Parasubthalamic Glutamatergic Neurons Coordinate Cardiovascular Homeostasis and Locomotion in Mice

Figures

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  Figure 1. Chemogenetic manipulation of PSTN neurons alters heart rate and blood pressure. A) Schematic of microinjection into the PSTN of the C57BL/6J mice. B) Representative photomicrographs of the PSTN depicting mCherry expression from a C57BL/6J mice. PSTN: parasubthalamic nucleus. STN: subthalamic nucleus. cp: cerebral peduncle. Scale bar: 100 µm. C) Representative voltage traces recorded from an mCherry, hM3Dq or hM4Di ‐expressing during the application of CNO. D) Schematic of the protocol in experiments. E,F,I,J) Summary of changes in heart rate and blood pressure in C57BL/6J mice after administration of CNO, mCherry mice: n = 7; hM3Dq mice: n = 6. E) ∆Heart rate (Two‐way ANOVA F1,11 = 8.504, p = 0.014). F) ∆Systolic pressure I) ∆Diastolic pressure J) ∆Mean pressure (Two‐way ANOVA; not statistically significant). G,H K, L) Summary of changes in heart rate and blood pressure in C57BL/6J mice after administration of CNO, mCherry mice: n = 7; hM4Di mice: n = 6. G) ∆Heart rate (Two‐way ANOVA; not statistically significant). H) ∆Systolic pressure (Two‐way ANOVA F1,11 = 8.224, p = 0.0153). K) ∆Diastolic pressure (Two‐way ANOVA; F1,11 = 19.36, p = 0.0011). L) ∆Mean pressure (Two‐way ANOVA;…

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  Figure 2. Chemogenetic manipulation of PSTN neurons alters locomotor activity. A,B) Summary of distance and maximum speed in 1‐h wheel running in mice (mCherry mice: n = 7, hM3Dq mice: n = 7; hM4Di mice: n = 6; paired two‐sided t‐test; mCherry vs hM4Di; Total distance: t5 = 5.065 p = 0.0039; Maximum speed: t5 = 4.944, p = 0.0043). C) Summary of 1‐h speed composition in mice (mCherry mice: n = 7, hM3Dq mice: n = 7; hM4Di mice: n = 6; One‐way ANOVA; high‐speed locomotion time: F2,17 = 13.49, p = 0.0003; mCherry vs hM4Di, p = 0.0030; medium‐speed locomotion time: F2,17 = 3.014, p = 0.1455; mCherry vs hM4Di, p = 0.0616, slow‐speed locomotion time: F2,17 = 21.31, p < 0.0001; mCherry vs hM3Dq, p = 0.0489; mCherry vs hM4Di, p = 0.0013). D) Pie charts showing 1‐h speed composition in the mCherry, hM3Dq and hM4Di, excluding immobility data. E) Correlations of total distance and |ΔMBP|, Pearson's r = −0.4861, p = 0.0348. F) Correlations of time spent in high‐speed locomotion and |ΔMBP|, Pearson's r = −0.4778, p = 0.0387. G) Correlations of maximum speed and |ΔMBP|, Pearson's r = −0.3869, p = 0.1018. |ΔMBP|, average amplitude of mean pressure changes. Each circle represents results from one mouse.…

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  Figure 3. Population activity of PSTN Vglut2 neurons during acute hypertension. A) Representative photomicrographs of the PSTN depicting Hoechst (blue), Vglut2+ neurons (green), c‐Fos+ (magenta) and merge (white) images of Vglut2::H2B mice after administration of saline or NE. B) Quantitative analysis of c‐Fos+ neurons in the PSTN. (Saline n = 4, NE n = 4, unpaired two‐sided t‐test; t6 = 9.935, p<0.0001) C Percentage of c‐Fos cells expressing Vglut2 (n = 4). D) Schematic of the in vivo recording configuration. E) Representative image showing GCaMP6s‐EGFP‐labeled PSTN Vglut2 neurons and the position of the tip of the fiber optic in the PSTN (n = 6). The position of the tip of the fiber optic in the PSTN. F) Heatmaps of all recorded trials showing the changes in calcium signals of PSTN Vglut2 neurons before and following vehicle or NE administration. G) The calcium signals of PSTN Vglut2 neurons before and after saline (gray trace) ± SEM (gray shading) or NE (red trace) ± SEM (red shading) administration. Arow heads indicate time of injection. H) AUC of calcium signals following saline/NE injection (n = 6, unpaired two‐sided t‐test; t10 = 3.094, p<0.0114). PSTN: parasubthalamic nucleus, STN:…

