Phrenic long-term facilitation after acute intermittent hypoxia requires spinal ERK activation but not TrkB synthesis

Abstract

Acute intermittent hypoxia (AIH) elicits a form of spinal respiratory plasticity known as phrenic long-term facilitation (pLTF). pLTF requires spinal serotonin receptor-2 activation, the synthesis of new brain-derived neurotrophic factor (BDNF), and the activation of its high-affinity receptor tyrosine kinase, TrkB. Spinal adenosine 2A receptor activation elicits a distinct pathway to phrenic motor facilitation (pMF); this BDNF synthesis-independent pathway instead requires new synthesis of an immature TrkB isoform. Since hypoxia increases extracellular adenosine levels, we tested the hypothesis that new synthesis of TrkB and BDNF contribute to AIH-induced pLTF. Furthermore, given that signaling mechanisms "downstream" from TrkB are unknown in either mechanism, we tested the hypothesis that pLTF requires MEK/ERK and/or phosphatidylinositol 3-kinase (PI3K)/Akt activation. In anesthetized Sprague-Dawley rats, an intrathecal catheter at cervical level 4 was used to deliver drugs near the phrenic motor nucleus. Since pLTF was blocked by spinal injections of small interfering RNAs targeting BDNF mRNA but not TrkB mRNA, only new BDNF synthesis is required for AIH-induced pLTF. Pretreatment with a MEK inhibitor (U0126) blocked pLTF, whereas a PI3K inhibitor (PI-828) had no effect. Thus, AIH-induced pLTF requires MEK/ERK (not PI3K/AKT) signaling pathways. When U0126 was injected post-AIH, pLTF development was halted but not reversed, suggesting that ERK is critical for the development but not maintenance of pLTF. Thus, there are clear mechanistic distinctions between AIH-induced pLTF (i.e., BDNF synthesis and MEK/ERK dependent) versus adenosine 2A receptor-induced pMF (i.e., TrkB synthesis and PI3K/Akt dependent).

Fig. 1.

Phrenic hypoxic responses during episodes of hypoxia. Changes in phrenic burst amplitudes during 5-min of hypoxic exposures (average of 3 episodes) from rats that received spinal injections before acute intermittent hypoxia (AIH) or vehicle (siBuffer; n = 7), small interfering (si)RNAs targeting TrkB (siTrkB; n = 8), siRNA targetting brain-derived neurotrophic factor (siBDNF; n = 9), MEK inhibitor (U0126, n = 9), or phosphatidylinositol 3-kinase (PI3K) inhibitor (PI-828; n = 6) and rats that received post-AIH injections of vehicle (n = 8) and U0126 (n = 8) are shown. Phrenic burst amplitudes increased from baseline in all groups, although no between-group differences were detected. ∫Phr, integrated phrenic. Values are means ± SE. #Significantly ifferent from baseline (P < 0.05).

Fig. 2.

Representative phrenic neurograms depicting experimental protocols in rats treated before AIH. A: intrathecal vehicle [RNA inhibition (RNAi) buffer] before (∼2 h) AIH. B: intrathecal siTrkB before AIH. C: intrathecal siBDNF before AIH. D: time control rats that received intrathecal vehicle without AIH. Pretreatment with siTrkB did not inhibit phrenic long-term facilitation (pLTF) compared with vehicle-treated rats exposed to AIH. siBDNF inhibited pLTF versus vehicle, time control experiments. MAP, mean arterial pressure.

Fig. 3.

siTrkB RNA does not block BDNF synthesis-dependent pLTF. Comparisons were made for ∫Phr amplitudes from rats spinally injected with siTrkB RNA (n = 8) versus siBDNF RNA (n = 9). ∫Phr amplitudes from siTrkB-treated rats were not different from vehicle-treated rats (RNAi buffer, n = 7) at 60 min post-AIH, whereas siBDNF treatment significantly inhibited pLTF at 60 min post-AIH. A group of rats treated with vehicle did not receive AIH (i.e., time controls, n = 5). ∫Phr amplitude in vehicle-treated control rats was not different than baseline at any time point. Values are means ± SE. #Significantly different from vehicle control; †significantly different from siBDNF treatment (P < 0.05).

Fig. 4.

Spinal MEK inhibition blocks pLTF. A: representative phrenic neurogram after intrathecal MEK inhibitor (U0126) before AIH. B: representative phrenic neurogram after intrathecal PI3K inhibitor (PI-828) before AIH. C: comparisons were made for ∫Phr burst amplitudes from spinally treated rats (expressed as the percent change from baseline). ∫Phr burst amplitudes from rats pretreated (20 min before) with a MEK inhibitor (U0126, n = 9) were decreased compared with vehicle treatment (n = 7) at 60 min post-AIH. In rats pretreated with PI3K inhibitor (PI-828, n = 6), ∫Phr burst amplitudes increased over baseline at 60 min post-AIH and were not different than vehicle treatment. Hence, MEK activity, not PI3K, is required for pLTF. Values are means ± SE. #Significantly different from vehicle control; †significantly different from MEK inhibitor (P < 0.05).

Fig. 5.

Representative phrenic neurograms depicting experimental protocols in rats treated post-AIH. Spinal injection post-AIH (<5 min) of vehicle (12 μl; A), vehicle control (no AIH; B), MEK inhibitor (U0126, 12 μl; C), and U0126 control (no AIH; D) treatments are shown. Post-AIH U0126 attenuated pLTF at 60 and 90 min compared with vehicle-treated rats exposed to AIH.

Fig. 6.

Spinal MEK inhibition post-AIH attenuates pLTF expression. Comparisons were made for ∫Phr burst amplitudes from spinally treated rats (expressed as the percent change from baseline). ∫Phr burst amplitudes from rats treated with vehicle (n = 8) immediately after AIH (<5 min) exhibited significant pLTF up to 90 min post-AIH. To test the hypothesis that pLTF maintanence requires MEK activity, rats were given a spinal injection of a selective inhibitor (U0126, 100 μM, n = 8) immediately after AIH. U0126 attenuated ∫Phr burst amplitudes at 60 min post-AIH and beyond, suggesting that continued MEK/ERK activity is required to maintain full pLTF expression. Control rats that received either vehicle (n = 8) or U0126 (n = 8) without AIH exhibited facilitation. Values are means ± SE. *Significantly different from MEK inhibitor; #significantly different from vehicle control; †significantly different from MEK inhibitor control (P < 0.05).

Fig. 7.

Changes in phrenic (ΔPhr) burst frequency. A–C: ΔPhr nerve burst frequency from baseline (in bursts/min) in rats that received a pre-AIH spinal injection of vehicle (siBuffer), siTrkB, siBDNF, and vehicle time control (A). B: comparisons from spinal MEK inhibitor (U0126) and PI3K inhibitor (PI-828). C: comparisons from groups that received post-AIH injections of vehicle, U0126, and time control treatments (vehicle and U0126). Values are means ± SE. #Significantly different than baseline; ‡significantly different than siBDNF; †significantly different than vehicle (pre-AIH treatment); ζsignificantly different than MEK inhibitor (pre-AIH); $significantly different than vehicle control (post-AIH); &significantly different than MEK inhibitor control (post-AIH) (P < 0.05).