Catecholaminergic neurons projecting to the paraventricular nucleus of the hypothalamus are essential for cardiorespiratory adjustments to hypoxia

Abstract

Brainstem catecholamine neurons modulate sensory information and participate in control of cardiorespiratory function. These neurons have multiple projections, including to the paraventricular nucleus (PVN), which contributes to cardiorespiratory and neuroendocrine responses to hypoxia. We have shown that PVN-projecting catecholaminergic neurons are activated by hypoxia, but the function of these neurons is not known. To test the hypothesis that PVN-projecting catecholamine neurons participate in responses to respiratory challenges, we injected IgG saporin (control; n = 6) or anti-dopamine β-hydroxylase saporin (DSAP; n = 6) into the PVN to retrogradely lesion catecholamine neurons projecting to the PVN. After 2 wk, respiratory measurements (plethysmography) were made in awake rats during normoxia, increasing intensities of hypoxia (12, 10, and 8% O2) and hypercapnia (5% CO2-95% O2). DSAP decreased the number of tyrosine hydroxylase-immunoreactive terminals in PVN and cells counted in ventrolateral medulla (VLM; -37%) and nucleus tractus solitarii (nTS; -36%). DSAP produced a small but significant decrease in respiratory rate at baseline (during normoxia) and at all intensities of hypoxia. Tidal volume and minute ventilation (VE) index also were impaired at higher hypoxic intensities (10-8% O2; e.g., VE at 8% O2: IgG = 181 ± 22, DSAP = 91 ± 4 arbitrary units). Depressed ventilation in DSAP rats was associated with significantly lower arterial O2 saturation at all hypoxic intensities. PVN DSAP also reduced ventilatory responses to 5% CO2 (VE: IgG = 176 ± 21 and DSAP = 84 ± 5 arbitrary units). Data indicate that catecholamine neurons projecting to the PVN are important for peripheral and central chemoreflex respiratory responses and for maintenance of arterial oxygen levels during hypoxic stimuli.

Keywords: anti-dopamine β-hydroxylase saporin; blood pressure; brainstem; chemoreflex; ventilation.

Fig. 1.

Anti-dopamine β-hydroxylase conjugated to the ribosomal-inactivating protein saporin (DSAP) injected into the PVN reduces tyrosine hydroxylase-immunoreactive (TH-IR) terminals in the paraventricular nucleus (PVN) and catecholaminergic neurons in both nucleus tractus solitarii (nTS) and ventrolateral medulla (VLM) regions. A: schematic representation (top) and photomicrographs of TH-IR terminals in the boxed region of PVN from an IgG- (middle) and a DSAP-treated rat (bottom). DSAP treatment reduced TH-IR in the PVN. B and C: schematic representations (top) of the areas corresponding to photomicrographs (bottom) containing the nTS (Bregma −14.10 mm; B) and VLM (Bregma −13.74 mm; C). Boxes indicate the regions represented in the images. Photomicrographs show TH-positive cells in representative IgG- and DSAP-treated animals. DSAP lesions reduced TH-IR in both regions. Dashed area represents the region counted. D and E: total no. of TH-positive neurons counted within the nTS (D) and VLM (E) in IgG- (open bars) and DSAP-treated rats (filled bars). F and G: caudal to rostral extent (relative to Bregma) of the nTS (F) and VLM (G) illustrating the loss of catecholaminergic cells. DSAP lesions significantly reduced the number of catecholaminergic neurons within both the nTS and VLM. P ≤ 0.05. *Main effect of treatment (DSAP < IgG controls). There also was a main effect of caudal to rostral distribution in both nTS and VLM. nTS (F): a > b and −14.64, −14.46, and −13.92; b > −13.74 and −14.82. VLM (G): a > b and −14.82, −13.02, −13.20, and −13.38; b > c and −15.00 and −13.56; c > d and −14.1; d > e and −14.46, −14,28, −14.64, and −13.74; e > f and −13.92; f > −12.12. 10, dorsal motor nucleus of the vagus; AP, area postrema; CC, central canal; TS, tractus solitarii.

Fig. 2.

Lesions of PVN-projecting catecholaminergic neurons blunted ventilatory responses to hypoxia. A: plethysmography traces showing changes in breathing in individual awake IgG control (top) and DSAP-injected rats (bottom) during normoxia and 3 intensities of hypoxia (10 s each). B–G: group data (n = 6 each) showing effect of graded hypoxia on oxygen saturation (B), respiratory rate (C), tidal volume index (D), minute ventilation index (E), mean arterial pressure (F), and heart rate (G) in IgG (○) vs. DSAP-treated animals (●) animals. Two-way repeated-measures ANOVA indicated significant interactions of hypoxia intensity and group in all respiratory variables and heart rate and a main effect of hypoxia for mean arterial pressure. DSAP injections significantly decreased respiratory rate during normoxia and blunted ventilatory responses to increasing intensities of hypoxia despite greater decreases in oxygen saturation. *P ≤ 0.05 vs. IgG; †P ≤ 0.05 vs. 21% O₂; ††P ≤ 0.05 vs. 21 and 12% O₂; †††P ≤ 0.05 vs. 21, 12, and 10% O₂ within a group.

Fig. 3.

Correlation of ventilatory responses and oxygen saturation in IgG vs. DSAP-injected animals. Respiratory rate (A), tidal volume index (B), and minute ventilation index (C) responses relative to oxygen saturation in IgG- (○) vs. DSAP-injected animals (●). The slope of each correlation (Table 2) was significantly less in DSAP-injected animals compared with control. *P ≤ 0.05 vs. IgG.

Fig. 4.

Lesion of PVN-projecting catecholaminergic neurons reduced the number of augmented breaths during hypoxia. A: representative flow traces from a control (top) and DSAP-lesioned (bottom) animal while breathing 8% O₂. Augmented breaths appear as large amplitude “spikes” in the traces with compressed time scales (5 min). The top trace is an expanded time scale of the region in gray for an augmented breath. There was a decreased prevalence of augmented breaths in the DSAP compared with the IgG-treated animal. B: mean data indicate that with decreasing O₂, control animals exhibit a progressive increase in the number of augmented breaths. In DSAP animals, there were significantly fewer augmented breaths at 10 and 8% O₂ (2-way repeated-measures ANOVA). C: DSAP decreased the amplitude of augmented breaths (main effect, 2-way repeated-measures ANOVA). *P ≤ 0.05 vs. IgG; †P ≤ 0.05 vs. 12% O₂; ††P ≤ 0.05 vs. 12 and 10% O₂.

Fig. 5.

The central CO₂ chemoreflex response is blunted after lesion of catecholaminergic cells projecting to the PVN. Plethysmography traces showing breathing during 100% O₂ (left) and 5% CO₂-95% O₂ (right) in individual IgG control (top) vs. DSAP animals (bottom). Group data (n = 6) showing effect of 100% O₂ compared with 5% CO₂-95% O₂ on respiratory rate (B), tidal volume index (C), and minute ventilation index (D) in control (open bars) vs. DSAP animals (black bars). There was a significant increase in all ventilatory parameters between 100% O₂ and 5% CO₂-95% O₂ in both groups. However, the response was blunted in DSAP- vs. IgG-injected animals. *P ≤ 0.05, DSAP vs. IgG; †P ≤ 0.05 vs. 100% within a group (IgG or DSAP).