST neurons. The AAV-hSyn-FLEx-mGFP-2A-synaptophysin-mRuby was injected into the BNST of TRAP2 mice. (B) Representative image for GFP+ axons and mRuby+ terminals in the PVN of PBS-TRAPed or IL-1β–TRAPed mice. The rightmost panel shows a higher-magnification view of the PVN in IL-1β–TRAPed mice. Arrowheads show regions with co-localization of mRuby and EYFP. Scale bars, 100 μm. (C) Schematic for anterograde tracing of IL-1β–TRAPed BNST neurons connected with PVN neurons. The AAV-pEF1a-DIO-FLPo-WPRE-hGHpA was injected into the BNST, and the AAV-Ef1a-fDIO-EYFP was injected into the PVN of TRAP2 mice. (D) Representative image for EYFP expression in the PVN, RVLM, and NTS. Arrowheads show neurons, and arrows show axonal projections with expression of EYFP. Scale bar, 100 μm for the PVN and RVLM. Scale bar, 200 μm for the NTS. (E) c-Fos expression in the RVLM after reactivation with saline as a control or CNO of IL-1β–responsive BNST neurons. Scale bar, 100 μm. (F) Schematic for activating the BNST–PVN neural pathway. The AAV-pEF1a-DIO-FLPo-WPRE-hGHpA was injected into the BNST, and the AAV-hSyn-fDIO-hM3D(Gq)-mCherry-WPREpA was injected into the PVN of TRAP2 mice. (G) Representative image of PVN showing Gq-DREADD-mCherry–expressing cells (red). Scale bar, 100 μm. (H) Serum IL-6 levels at 2 h after reactivation with saline as a control or CNO of the BNST–PVN neuronal pathway. Data are represented as individual mouse data points pooled from two independent experiments. Unpaired t test. (I) ΔHR for 60 min after reactivation of the BNST–PVN neuronal pathway: saline (black) or CNO (red) (saline, n = 7 mice; CNO, n = 10 mice, mixed-effects analysis with Šidák correction). (J) AUC of ΔHR after reactivation. Data are represented as individual mouse data points pooled from two independent experiments. Unpaired t test. (K) Serum corticosterone levels at 2 h after reactivation of the BNST–PVN neuronal pathway. Data are represented as individual mouse data points pooled from two independent experiments. Unpaired t test. *P < 0.05; **P < 0.01.

BNST–PVN-RVLM neural signaling mediates IL-1β–induced changes in heart rate and IL-6. (A) Schematic for anterograde tracing of axonal projections and terminals from PBS-TRAPed or IL-1β–TRAPed BNST neurons. The AAV-hSyn-FLEx-mGFP-2A-synaptophysin-mRuby was injected into the BNST of TRAP2 mice. (B) Representative image for GFP+ axons and mRuby+ terminals in the PVN of PBS-TRAPed or IL-1β–TRAPed mice. The rightmost panel shows a higher-magnification view of the PVN in IL-1β–TRAPed mice. Arrowheads show regions with co-localization of mRuby and EYFP. Scale bars, 100 μm. (C) Schematic for anterograde tracing of IL-1β–TRAPed BNST neurons connected with PVN neurons. The AAV-pEF1a-DIO-FLPo-WPRE-hGHpA was injected into the BNST, and the AAV-Ef1a-fDIO-EYFP was injected into the PVN of TRAP2 mice. (D) Representative image for EYFP expression in the PVN, RVLM, and NTS. Arrowheads show neurons, and arrows show axonal projections with expression of EYFP. Scale bar, 100 μm for the PVN and RVLM. Scale bar, 200 μm for the NTS. (E) c-Fos expression in the RVLM after reactivation with saline as a control or CNO of IL-1β–responsive BNST neurons. Scale bar, 100 μm. (F) Schematic for activating the BNST–PVN neural pathway. The AAV-pEF1a-DIO-FLPo-WPRE-hGHpA was injected into the BNST, and the AAV-hSyn-fDIO-hM3D(Gq)-mCherry-WPREpA was injected into the PVN of TRAP2 mice. (G) Representative image of PVN showing Gq-DREADD-mCherry–expressing cells (red). Scale bar, 100 μm. (H) Serum IL-6 levels at 2 h after reactivation with saline as a control or CNO of the BNST–PVN neuronal pathway. Data are represented as individual mouse data points pooled from two independent experiments. Unpaired t test. (I) ΔHR for 60 min after reactivation of the BNST–PVN neuronal pathway: saline (black) or CNO (red) (saline, n = 7 mice; CNO, n = 10 mice, mixed-effects analysis with Šidák correction). (J) AUC of ΔHR after reactivation. Data are represented as individual mouse data points pooled from two independent experiments. Unpaired t test. (K) Serum corticosterone levels at 2 h after reactivation of the BNST–PVN neuronal pathway. Data are represented as individual mouse data points pooled from two independent experiments. Unpaired t test. *P < 0.05; **P < 0.01.

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