Angiotensin II stimulates spinally projecting paraventricular neurons through presynaptic disinhibition

Abstract

Paraventricular nucleus (PVN) neurons that project to the spinal cord are important in the control of sympathetic outflow. Angiotensin II (Ang II) can stimulate PVN neurons, but its cellular mechanisms are not clear. In this study, we determined the effect of Ang II on the excitatory and inhibitory synaptic inputs to spinally projecting PVN neurons. Whole-cell patch-clamp recordings were performed on PVN neurons labeled by a retrograde fluorescence tracer injected into the thoracic spinal cord of rats. Immunocytochemistry labeling revealed that the immunoreactivity of angiotensin type 1 (AT1) receptors was colocalized with a presynaptic marker, synaptophysin, in the PVN. Application of 0.1-5 microm Ang II significantly decreased the amplitude of evoked GABAergic IPSCs in a concentration-dependent manner. Also, Ang II decreased the frequency of miniature IPSCs from 2.56 +/- 0.45 to 1.05 +/- 0.20 Hz (p < 0.05; n = 12), without affecting the amplitude and the decay time constant. The effect of Ang II on miniature IPSCs was blocked by losartan but not PD123319. However, Ang II had no effect on the evoked glutamatergic EPSCs and did not alter the frequency and amplitude of miniature EPSCs at concentrations that attenuated IPSCs. Furthermore, Ang II increased the firing rate of PVN neurons from 3.75 +/- 0.36 to 7.89 +/- 0.85 Hz (p < 0.05; n = 9), and such an effect was abolished by losartan. In addition, Ang II failed to excite PVN neurons in the presence of bicuculline. Thus, this study provides substantial new evidence that Ang II excites spinally projecting PVN neurons by attenuation of GABAergic synaptic inputs through activation of presynaptic AT1 receptors.

Figure 1.

Identification of a retrogradely labeled spinally projecting PVN neuron. A, A FluoSphere-labeled PVN neuron in the slice viewed with fluorescence illumination. B, Photomicrograph of the same neuron (^*) shown in A with an attached recording electrode (Λ) in the slice viewed with differential interference contrast optics. C, Photomicrograph showing the morphology of a recorded PVN neuron labeled with biocytin. 3V, Third ventricle. Scale bars, 50 μm.

Figure 2.

Evoked synaptic responses in labeled PVN neurons at different holding potentials. A, Focal stimulation-evoked synaptic currents under different holding potentials (from -80 to 0 mV). Synaptic responses were also recorded in the presence of 20 μm CNQX or 20 μm CNQX plus 20 μm bicuculline. B, Peak I—V relationships of the synaptic currents. The peak synaptic currents were plotted as a function of the holding potential. CNQX-sensitive currents (▴) were obtained by subtracting peak synaptic currents measured in CNQX solution from those in control (•). Bicuculline-sensitive currents (▪) were obtained by subtracting synaptic currents measured in CNQX from those in CNQX plus bicuculline solution. C, Summary data showing the difference of peak synaptic currents measured at holding potentials of -70 mV (eEPSCs) and 0 mV (eIPSCs). Data are presented as means ± SEM (^*p < 0.05 compared with eEPSCs; n = 9; Wilcoxon signed rank test).

Figure 3.

Differential effects of Ang II on evoked IPSCs and EPSCs. A, Original recordings showing that Ang II concentration dependently inhibited eIPSCs. The eIPSCs were elicited at a holding potential of 0 mV and abolished by 20 μm bicuculline. B, Ang II failed to inhibit eEPSCs, which were recorded at a holding potential of -70 mV and abolished by 20 μm CNQX. C, Summary data showing differential effect of 0.5–5 μm Ang II on eIPSCs and eEPSCs. Data are presented as means ± SEM (^*p < 0.05 compared with control; n = 9; Kruskal—Wallis ANOVA, followed by Dunn's post hoc test).

Figure 4.

Effect of Ang II on mIPSCs in labeled PVN neurons. A, Representative tracings from a FluoSphere-labeled neuron in the PVN showing mIPSCs recorded during control, application of 2 μm Ang II, washout, and application of 20 μm bicuculline. Note that bicuculline completely eliminated mIPSCs. B, C, Cumulative probability plot analysis of mIPSCs of the same neuron showing the distribution of the interevent interval (B) and peak amplitude (C) during control, Ang II application, and washout. Ang II increased the interevent interval of mIPSCs (p < 0.05; Kolmgorov—Smirnov test) without changing the distribution of the amplitude. D, Superimposed averages of 100 consecutive mIPSCs obtained during control and Ang II application. The decay phase of mIPSCs was best fitted with a double-exponential function. Both fast (τ = 5.69 msec) and slow(τ =16.71 msec) components of the decay phase during control and Ang II administration were similar. E,F, Summary data showing the effect of 2 μm Ang II on the frequency (E) and amplitude (F) of mIPSCs of nine labeled PVN neurons. Data are presented as means ± SEM (^*p < 0.05 compared with the control; Kruskal—Wallis ANOVA, followed by Dunn's post hoc test).

