Exercise training increases GAD65 expression, restores the depressed GABA A receptor function within the PVN and reduces sympathetic modulation in hypertension

Figures

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  Figure 1. Timeline of the experimental design used in protocols I and II after a 2 weeks adaptation period to the treadmill. SHR and WKY rats were submitted to training (T) or sedentary (S) protocols for 8 (Protocol I) or 4 weeks (Protocol II). Maximal exercise tests (MET) to allocate rats with similar exercise capacity to T and S groups and establish the training intensity and to calculate the performance gain were performed at the beginning and end of protocols, respectively. In protocol I another MET was performed at the fourth experimental week for adjustment of training intensity in T rats. Functional recordings followed by brain harvesting were made at weeks 0, 1, 2, 4, and 8 (red and yellow arrows). Rats allocated to Protocol II were previously submitted to bilateral PVN cannulation, followed by 1‐week recovery before starting T and S protocols. Functional recordings, bicuculline administration and brain harvesting (red and yellow arrows) were performed at the end of experiments.

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  Figure 2. Temporal changes on GAD65 (panel A) and GAD67 (panel B) gene expression within the PVN of spontaneously hypertensive (SHR) and normotensive rats (WKY) submitted to exercise training (T) or sedentary (S) protocols for 8 weeks. Values are means of 6–8 rats/subgroup. GAD65 – training effect: group F 4,69 = 3.64, P = 0.060; weeks F 4,69 = 4.77, P = 0.002; group × week F 4,69 = 0.30, P = 0.879; groups comparison: group F 1,53 = 3.21, P = 0.079; condition F 1,53 = 5.43, P = 0.023; weeks F 1,53 = 9.04, P = 0.004; group × condition F 1,53 = 0.01, P = 0.977; group × week F 1,53 = 0.05, P = 0.825; condition × week F 1,53 = 5.43, P = 0.024; group × condition × week F 1,53 = 0.01, P = 0.977. GAD67 – training effect: group F 4,69 = 1.12 P = 0.294; weeks F 4,69 = 1.03, P = 0.397; group × week F 4,69 = 0.18, P = 0.948; groups comparison: group F 1,55 = 0.65, P = 0.425; condition F 1,55 = 0.03, P = 0.856; weeks F 1,55 = 2.99, P = 0.089; group × condition F 1,55 = 0.01, P = 0.971; group × week F 1,55 = 0.02, P = 0.897; condition × week F 1,55 = 0.03, P = 0.856; group × condition × week F 1,55 = 0.01, P = 0.971. Significance (P < 0.05) * versus respective week 0; † versus respective S control.

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  Figure 3. Temporal changes on hemodynamic parameters (panels A and B) and correlations between resting mean arterial pressure (MAP, panel C) and heart rate (HR, panel D) with GAD65 gene expression within the PVN of spontaneously hypertensive (SHR) and normotensive rats (WKY) submitted to exercise training (T) or sedentary (S) protocols for 8 weeks. n = 6–8 rats/subgroup. MAP – training effect: group F 4,98 = 1170.43, P < 0.001; week F 4,98 = 1.86, P = 0.165; group × week F 4,98 = 3.31, P = 0,045; groups comparison: group F 1,61 = 996.64, P < 0.001; condition F 1,61 = 1.67, P = 0.201; weeks F 1,61 = 0.62, P = 0.433; group × condition F 1,61 = 3.61, P = 0.050; group × week F 1,61 = 0.12, P = 0.728; condition × week F 1,61 = 4.19, P = 0.044; group × condition × week F 1,61 = 2.86, P = 0.096. HR – training effect: group F 4,98 = 36.47, P < 0.001; week F 4,98 = 3.10, P = 0.019; group × week F 4,98 = 0.68, P = 0,605; groups comparison: group F 1,61 = 50.35, P < 0.001; condition F 1,61 = 0.51, P = 0.476; weeks F 1,61 = 7.16, P = 0.009; group × condition F 1,61 = 2.98, P = 0.049; group × week F 1,61 = 0.34, P = 0.564; condition × week F 1,61 = 2.60, P = 0.052; group × condition × week F 1,61 =…

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  Figure 4. (A) Photomicrographs comparing the effects of hypertension and exercise training in GAD65 immunoreactivity (ir) within the PVN of spontaneously hypertensive (SHR) and normotensive rats (WKY) submitted to exercise training (T) or sedentary (S) protocols for 4 weeks. In the presence of Triton, a very dense labeling of GAD65‐immunoreactive terminals is observed in trained groups. Scale bar = 25 mm. (B) Quantification of GAD65 density within the PVN in the 4 groups of rats. Values are means of 8–9 slices/rat, 4–5 rats/group. GAD65 ir – groups comparison: group F 1,13 = 2.63, P = 0.129; condition F 1,13 = 21.43, P < 0.001; group × condition F 1,13 = 0.33, P = 0.574. Significance (P < 0.05): † versus respective S control.

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  Figure 5. Raw tracings of pulsatile (PAP) and mean arterial pressure (MAP) and heart rate (HR) in sedentary (S) and trained (T) conscious normotensive (WKY) and spontaneously hypertensive rats (SHR) before and after bilateral bicuculline microinjection into the PVN (arrow).

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  Figure 6. (A–D) Comparison of baseline values of mean arterial pressure (MAP, panel A) and heart rate (HR, panel B) and MAP (panel C) and HR (panel D) responses to bilateral bicuculline administration into the PVN of SHR and WKY submitted to training (T) or sedentary (S) protocol for 4 weeks. n = 5–6 rats/group MAP – groups comparison: group F 1,16 = 80.54, P < 0.001; condition F 1,16 = 10.19, P = 0.006; group × condition F 1,16 = 1.33, P = 0.265. HR – groups comparison: group F 1,16 = 20.71, P < 0.001, condition F 1,16 = 14.14, P = 0.002; group × condition F 1,16 = 0.58, P = 0.456. ∆MAP – groups comparison: group F 1,16 = 4.37, P = 0.053; condition F 1,16 = 14.55, P = 0.002; group × condition F 1,16 = 0.06, P = 0.805. ∆HR – groups comparison: group F 1,16 = 0.58, P = 0.459, condition F 1,16 = 12.87, P = 0.003; group × condition F 1,16 = 4.58, P = 0.049. Significances (P < 0.05) are * versus WKY, † versus respective S control. (E) Localization of the positive bilateral microinjections of bicuculline within the ventromedial and posterior PVN nuclei. ∆ represents the WKY and ○ the SHR rats; white symbols correspond to the sedentary and dark ones to the trained rats. Schematic diagrams are…

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  Figure 7. Changes in autonomic control of the heart induced by bilateral bicuculline administration into the PVN of spontaneously hypertensive (SHR) and normotensive rats (WKY) submitted to exercise training (T) or sedentary (S) protocols for 4 weeks. Graphs represent the changes in the sympathetic activity (LF‐PI), parasympathetic activity (HF‐PI) in normalized units (nu), ratio between both (LF/HF), and the spontaneous baroreflex sensitivity (sBRS) before and after bicuculline administration. n = 5–6 rats/group. Statistical values for groups’ comparison and bicuculline effect are shown in Table 2. Significances (P < 0.05) are * versus before, # versus respective WKY, † versus respective S control.