Chronic intermittent hypoxia affects endogenous serotonergic inputs and expression of synaptic proteins in rat hypoglossal nucleus

Abstract

Evidence has shown that hypoxic episodes elicit hypoglossal neuroplasticity which depends on elevated serotonin (5-HT), in contrast to the rationale of obstructive sleep apnea (OSA) that deficient serotonergic input to HMs fails to keep airway patency. Therefore, understanding of the 5-HT dynamic changes at hypoglossal nucleus (HN) during chronic intermittent hypoxia (CIH) will be essential to central pathogenic mechanism and pharmacological therapy of OSA. Moreover, the effect of CIH on BDNF-TrkB signaling proteins was quantified in an attempt to elucidate cellular cascades/synaptic mechanisms following 5-HT alteration. Male rats were randomly exposed to normal air (control), intermittent hypoxia of 3 weeks (IH3) and 5 weeks (IH5) groups. Through electrical stimulation of dorsal raphe nuclei (DRN), we conducted amperometric technique with carbon fiber electrode in vivo to measure the real time release of 5-HT at XII nucleus. 5-HT_2A receptors immunostaining measured by intensity and c-Fos quantified visually were both determined by immunohistochemistry. CIH significantly reduced endogenous serotonergic inputs from DRN to XII nucleus, shown as decreased peak value of 5-HT signals both in IH3 and IH5groups, whereas time to peak and half-life period of 5-HT were unaffected. Neither 5-HT_2A receptors nor c-Fos expression in HN were significantly altered by CIH. Except for marked increase in phosphorylation of ERK in IH5 rats, BDNF-TrkB signaling and synaptophys consistently demonstrated downregulated levels. These results suggest that the deficiency of 5-HT and BDNF-dependent synaptic proteins in our CIH protocol contribute to the decompensated mechanism of OSA.

Keywords: Obstructive sleep apnea; chronic intermittent hypoxia; hypoglossal nucleus; serotonin.

Figure 1

Experimental schema. A. A rat model of CIH resembling sleep apnea. Time course of inspired fraction of oxygen (FiO₂) level changes in CIH chambers during one successive cycles of intermittent hypoxia, as sampled at 5-second intervals. B. Amperometric detection of serotonin 5-HT release evoked by electrical stimulation in DRN and recorded in XII nucleus by carbon fiber electrode in vivo. DRN located at bregma -7.44 mm and XII nucleus at bregma -13.68 mm according to the atlas. Aq = aqueduct, DR = dorsal raphe, 4V =4th ventricle, cc = central canal, 12N = XII nucleus.

Figure 2

CIH significantly decreased serotonergic inputs from DRN to XII nucleus in vivo. A. Representative traces of typical 5-HT signals with the peak value, time to peak and half-life period separately labeled (left image). Representative raw data of 5-HT signals recorded from control, IH3 and IH5 group rats merged as right image. B. Histogram of the peak value (a), time to peak (b), half-life period (c) of 5-HT release analyzed respectively from control (normal room air), IH3 (exposed to 3 weeks of CIH) and IH5 (exposed to 5 weeks of CIH) group rats. C. Blood gas analysis and respiratory rate of control, IH3 and IH5 group rats. D. Example of a unilateral lesion site made by stimulus electrode in DRN shown as Nissl staining. Scale bar =150 μm. Data were presented as mean ± S.E.M (n=6 rats/group), #P<0.05, *P<0.01 versus control group.

Figure 3

Effect of CIH on 5-HT_2A receptor immunostaining in the XII nucleus. A. Representative photomicrograph of 5-HT_2A receptor immunostaining with white line delimiting the entire XII nucleus and dotted lines indicating the borders of dorsal and ventral halves of the XII nucleus. The background region encircled used for measurement of ratio of staining intensity. cc = central canal. Scale bar =100 μm. B. The average intensity of 5-HT_2A receptor within the dorsal and ventral XII nucleus measured by densitometry from control (normal room air), IH3 (exposed to 3 weeks of CIH) and IH5 (exposed to 5 weeks of CIH) group rats. C. The ratio intensity of 5-HT_2A receptor staining within the dorsal and ventra XII nucleus to the background measured by densitometry from control, IH3 and IH5 group rats. D. Intensity of 5-HT_2A receptor staining positively correlated with background separately analyzed within the dorsal and ventral XII nucleus. Blue circle = control group; green circle = IH3 group; yellow circle = IH5 group. Data were presented as mean ± S.E.M (n=6 rats/group).

Figure 4

Effect of CIH on c-Fos expression in the XII nucleus. A. Representative photomicrographs for c-Fos expression in the XII nucleus from control (normal room air), IH3 (exposed to 3 weeks of CIH) and IH5 (exposed to 5 weeks of CIH) group rats. Coordinating distance to bregma according to rat brain atlas of Paxinos and Watson illustrated as rostral and caudal XII nucleus. Black arrow indicates nuclear c-Fos immunoreactivity and a magnified view (400×) of the area indicated by the inset boxed. B. Numbers of c-Fos-positive neurons in XII nucleus of the three groups. Data were presented as mean ± S.E.M (n=6 rats/group). cc = central canal, Sol = nucleus of the solitary tract. Scale bar =100 μm.

Figure 5

Representative western blots with summary quantifications of BDNF (A), TrkB (B), synaptophysin (C) and phosphorylation of ERK (D) protein expression in XII nucleus. GAPDH was analyzed as a loading control. Data were presented as mean ± S.E.M (n=6 rats/group), #P<0.05, *P<0.01 versus control group.