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. 2015 Sep;114(3):1784-91.
doi: 10.1152/jn.00539.2015. Epub 2015 Jul 29.

Mammalian target of rapamycin is required for phrenic long-term facilitation following severe but not moderate acute intermittent hypoxia

Brendan J Dougherty  1 , Daryl P Fields  2 , Gordon S Mitchell  3
Affiliations

Mammalian target of rapamycin is required for phrenic long-term facilitation following severe but not moderate acute intermittent hypoxia

Brendan J Dougherty et al. J Neurophysiol. 2015 Sep.

Abstract

Phrenic long-term facilitation (pLTF) is a persistent increase in phrenic nerve activity after acute intermittent hypoxia (AIH). Distinct cell-signaling cascades give rise to pLTF depending on the severity of hypoxemia within hypoxic episodes. Moderate AIH (mAIH; three 5-min episodes, PaO2 ∼35-55 mmHG) elicits pLTF by a serotonin (5-HT)-dependent mechanism that requires new synthesis of brain-derived neurotrophic factor (BDNF), activation of its high-affinity receptor (TrkB), and ERK MAPK signaling. In contrast, severe AIH (sAIH; three 5-min episodes, PaO2 ∼25-30 mmHG) elicits pLTF by an adenosine-dependent mechanism that requires new TrkB synthesis and Akt signaling. Although both mechanisms require spinal protein synthesis, the newly synthesized proteins are distinct, as are the neurochemicals inducing plasticity (serotonin vs. adenosine). In many forms of neuroplasticity, new protein synthesis requires translational regulation via mammalian target of rapamycin (mTOR) signaling. Since Akt regulates mTOR activity, we hypothesized that mTOR activity is necessary for sAIH- but not mAIH-induced pLTF. Phrenic nerve activity in anesthetized, paralyzed, and ventilated rats was recorded before, during, and 60 min after mAIH or sAIH. Rats were pretreated with intrathecal injections of 20% DMSO (vehicle controls) or rapamycin (0.1 mM, 12 μl), a selective mTOR complex 1 inhibitor. Consistent with our hypothesis, rapamycin blocked sAIH- but not mAIH-induced pLTF. Thus spinal mTOR activity is required for adenosine-dependent (sAIH) but not serotonin-dependent (mAIH) pLTF, suggesting that distinct mechanisms regulate new protein synthesis in these forms of spinal neuroplasticity.

Keywords: hypoxia; long-term facilitation; mTOR; motor neuron; phrenic; plasticity; rapamycin; spinal; translational regulation.

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Figures

Fig. 1.
Fig. 1.
Moderate acute intermittent hypoxia (mAIH)-induced phrenic long-term facilitation (pLTF) does not require mammalian target of rapamycin complex 1 (mTORC1) activity. A–C: representative traces of integrated phrenic neurograms during and after mAIH in vehicle control (20% DMSO in saline; A), rapamycin-treated (0.1 mM, 12 μl; B), and time control rats (no AIH; C). The dashed line indicates baseline (BL) phrenic amplitude in each trace. In A and B, typical pLTF in response to mAIH is shown, demonstrating that mTORC1 signaling is not necessary for mAIH-induced pLTF. In C, no time-dependent change in phrenic burst amplitude is shown, demonstrating experimental preparation stability. D: phrenic burst amplitude (percent change above BL) in vehicle controls (n = 7, filled circles), rapamycin-treated (n = 5, open circles), and combined time control rats (n = 8, triangles). pLTF is significant in vehicle control and rapamycin-treated rats at 30 and 60 min post-mAIH compared with time controls (both P < 0.001). E: frequency of phrenic bursting was unaffected by mAIH or rapamycin. Burst frequency remained consistent across groups at all time points (P > 0.05). ***P < 0.001 vs. time controls.
Fig. 2.
Fig. 2.
Severe AIH (sAIH)-induced pLTF requires mTOR activity. A–C: representative traces of integrated phrenic neurograms during and after sAIH in vehicle control (20% DMSO in saline; A), rapamycin-treated (mTORC1 inhibitor, 0.1 mM, 12 μl; B), and time control rats (no AIH; C). The dashed line indicates BL phrenic amplitude. In A, robust pLTF is shown following sAIH in control rats. As shown in B, rapamycin pretreatment abolishes sAIH-induced pLTF, demonstrating that mTORC1 signaling is necessary. In C, no time-dependent change in phrenic burst amplitude is shown without AIH (i.e., time controls). D: phrenic burst amplitude (percent change above BL) in vehicle control (n = 8, filled circles), rapamycin-pretreated (n = 6, open circles), and combined time control rats (n = 8, triangles). pLTF is significantly elevated in vehicle control rats compared with rapamycin-pretreated and time control rats at 15, 30, and 60 min post-sAIH (P < 0.05 for each). E: phrenic burst frequency was elevated in vehicle- and rapamycin-treated rats at 30 min post sAIH (P < 0.05), although frequency had returned to BL by 60 min post-sAIH. No differences in burst frequency were observed between treatment groups (P > 0.05), indicating the effect on burst frequency was a result of sAIH vs. rapamycin. *P < 0.05; ***P < 0.001 vs. time controls. #P < 0.01; ###P < 0.001 vs. rapamycin-treated rats.
Fig. 3.
Fig. 3.
Working model of convergent pLTF pathways. The Q pathway to phrenic motor facilitation (pMF) is initiated by episodic activation of Gq protein-coupled 5-HT2 receptors (MacFarlane and Mitchell 2009; MacFarlane et al. 2011) followed by PKCθ activation (Devinney et al. 2015), new brain-derived neurotrophic factor (BDNF) synthesis (Baker-Herman et al. 2004), TrkB (Baker-Herman et al. 2004; Dale EA and Mitchell GS, unpublished observations), and ERK/MAP kinase activation (Hoffman et al. 2012). The mechanism whereby ERK elicits pLTF remains unknown. We propose a newly organized “S” pathway (right) to pLTF initiated by adenosine 2A (A2A) receptors (Golder et al. 2008), followed by cAMP production, EPAC, Akt, and mTOR activation (Fields et al. 2015) and new synthesis of immature TrkB isoforms (Golder et al. 2008). Mechanisms downstream of iTrkB are unknown. S-to-Q pathway inhibition via PKA (Hoffman and Mitchell 2013) diverges at cAMP based on recent observations (Fields et al. 2015).
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