Inactivity-induced Phrenic Motor Facilitation Research Articles

Inactivity-induced phrenic motor facilitation requires PKCζ activity within phrenic motor neurons.

Prolonged inhibition of respiratory neural activity elicits a long-lasting increase in phrenic nerve amplitude, known as inactivity-induced phrenic motor facilitation (iPMF). Facilitation also occurs transiently in inspiratory intercostal nerve activity following inactivity (iIMF). Atypical PKC activity in the cervical spinal cord is necessary for iPMF and iIMF, but the site and relevant PKC isoform are unknown. Here, we used RNA interference to test the hypothesis that the atypical PKCζ isoform within phrenic motor neurons is necessary for iPMF, but PKCζ within intercostal motor neurons is unnecessary for transient iIMF. Intrapleural siRNA injections were made in rats to knock down phrenic and intercostal motor neuron PKCζ mRNA (siPKCζ). Control rats received non-targeting siRNA (NTsi) or siPKCθ; PKCθ is required for other forms of respiratory motor plasticity. Phrenic nerve and external intercostal (T2) EMG activity were measured in anesthetized, mechanically ventilated rats exposed to 30 min of respiratory neural inactivity (i.e. neural apnea) from modest hypocapnia, or a similar duration without neural apnea (time control). Phrenic burst amplitude increased from baseline with NTsi (68±10%) and siPKCθ (57±8%) 60 min post-neural apnea versus time controls (-3±3%). In contrast, siPKCζ virtually abolished iPMF (5±4%). While iIMF was transient in all groups, siPKCζ attenuated iIMF 5 min post-neural apnea (50±21%) vs NTsi (97±22%) and siPKCθ (103±20%). Neural inactivity elevated phrenic, but not intercostal responses to hypercapnia--an effect blocked by siPKCζ. We conclude phrenic motor neuron PKCζ is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for transient iIMF.

Gestational intermittent hypoxia induces neuroinflammation and impairs compensatory respiratory plasticity in adult offspring

Sleep disordered breathing during pregnancy is increasing at an alarming rate, but virtually nothing is known about long‐lasting effects on adult offspring physiology. Preliminary data suggest that microglia isolated from adult rat offspring exposed to in utero intermittent hypoxia exhibit enhanced inflammatory gene expression and primed responses to inflammatory stimuli. Since neuroinflammation abrogates some forms of respiratory plasticity, we tested the hypothesis that gestational intermittent hypoxia (GIH) impairs respiratory plasticity in adult offspring. Pregnant rat dams were exposed to chronic intermittent hypoxia (8 hrs/day, 2 min 10.5% O2 separated by 2 min of 21% O2) or intermittent normoxia from gestation days 10–21 (GIH and GNX, respectively). Respiratory plasticity was assessed by measuring the response to recurrent neural apnea in urethane‐anesthetized, mechanically ventilated male and female GNX and GIH offspring. Recurrent neural apnea was induced by briefly hyperventilating below the CO2 apneic threshold for breathing (5, ~1 min neural apnea episodes, separated by 5 min). In GNX offspring, recurrent neural apnea triggered a significant increase in phrenic burst amplitude (45±3 %baseline, n=6, p<0.05), a form of plasticity known as inactivity‐induced phrenic motor facilitation (iPMF). By contrast, intermittent neural apnea did not trigger increased phrenic burst amplitude in GIH offspring (7±8 %baseline, n=10, p>0.05), suggesting that in utero intermittent hypoxia impairs the capacity to elicit iPMF. Further, the phrenic response to intermittent neural apnea was significantly different in GIH and GNX rats (p<0.05). A critical role for microglial neuroinflammation is suggested by preliminary data indicating that the capacity to elicit iPMF can be restored in adult GIH offspring in which microglia are depleted with Pexidartinib (PLX3397) or following administration of the non‐steroidal anti‐inflammatory drug ketoprofen. Since iPMF is hypothesized to be a compensatory form of respiratory plasticity that prevents recurrent apneas, the impact of impaired iPMF on breathing stability in adult GIH offspring during sleep is currently being investigated.Support or Funding InformationHL105511, NS085226This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intermittent apnea elicits inactivity-induced phrenic motor facilitation via a retinoic acid- and protein synthesis-dependent pathway.

