Intermittent spinal serotonin receptor activation elicits phrenic motor facilitation (pMF), a form of spinal respiratory motor plasticity. Episodic activation of either serotonin type 2 (5-HT) or type 7 (5-HT) receptors elicits pMF, although they do so via distinct cellular mechanisms known as the Q (5-HT) and S (5-HT) pathways to pMF. When coactivated, these pathways interact via mutual cross-talk inhibition. Although we have a rudimentary understanding of mechanisms mediating cross-talk interactions between spinal 5-HT subtype A (5-HT) and 5-HT receptor activation, we do not know if similar interactions exist between 5-HT subtype B (5-HT) and 5-HT receptors. We confirmed that either spinal 5-HT or 5-HT receptor activation alone elicits pMF and tested the hypotheses that 1) concurrent activation of both receptors suppresses pMF due to cross-talk inhibition; 2) 5-HT receptor inhibition of 5-HT receptor-induced pMF requires protein kinase A (PKA) activity; and 3) 5-HT receptor inhibition of 5-HT receptor-induced pMF requires NADPH oxidase (NOX) activity. Selective 5-HT and 5-HT receptor agonists were administered intrathecally at C4 (3 injections, 5-min intervals) to anesthetized, paralyzed, and ventilated rats. Whereas integrated phrenic nerve burst amplitude increased after selective spinal 5-HT or 5-HT receptor activation alone (i.e., pMF), pMF was no longer observed with concurrent 5-HT and 5-HT receptor agonist administration. With concurrent receptor activation, pMF was rescued by inhibiting either NOX or PKA activity, demonstrating their roles in cross-talk inhibition between these pathways to pMF. This report demonstrates cross-talk inhibition between 5-HT- and 5-HT receptor-induced pMF and that NOX and PKA activity are necessary for that cross-talk inhibition.

Cervical spinal adenosine 2A (A ) receptor activation elicits a prolonged increase in phrenic nerve activity, an effect known as phrenic motor facilitation (pMF). The specific cervical spinal cells expressing the relevant A receptors for pMF are unknown. This is an important question since the physiological outcome of A receptor activation is highly cell type specific. Thus, we tested the hypothesis that the relevant A receptors for pMF are expressed in phrenic motor neurons per se versus non-phrenic neurons of the cervical spinal cord. A receptor immunostaining significantly colocalized with NeuN-positive neurons (89 ± 2%). Intrapleural siRNA injections were used to selectively knock down A receptors in cholera toxin B-subunit-labelled phrenic motor neurons. A receptor knock-down was verified by a ∼45% decrease in A receptor immunoreactivity within phrenic motor neurons versus non-targeting siRNAs (siNT; P < 0.05). There was no evidence for knock-down in cervical non-phrenic motor neurons. In rats that were anaesthetized, subjected to neuromuscular blockade and ventilated, pMF induced by cervical (C3-4) intrathecal injections of the A receptor agonist CGS21680 was greatly attenuated in siA (21%) versus siNT treated rats (147%; P < 0.01). There were no significant effects of siA on phrenic burst frequency. Collectively, our results support the hypothesis that phrenic motor neurons express the A receptors relevant to A receptor-induced pMF.

Background Inflammation undermines multiple forms of neuroplasticity. Although inflammation and its influence on plasticity in multiple neural systems has been extensively studied, its effects on plasticity of neural networks controlling vital life functions, such as breathing, are less understood. In this study, we investigated the signaling mechanisms whereby lipopolysaccharide (LPS)-induced systemic inflammation impairs plasticity within the phrenic motor system—a major spinal respiratory motor pool that drives contractions of the diaphragm muscle. Here, we tested the hypotheses that lipopolysaccharide-induced systemic inflammation (1) blocks phrenic motor plasticity by a mechanism that requires cervical spinal okadaic acid-sensitive serine/threonine protein phosphatase (PP) 1/2A activity and (2) prevents phosphorylation/activation of extracellular signal-regulated kinase 1/2 mitogen activated protein kinase (ERK1/2 MAPK)—a key enzyme necessary for the expression of phrenic motor plasticity. Methods To study phrenic motor plasticity, we utilized a well-characterized model for spinal respiratory plasticity called phrenic long-term facilitation (pLTF). pLTF is characterized by a long-lasting, progressive enhancement of inspiratory phrenic nerve motor drive following exposures to moderate acute intermittent hypoxia (mAIH). In anesthetized, vagotomized and mechanically ventilated adult Sprague Dawley rats, we examined the effect of inhibiting cervical spinal serine/threonine PP 1/2A activity on pLTF expression in sham-vehicle and LPS-treated rats. Using immunofluorescence optical density analysis, we compared mAIH-induced phosphorylation/activation of ERK 1/2 MAPK with and without LPS-induced inflammation in identified phrenic motor neurons. Results We confirmed that mAIH-induced pLTF is abolished 24 h following low-dose systemic LPS (100 μg/kg, i.p.). Cervical spinal delivery of the PP 1/2A inhibitor, okadaic acid, restored pLTF in LPS-treated rats. LPS also prevented mAIH-induced enhancement in phrenic motor neuron ERK1/2 MAPK phosphorylation. Thus, a likely target for the relevant okadaic acid-sensitive protein phosphatases is ERK1/2 MAPK or its upstream activators. Conclusions This study increases our understanding of fundamental mechanisms whereby inflammation disrupts neuroplasticity in a critical population of motor neurons necessary for breathing, and highlights key roles for serine/threonine protein phosphatases and ERK1/2 MAPK kinase in the plasticity of mammalian spinal respiratory motor circuits.

