Key Brainstem Structures Activated during Hypoxic Exposure in One-day-old Mice Highlight Characteristics for Modeling Breathing Network in Premature Infants

Abstract

We mapped and characterized changes in the activity of brainstem cell groups under hypoxia in one-day-old newborn mice, an animal model in which the central nervous system at birth is particularly immature. The classical biphasic respiratory response characterized by transient hyperventilation, followed by severe ventilation decline, was associated with increased c-FOS immunoreactivity in brainstem cell groups: the nucleus of the solitary tract, ventral reticular nucleus of the medulla, retrotrapezoid/parafacial region, parapyramidal group, raphe magnus nucleus, lateral, and medial parabrachial nucleus, and dorsal subcoeruleus nucleus. In contrast, the hypoglossal nucleus displayed decreased c-FOS immunoreactivity. There were fewer or no activated catecholaminergic cells activated in the medulla oblongata, whereas ~45% of the c-FOS-positive cells in the dorsal subcoeruleus were co-labeled. Approximately 30% of the c-FOS-positive cells in the parapyramidal group were serotoninergic, whereas only a small portion were labeled for serotonin in the raphe magnus nucleus. None of the c-FOS-positive cells in the retrotrapezoid/parafacial region were co-labeled for PHOX2B. Thus, the hypoxia-activated brainstem neuronal network of one-day-old mice is characterized by (i) the activation of catecholaminergic cells of the dorsal subcoeruleus nucleus, a structure implicated in the strong depressive pontine influence previously reported in the fetus but not in newborns, (ii) the weak activation of catecholaminergic cells of the ventral reticular nucleus of the medulla, an area involved in hypoxic hyperventilation, and (iii) the absence of PHOX2B-positive cells activated in the retrotrapezoid/parafacial region. Based on these results, one-day-old mice could highlight characteristics for modeling the breathing network of premature infants.

Keywords: PHOX2B; c-FOS; catecholamine; hypoxic ventilatory depression; serotonin; subcoeruleus nucleus.

Figure 1

One-day-old mice displayed a ventilatory depression in response to hypoxia. Average percent change in $\dot{V}$_E from baseline at 2 min (peak HVR, black bar) and 25 min (white bar) of hypoxic exposure in one-day-old mice (n = 8). ^*p < 0.05 vs. baseline, ^#p < 0.05 vs. peak HVR.

Figure 2

c-FOS-positive cells in the medulla oblongata and pons of one-day-old mice under hypoxia. Drawings of representative sections from the medulla oblongata (A–D) and pons (E,F) under normoxia (A,C,E) and hypoxia (B,D,F). Scale bar = 500 μm. Photomicrographs of c-FOS immunoreactivity in the mNTS (G,H), the RPa and ROb (I,J), the PP (K,L), and the dSubC (M,N) under normoxia (G,I,K,M) and hypoxia (H,J,L,N). Scale bar = 100 μm. 7N, facial nucleus; 10N, dorsal motor nucleus of the vagus 12N, hypoglossal nucleus; A5, A5 region; Amb: ambiguus nucleus; AP, area postrema; dSubC, dorsal part of the subcoeruleus nucleus; mNTS, median part of the nucleus of the tractus solitarius; cVLM, caudal part of the ventrolateral medullary reticular nucleus; PP, parapyramidal group; Py, pyramidal tract; RPa, raphe pallidus nucleus; RMg, raphe magnus nucleus; RTN/pFRG, retrotrapezoid nucleus/parafacial respiratory group; vlNTS, ventrolateral part of the nucleus of the tractus solitarius; vSubC, ventral part of the subcoeruleus nucleus.

Figure 3

Catecholaminergic character of hypoxic c-FOS-positive cells of one-day-old mice. Drawings illustrating the distribution of cells immunoreactive for c-FOS (white points) or both c-FOS and TH (black points) in the mNTS, vlNTS (A), VLM (B), and dSubC and vSubC (C) under hypoxic conditions. Scale bar = 100 μm. Photomicrographs of sections double-immunolabeled for c-FOS (gray) and TH (brown) in the mNTS (D), VLM (E), and dSubC (F) corresponding to the regions outlined by the black rectangles in (A–C), respectively. Scale bar = 100 μm. (G) photomicrograph representing an enlargement of the black rectangle in (F) Scale bar = 10 μm. Black arrows indicate c-FOS-positive neurons that are also immunoreactive with TH. Amb: ambiguus nucleus; cc: central canal; DMX, dorsal motor nucleus of vagus; dSubC, dorsal part of the subcoeruleus nucleus; mNTS, median part of the nucleus of the tractus solitarius; Mo5, motor trigeminal nucleus; VLM, ventrolateral medullary reticular nucleus; vlNTS, ventrolateral part of the nucleus of the tractus solitarius; vSubC, ventral part of the subcoeruleus nucleus.

Figure 4

Hypoxic c-FOS-positive cells of the RTN/pFRG in one-day-old mice are not immunolabeled for PHOX2B. Drawing illustrating the distribution of cells immunoreactive for c-FOS and PHOX2B in the RTN/pFRG (A). Scale bar = 100 μm. Solid black triangles indicate neurons that express PHOX2B and white points indicate c-FOS-positive neurons. Photomicrographs of a section double-immunolabeled for c-FOS (in red, B,D) and PHOX2B (in green, C,D) in the RTN/pFRG corresponding to the region outlined by the black rectangle (A). Scale bar = 50 μm. n7, facial nucleus; RTN/pFRG, retrotrapezoid nucleus/parafacial respiratory group.

Figure 5

Serotoninergic character of hypoxic c-FOS-positive cells of one-day-old mice. Drawings illustrating the distribution of cells immunoreactive for c-FOS (white points) or both c-FOS and 5-HT (black points) in the PP (A) and RMg (B) under hypoxic conditions. Scale bar = 100 μm. Photomicrographs of sections double-immunolabeled for c-FOS (gray) and 5-HT (brown) in the PP (C) and RMg (D) corresponding to the regions outlined by the black rectangles of (A,B), respectively. Scale bar = 100 μm. (E,F) photomicrographs representing an enlargement of the black rectangles in (C,D), respectively. Scale bar = 50 μm. Black arrows indicate c-FOS-positive neurons that are also immunoreactive for 5-HT. RPa, raphe pallidus nucleus; RMg, raphe magnus nucleus; PP, parapyramidal group; Py, pyramidal tract.