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  Figure 4. Chemogenetic manipulation of PSTN Vglut2 neurons alters heart rate and blood pressure. A) Schematic of microinjection into the PSTN of the Vglut2‐Cre mice. B) Representative photomicrographs of the PSTN depicting EGEP expression from a Vglut2‐Cre mice. PSTN: parasubthalamic nucleus. cp: cerebral peduncle. Scale bar: 100 µm. C) Representative voltage traces recorded from an mCherry, hM3Dq or hM4Di ‐expressing during the application of CNO. D) Schematic of the protocol in experiments. E,F,I,J) Summary of changes in heart rate and blood pressure in Vglut2‐Cre mice after administration of CNO, EGFP mice: n = 7; hM3Dq mice: n = 6 E) ∆Heart rate (Two‐way ANOVA F1,11 = 45.69, p < 0.0001). F) ∆Systolic pressure I) ∆Diastolic pressure J) ∆Mean pressure (Two‐way ANOVA; not statistically significant). G,H,K,L) Summary of changes in heart rate and blood pressure in Vglut2‐Cre mice after administration of CNO, EGFP mice: n = 6; hM4Di mice: n = 6. G) ∆Heart rate (Two‐way ANOVA; not statistically significant). H) ∆Systolic pressure (Two‐way ANOVA F1,11 = 11.24, p = 0.0065). K) ∆Diastolic pressure (Two‐way ANOVA; F1,11 = 18.42, p = 0.0013). L) ∆Mean pressure (Two‐way ANOVA; F1,11 = 35.06, p <…

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  Figure 5. Chemogenetic manipulation of PSTN Vglut2 neurons alters locomotor activity. A,B) Summary of distance and maximum speed in 1‐h wheel running in mice (EGFP mice: n = 6, hM3Dq mice: n = 6; hM4Di mice: n = 6; paired two‐sided t‐test; Total distance: EGFP mice: n = 6, not statistically significant; hM3Dq mice: n = 6, t5 = 2.637, p = 0.0461; hM4Di mice: n = 6; t5 = 10.13, p = 0.0002; Maximum speed: EGFP mice: n = 6, not statistically significant; hM3Dq mice: n = 6, not statistically significant; hM4Di mice: n = 6; t5 = 8.298, p = 0.0004; C) Summary of 1‐h speed composition in mice (EGFP mice: n = 6, hM3Dq mice: n = 6; hM4Di mice: n = 6; One‐way ANOVA; high‐speed locomotion time: F2,15 = 46.50, p < 0.0001 ; EGFP vs hM4Di, p < 0.0001; medium‐speed locomotion time: F2,15 = 22.02, p <0.0001; EGFP vs hM4Di, p = 0.0004; slow‐speed locomotion time: F2,17 = 10.18, p = 0.0016; EGFP vs hM4Di, p = 0.004; immobility time: F2,15 = 40.46, p < 0.0001). D) Pie charts showing 1‐h speed composition in EGFP, hM3Dq, and hM4Di mice, with immobility data excluded for hM3Dq mice. E) Correlations of total distance and |ΔMBP|, Pearson's r =−0.8005, p < 0.0001. F) Correlations of time spent in high‐speed…