Chronic intermittent hypoxia (CIH) and one-kidney, one-clip (1K, 1C) experimental models lead to sympathetic overactivity and hypertension. We hypothesized that previous exposure to CIH potentiates the development of 1K, 1C renal hypertension. Male rats were divided into the following four groups: Control-1K, 1C, maintained under normoxia followed by 1K, 1C surgery (n = 19); Control-Sham, maintained under normoxia, followed by sham surgery (n = 19); CIH-1K, 1C, exposed to CIH (10 days) and 1K, 1C surgery (n = 19); and CIH-Sham, exposed to CIH and sham surgery (n = 18). Animals were catheterized 8 days after 1K, 1C or Sham surgeries and cardiovascular and respiratory parameters recorded on the following day. Baseline mean arterial pressure was higher in Control-1K, 1C than in Control-Sham rats (P < 0.05) and was higher in CIH-1K, 1C than in CIH-Sham rats (P < 0.05). However, the increase in mean arterial pressure in CIH-1K, 1C animals was significantly blunted in comparison to Con-1K, 1C rats (P < 0.05), indicating that previous exposure to CIH attenuates the development of renal hypertension. Systemic administration of hexamethonium, a ganglionic blocker, promoted a larger hypotensive response in Con-1K, 1C compared with CIH-1K, 1C rats (P < 0.05), suggesting that sympathetic activity was attenuated in rats previously exposed to the CIH protocol. In addition, removal of the carotid bodies before 1K, 1C renal hypertension eliminated the protective effect of CIH preconditioning on the development of the 1K, 1C hypertension. We conclude that previous exposure to CIH attenuates the development of renal hypertension via a carotid body-dependent mechanism.

Plasticity is a fundamental property of the neural system controlling breathing. One key example of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a persistent increase in phrenic nerve activity elicited by acute intermittent hypoxia (AIH). pLTF can arise from distinct cell signaling cascades initiated by serotonin versus adenosine receptor activation, respectively; these signaling cascades interact via powerful cross-talk inhibition. Here, we demonstrate that the daily rest versus active phase and the duration of hypoxic episodes within an AIH protocol have profound impact on the magnitude and even mechanism of pLTF due to shifts in the serotonin/adenosine balance. Using the historical “standard” AIH protocol (3, 5 min moderate hypoxic episodes), we demonstrate that pLTF magnitude is unaffected by exposure in the mid-active versus mid-rest phase, yet the mechanism driving pLTF shifts from serotonin-dominant during mid-rest to adenosine-dominant in the mid-active phase. This mechanistic “flip” results from combined influences of hypoxia-evoked adenosine release and normal cycles in basal spinal adenosine between the rest versus active phase. Since AIH consisting of shorter hypoxic episodes but the same cumulative duration of hypoxia (15, 1 min episodes) elicits less adenosine release during hypoxic episodes, mid-rest pLTF is amplified due to a diminished adenosine constraint to serotonin-driven plasticity; on the other hand, this same 15 × 1 AIH protocol delivered in the mid-active phase suppresses serotonin-dominant pLTF due to elevated background adenosine levels but low hypoxia-evoked adenosine release. These findings demonstrate the importance of the serotonin/adenosine balance in regulating the amplitude and even mechanism of AIH-induced pLTF. Since AIH is emerging as a promising therapeutic modality to restore respiratory and non-respiratory movements in people with spinal cord injury or ALS, knowledge of how time-of-day and hypoxic episode duration impact the serotonin/adenosine balance and the magnitude and mechanism of pLTF has profound biological, experimental and translational implications.