Figure 5.

Summary data showing the effect of losartan on the Ang II-induced inhibition on mIPSCs. A, B, The effect of 2 μm Ang II on the frequency (A) and amplitude (B) of mIPSCs of nine labeled PVN neurons. GDP-β-s was included in the recording electrode internal solution. C, D, The blockade effect of losartan on Ang II-induced inhibition of mIPSCs. Data are presented as means ± SEM (^*p < 0.05 compared with the control; Kruskal—Wallis ANOVA, followed by Dunn's post hoc test). Los, Losartan.

Figure 6.

Lack of effect of Ang II on mEPSCs in labeled PVN neurons. A, Representative tracings from a labeled neuron in the PVN showing spontaneous mEPSCs during control, application of 2 μm Ang II, and application of 20 μm CNQX. B, C, Cumulative plot analysis of mEPSCs of the same neuron showing the distribution of the interevent interval (B) and amplitude (C) during control and application of 2 μm Ang II. Neither the interevent interval nor the amplitude of mEPSCs was affected by Ang II. D, Superimposed averages of 100 consecutive mEPSCs obtained during control and Ang II application. The decay phase of mEPSCs was best fitted with a single-exponential function. The decay time constant was similar during control (τ = 2.87 msec) and Ang II application (τ = 2.74 msec). E, F, Summary data showing the effect of 2 μm Ang II on the frequency (E) and amplitude (F) of mEPSCs in nine labeled PVN neurons. Data are presented as means ± SEM.

Figure 7.

Confocal images showing the colocolization of synaptophysin and AT₁ receptor immunoreactivities in the PVN. A,B, Synaptophysin(A, red) and AT₁ receptor (B, green) immunoreactivities in the PVN viewed under a confocal microscope. C, Digitally merged images from A and B. Note that the synaptophysin and AT ₁ receptor immunoreactivities are colocalized in the PVN (yellow). Magnification: A—C, 800×. D—F, Higher-magnification confocal images showing that AT₁-containing varicosities (green) are in close apposition to the soma or dendrites of three biocytin-filled (red) cells. Magnification: D—F, 5040×. All images are single confocal optical sections.

Figure 8.

Excitatory effect of Ang II and losartan on the firing activity of labeled PVN neurons. A, Top, Histogram showing the reproducible effect of 2 μm Ang II on the firing activity of a labeled PVN neuron. Bottom, Raw tracings showing the spontaneous activity of the same cell during control, application of Ang II, and washout. Note that Ang II increased the discharge activity of the neuron in a reproducible manner. B, Top, Histogram showing the effect of losartan on the Ang II-induced excitation of a PVN neuron. Bottom, Raw tracings showing the spontaneous activity of the same cell during control, application of 2 μm Ang II, and 2 μm Ang II plus 2 μm losartan. C, Effects of 2 μm Ang II and 2 μm Ang II plus 2 μm losartan on the firing activity of eight FluoSphere-labeled PVN neurons. Note that losartan completely eliminated the effect of Ang II. Data are presented as means ± SEM (^*p < 0.05 compared with control; Kruskal—Wallis ANOVA, followed by Dunn's post hoc test). Los, Losartan.

Figure 9.

Effects of Ang II and bicuculline on the discharge activity of labeled PVN neurons.A, Histogram showing the effect of 2 μm Ang II, 20 μm bicuculline, and 2 μm Ang II plus 20 μm bicuculline on the firing activity of a labeled PVN neuron. B, Original tracings recorded during control, application of Ang II, bicuculline alone, and Ang II plus bicuculline from the same neuron shown in A. Note that Ang II failed to increase the firing activity of this neuron in the presence of bicuculline. C, Effect of 2 μm Ang II and 2 μm Ang II plus 20 μm bicuculline on the firing activity of another group of nine labeled PVN neurons. Data are presented as means ± SEM (^*p < 0.05 compared with control; Kruskal—Wallis ANOVA, followed by Dunn's post hoc test). Bic, Bicuculline.