High Salt Intake Augments Excitability of PVN Neurons in Rats: Role of the Endoplasmic Reticulum Ca2+ Store

Figures

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  Figure 1. Representative raw tracings of splanchnic sympathetic nerve activity (SSNA), renal sympathetic nerve activity (RSNA) and arterial blood pressure (ABP) in response to paraventricular nucleus (PVN) microinjection of graded doses (vehicle, 0.15, 0.30, 0.75, 1.5 nmol) of the ER Ca2+ ATPase inhibitor thapsigargin (TG); HS-high salt diet (2%).

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  Figure 2. Summary data showing changes in SSNA, RSNA, mean arterial pressure (MAP), and heart rate (HR) in response to bilateral microinjections of varying doses of TG (vehicle, n = 3; 0.15 nmol, n = 4; 0.3 nmol, n = 5; 0.75 nmol, n = 6; 1.5 nmol, n = 5) into the PVN. *P < 0.05 vs. vehicle; †P < 0.05 vs. 0.3 nmol (1-way ANOVA Newman-Keuls multiple-comparison test).

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  Figure 3. (A) Representative traces showing HR, SSNA, RSNA, and ABP responses to bilateral PVN microinjection of TG (0.75 nmol) in a rat on a 0.4% normal salt (NS) diet (left), and a 2% high salt (HS) (right). Bilateral PVN microinjection (100 nl each) of TG (arrowheads) markedly increased HR, SSNA, RSNA and ABP in a NS diet rat, whereas responses were attenuated in a rat fed a HS diet. (B) Summary data showing peak changes in SSNA, RSNA, MAP, and HR after bilateral PVN microinjection of TG (0.75 nmol) in normal salt (NS, n = 6) and high salt (HS, n = 6) rats. Note that SSNA and RSNA responses to PVN TG were significantly attenuated in HS rats compared to NS. *P < 0.05 HS vs. NS (unpaired student t-test). Avg, average; ∫, integrated.

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  Figure 4. Effect of ER Ca2+ uptake inhibition with TG on excitability of PVN-RVLM neurons from NS and HS rats. (A) Voltage traces illustrating neuronal excitability in response to a 200 pA current injection in PVN-RVLM neurons from NS (left) and HS (right) rats in the absence (top, control) and presence (bottom left) of the ER Ca2+ ATPase inhibitor TG. (B) Linear response demonstrating the slope of firing frequency in response to graded current injection (0–200 pA) in PVN-RVLM neurons in the absence and presence of TG in NS (left) and HS (right) neurons. TG increased firing frequency in the NS group, but not HS. Note that firing frequency in response to 200 pA depolarizing current injection was increased in HS (right) compared to NS (left). (C) Summary data showing slope of firing frequency in response to graded current injection before and after bath application of TG in NS and HS rats. Inhibition of the ER Ca2+ store with TG augmented the slope in NS, but not HS neurons. Note that in control conditions, the slope was significantly greater in HS neurons. NS-normal salt; HS-high salt; TG-thapsigargin. †P < 0.05 NS vs. NS –TG; &P < 0.05 NS vs. HS; *P < 0.05 NS vs. NS-TG; #p < 0.05 NS vs. HS…

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  Figure 5. Effects of ER Ca2+ uptake inhibition with TG on spike-frequency adaptation in PVN-RVLM neurons. (A) Linear response of inter-spike interval (ISI)-ISI number over trains of action potentials in response to 200 pA current injection in PVN-RVLM neurons from NS left and HS right treatment groups before and after bath application of thapsigargin (TG). (B) Summary data showing slope of the ISI-ISI number response to 200 pA current injection was diminished in HS rats compared to NS revealing greater spike frequency adaptation in NS neurons. TG attenuated the slope of ISI in NS, but not HS neurons. NS-normal salt; HS-high salt; TG-thapsigargin. *P < 0.05 NS vs. NS-TG; #p < 0.05 NS vs. HS (1-way ANOVA Newman-Keuls multiple-comparison test).

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  Figure 6. (A) PVN-RVLM neuron with red fluorescence from retrograde label (top) and same neuron with patch pipette positioned on cell surface with DIC microscopy (bottom). (B) Schematic drawings of coronal sections throughout the rat hypothalamus. Shaded area indicates spread of injected dye used to mark the injection sites in the bilateral PVN. The shape of each area was determined by overlaying tracings of the outermost diffusion area of injected dye (100 nl) through the rostral-caudal plane of the PVN. (C) Representative coronal slice through the PVN demonstrating spread of injected dye. AH, anterior hypothalamic area; 3V, third cerebral ventricle; RCh, retrochiasmatic area; MPO, medial preoptic nucleus; opt, optic tract; SOX, supraopticdecussation; StHy, striohypothalamic nucleus.