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  Figure 6. Identification of PSTN‐NTS monosynaptic glutamatergic pathway. A) Schematic of unilateral AAV2/9‐hSyn‐DIO‐EGFP injection into the PSTN of the Vglut2‐Cre mice. B) Representative image of virus injection site in the PSTN, EGFP‐labeled. Scale bar: 500 µm. C) Quantitative analysis of projection intensity NTS and DMX (AP = −7.3 ≈ −7.4mm), n = 3. D) representative images of PSTN Vglut2 neurons projecting to NTS subregions (EGFP‐labeled) and Scale bar: 200 µm. E) Schematic of bilateral AAV‐ fDIO‐ EGFP injection into the PSTN and bilateral RetroAAV‐ FLEX‐ Flpo injection into the NTS of the Vglut2‐Cre mice. F) Representative photomicrographs of the PSTN depicting Hoechst (blue), NeuN (magenta), Vglut2+ neurons (green) and merge (white) images of Vglut2‐Cre mice. Scale bar: 100 µm. G) Statistical analysis of the percentage of PSTN Vglut2 neurons projecting to NTS, n = 3. H) Schematic of virus injection into the PSTN of the Vglut2::H2B mice and optical activation of PSTN Vglut2 terminals with simultaneous whole‐cell patch‐clamp recordings of EGFP+ NTS Vglut2 neurons. I) Representative image of virus injection site in the PSTN (mCherry‐labeled) and sample traces of action potentials evoked…

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  Figure 7. Chemogenetic manipulation of NTS‐projecting PSTN Vglut2 neurons alters heart rate and blood pressure. A) Schematic of microinjection into the PSTN of the Vglut2‐Cre mice. B) Representative photomicrographs of the PSTN depicting hoechst (blue), EGFP (cyan), c‐Fos+ (Magenta), and merge (white) images of PSTN‐EGFP, PSTN‐hM3Dq and PSTN‐hM4Di mice after administration of CNO. White arrows point to merged cell bodies. PSTN: parasubthalamic nucleus. Scale bar: 100 µm. C) Representative voltage traces recorded from an mCherry, hM3Dq or hM4Di ‐expressing during the application of CNO. D) Schematic of the protocol in experiments. E,F,I,J) Summary of changes in heart rate and blood pressure in Vglut2‐Cre mice after administration of CNO, EGFP mice: n = 6; hM3Dq mice: n = 6. E) ∆Heart rate (Two‐way ANOVA F1,10 = 5.763, p = 0.0373). F) ∆Systolic pressure (Two‐way ANOVA; not statistically significant) I) ∆Diastolic pressure (Two‐way ANOVA F1,10 = 5.134, p = 0.0469) J) ∆Mean pressure (Two‐way ANOVA; not statistically significant). G,H,K,L) Summary of changes in heart rate and blood pressure in Vglut2‐Cre mice after administration of CNO, EGFP mice: n = 6; hM4Di mice: n = 6. G) ∆Heart rate…

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  Figure 8. Chemogenetic manipulation of NTS‐projecting PSTN Vglut2 neurons alters locomotor activity. A,B) Summary of distance and maximum speed in 1‐h wheel running in mice (EGFP mice: n = 6, hM3Dq mice: n = 6; hM4Di mice: n = 6; paired two‐sided t‐test; Total distance: EGFP mice: n = 6, not statistically significant; hM3Dq mice: n = 6, t5 = 2.251, p = 0.0742; hM4Di mice: n = 6; t5 = 3.978, p = 0.0106; Maximum speed: EGFP mice: n = 6, hM3Dq mice: n = 6, not statistically significant; hM4Di mice: n = 6; t5 = 2.667, p = 0.0445); C) Summary of 1‐h speed composition in activation group (hM3Dq) mice (n = 6; paired two‐sided t‐test; high‐speed locomotion time: t5 = 2.326, p = 0.0675; medium‐speed locomotion time: not statistically significant; slow‐speed locomotion time: t5 = 3.653, p = 0.0147). D,F,H Pie charts showing 1‐h speed composition baseline and after CNO administration in hM3Dq, hM4Di and EGFP mice. E) Summary of 1‐h speed composition in inhibition group (hM4Di) mice (n = 6; paired two‐sided t‐test; high‐speed locomotion time: t5 = 7.290, p = 0.0008; medium‐speed locomotion time and slow‐speed locomotion time: not statistically significant). G) Summary of 1‐h speed composition in…