Expressions of 5-HT/5-HT2A receptors and phospho-protein kinase C theta in the pre-Bötzinger complex in normal and chronic intermittent hypoxic rats

Abstract

The pre-Bötzinger complex (pre-BötC), a functionally defined subregion in the ventrolateral medulla oblongata, is a presumed kernel of normal respiratory rhythmogenesis. However, less is known about the pre-BötC's contribution to respiratory neuroplasticity. The most frequently studied model for respiratory neuroplasticity is episodic hypoxia-induced phrenic long-term facilitation, which is 5-HT2A receptors (5-HT2AR)-dependent. We hypothesized that preconditioning with chronic intermittent hypoxic (CIH) would activate the 5-HT/5-HT2AR system and the downstream protein kinase C (PKC) pathway in the pre-BötC. Animals were exposed to alternating 5 min of hypobaric hypoxia and 5 min of normoxia for 10 h/day for 7 days. Hypobaric hypoxia was achieved by continuous air evacuation to reach a pressure of 210–220 mm Hg, corresponding to an altitude of 9000–10000 m. In contrast to the CIH model, a group of animals were pretreated with chronic sustaining hypoxia (CSH), a protocol of continuous hypobaric hypoxia at 360 mm Hg, corresponding to an altitude of about 6000 m, for 10 h/day for 7 days. Immunoreactivity of 5-HT and 5-HT2AR was examined in the pre-BötC, identified by the presence of neurokinin-1 receptor (NK1R). We found that 15.5% of 5-HT-immunoreactive (ir) terminals were in contact with NK1R-ir neurons. Asymmetric synapses could be identified between them. 38.7% of NK1R-ir dendrites were also immunoreactive for 5-HT2AR, which was distributed along the inner surface of the plasma membrane in control animals. CIH challenge increased the expressions of 5-HT and 5-HT2AR in the pre-BötC, an increase in the expressed 5-HT2AR that was not detected in this region in CSH animals. Specifically, 5-HT2AR was distributed not only along the inner surface, but also along the outer surface, or directly on the plasma membrane, a pattern not detectable in control animals. 5-HT2AR was also detectable in the invaginations of the plasma membrane, where receptor endocytosis or exocytosis might occur, indicating CIH-induced higher trafficking of 5-HT2AR. Concurrently, there was an up-regulation of phospho-PKC theta (P-PKCθ) in the pre-BötC, suggesting a 5-HT/5-HT2AR-activated PKC mechanism that may contribute to hypoxia-induced respiratory neuroplasticity in the pre-BötC. The close association of P-PKCθ with the postsynaptic density implicates a postsynaptic mechanism mediating respiratory neuroplasticity in the pre-BötC.