Reduced c-Fos expression in medullary catecholaminergic neurons in rats 20 h after exposure to chronic intermittent hypoxia

Abstract

Persons affected by obstructive sleep apnea (OSA) have increased arterial blood pressure and elevated activity in upper airway muscles. Many cardiorespiratory features of OSA have been reproduced in rodents subjected to chronic-intermittent hypoxia (CIH). We previously reported that, following exposure to CIH, rats have increased noradrenergic terminal density in brain stem sensory and motor nuclei and upregulated expression of the excitatory α(1)-adrenergic receptors in the hypoglossal motor nucleus. This suggested that CIH may enhance central catecholaminergic transmission. We now quantified c-Fos expression in different groups of pontomedullary catecholaminergic neurons as an indirect way of assessing their baseline activity in rats subjected to CIH or sham treatment (7 AM-5 PM daily for 35 days). One day after the last CIH exposure, the rats were gently kept awake for 2.5 h and then were anesthetized and perfused, and their pontomedullary brain sections were subjected to dopamine β-hydroxylase (DBH) and c-Fos immunohistochemistry. DBH-positive cells were counted in the A1/C1, A2/C2, A5, subcoeruleus (sub-C) and A7 groups of catecholaminergic neurons, and the percentages of those expressing c-Fos were determined. We found that fewer DBH cells expressed c-Fos in CIH- than in sham-treated rats in the medulla (significant in the A1 group). In the pons (rostral A5, sub-C, and A7), c-Fos expression did not differ between the CIH- and sham-treated animals. We suggest that, when measured 20 h after the last CIH exposure, catecholaminergic transmission is enhanced through terminal sprouting and receptor upregulation rather than through increased baseline activity in pontomedullary catecholaminergic neurons.

Fig. 1.

Time course of body weight changes during the exposure to chronic intermittent hypoxia (CIH) and sham treatments. CIH rats had a transient arrest of body weight gain on the first 2–3 days of exposure, after which they gained weight at a slower rate than the sham-treated group (4.3 vs. 5.2 g/day; see text for details). As a result, the mean body weight of the CIH group was significantly lower than for the sham-treated group throughout the exposure period (P = 0.007–0.05, unpaired t-tests).

Fig. 2.

Examples of dopamine β-hydroxylase (DBH)-positive cells with or without c-Fos-stained nuclei in the A1/C1 (A), A5 (B), and A7 (C) groups. In each column, the top shows a low-magnification image and the bottom shows an enlarged image of the detail framed in the top. The filled and open arrows in the high magnification images for the A1/C1 (a) and A5 (b) groups point to examples of DBH-positive cells with and without c-Fos-stained nuclei, respectively, and the arrowhead in a points to a c-Fos-labeled nucleus in a cell of an unidentified phenotype. The images of the A7 group (C and c) are shown at a lower magnification than in the other panels to visualize the location of the entire A7 group (C) and all its cells in this brain section (c). According to a rat brain atlas (44), the anteroposterior levels closest to these three examples are the following: −13.20 mm (A), −10.56 mm (B), and −8.52 mm (C) relative to bregma. 12n, roots of the hypoglossal nerve; IOD, inferior olive, dorsal division; IOPr, inferior olive, principal division; lfp, longitudinal fasciculus of the pons; Sp5, spinal trigeminal sensory nucleus.

Fig. 3.

Distribution of c-Fos-positive (c-Fos⁺) and c-Fos-negative (c-Fos⁻) DBH cells at two anteroposterior (A-P) levels corresponding to the medullary noradrenergic A1 group. Locations of all DBH cells found on one side at these two levels in all 6 sham-treated and 6 CIH-exposed rats were redrawn onto the corresponding standard cross-sections from a rat brain atlas (44). At both levels, the percentage of DBH cells that were c-Fos⁺ (filled circles) was higher in sham-treated than in CIH-exposed rats, with values similar to the average data for the entire A1 group (58.5% sham vs. 51.9% CIH at A-P −14.04 mm from bregma, and 59.4% sham vs. 55.2% CIH at A-P −14.28 mm from bregma; see Table 1 for the corresponding data for the entire A1 group). There was no obvious aggregation of c-Fos⁺ or c-Fos⁻ (open circles) DBH cells within the A1 region either within or between the treatment groups. Amb, nucleus ambiguus; IO, inferior olive; LRt, lateral reticular nucleus; py, pyramidal tract; rs, rubrospinal tract; Sp5C, spinal trigeminal sensory nucleus, caudal division; Sp5I, spinal trigeminal sensory nucleus, interpolar division.

Fig. 4.

Percentages of DBH cells that had c-Fos-stained nuclei in different groups of pontomedullary catecholaminergic neurons. The percentage of DBH-positive (DBH⁺) cells that were c-Fos-positive (c-Fos⁺) was significantly lower, or tended to be lower, in the CIH- than sham-treated rats in all medullary groups [A1, C1, A2/C2, and caudal A5 (A5-c); significant effect of the treatment by two-way ANOVA, 6 rats per treatment group]. The effect was also significant for the A1 group when tested by a paired t-test. In contrast, in the pons [rostral A5 (A5-r), sub-C and A7], CIH, and sham animals did not differ. All animals were perfused at the same time of the day 1 day after 35 days of either daily CIH exposure or sham treatment and with wakefulness maintained before perfusion by exposure to a novel environment.