Respiratory motoneuron pools must provide rhythmic inspiratory drive that is robust and reliable, yet dynamic enough to respond to respiratory challenges. One form of plasticity that is hypothesized to contribute to motor output stability by sensing and responding to inadequate respiratory neural activity is inactivity-induced phrenic motor facilitation (iPMF), an increase in inspiratory output triggered by a reduction in phrenic synaptic inputs. Evidence suggests that mechanisms giving rise to iPMF differ depending on the pattern of reduced respiratory neural activity (i.e., neural apnea). A prolonged neural apnea elicits iPMF via a spinal TNF-α-induced increase in atypical PKC activity, but little is known regarding mechanisms that elicit iPMF following intermittent neural apnea. We tested the hypothesis that iPMF triggered by intermittent neural apnea requires retinoic acid and protein synthesis. Phrenic nerve activity was recorded in urethane-anesthetized and -ventilated rats treated intrathecally with an inhibitor of retinoic acid synthesis (4-diethlyaminobenzaldehyde, DEAB), a protein synthesis inhibitor (emetine), or vehicle (artificial cerebrospinal fluid) before intermittent (5 episodes, ~1.25 min each) or prolonged (30 min) neural apnea. Both DEAB and emetine abolished iPMF elicited by intermittent neural apnea but had no effect on iPMF elicited by a prolonged neural apnea. Thus different patterns of reduced respiratory neural activity elicit phenotypically similar iPMF via distinct spinal mechanisms. Understanding mechanisms that allow respiratory motoneurons to dynamically tune their output may have important implications in the context of respiratory control disorders that involve varied patterns of reduced respiratory neural activity, such as central sleep apnea and spinal cord injury.NEW & NOTEWORTHY We identify spinal retinoic acid and protein synthesis as critical components in the cellular cascade whereby repetitive reductions in respiratory neural activity elicit rebound increases in phrenic inspiratory activity.

Reduced respiratory neural activity elicits a long-lasting decrease in the CO2 threshold for apnea in anesthetized rats

Two critical parameters that influence breathing stability are the levels of arterial pCO2 at which breathing ceases and subsequently resumes – termed the apneic and recruitment thresholds (AT and RT, respectively). Reduced respiratory neural activity elicits a chemoreflex-independent, long-lasting increase in phrenic burst amplitude, a form of plasticity known as inactivity-induced phrenic motor facilitation (iPMF). The physiological significance of iPMF is unknown. To determine if iPMF and neural apnea have long-lasting physiological effects on breathing, we tested the hypothesis that patterns of neural apnea that induce iPMF also elicit changes in the AT and RT. Phrenic nerve activity and end-tidal CO2 were recorded in urethane-anesthetized, ventilated rats to quantify phrenic nerve burst amplitude and the AT and RT before and after three patterns of neural apnea that differed in their duration and ability to elicit iPMF: brief intermittent neural apneas, a single brief “massed” neural apnea, or a prolonged neural apnea. Consistent with our hypothesis, we found that patterns of neural apnea that elicited iPMF also resulted in changes in the AT and RT. Specifically, intermittent neural apneas progressively decreased the AT with each subsequent neural apnea, which persisted for at least 60min. Similarly, a prolonged neural apnea elicited a long-lasting decrease in the AT. In both cases, the magnitude of the AT decrease was proportional to iPMF. In contrast, the RT was transiently decreased following prolonged neural apnea, and was not proportional to iPMF. No changes in the AT or RT were observed following a single brief neural apnea. Our results indicate that the AT and RT are differentially altered by neural apnea and suggest that specific patterns of neural apnea that elicit plasticity may stabilize breathing via a decrease in the AT.

Inactivity‐induced Phrenic Motor Facilitation Following Intermittent, but not Sustained, Neural Apnea Requires Retinoic Acid and Protein Synthesis

Phrenic motor output is enhanced following respiratory neural inactivity (neural apnea), a form of spinal plasticity called inactivity‐induced phrenic motor facilitation (iPMF). Mechanisms giving rise to iPMF differ depending on the pattern of neural apnea since TNFα is required to initiate iPMF following a sustained neural apnea, but not when neural apnea occurs intermittently. Some forms of inactivity‐induced plasticity in other neural networks require retinoic acid (RA) to promote translation of proteins that increase neuronal synaptic strength. Accordingly, we hypothesized that iPMF elicited by intermittent neural apnea requires spinal RA and protein synthesis. Anesthetized and ventilated rats were prepared for electrophysiological recording of phrenic burst amplitude. Rats received intrathecal injection (C4) of the RA synthesis inhibitor DEAB (100μM), the protein synthesis inhibitor emetine (70μM) or vehicle (aCSF) prior to intermittent neural apnea (5 ~1.25 min episodes), sustained neural apnea (30 min) or no neural apnea (time control). Following intermittent neural apnea, iPMF was impaired in rats receiving either DEAB (22±10 %baseline) or emetine (10±9 %baseline) since phrenic amplitude was decreased compared to aCSF (56±6 %baseline; p<0.05) and not different from time controls (7±7 %baseline; p>0.05). In contrast, DEAB and emetine did not impair iPMF following sustained neural apnea (87±18 and 56±12 %baseline, respectively) since phrenic amplitude was not different than aCSF (p>0.05) and increased from time controls (p<0.05). Together, these data indicate that mechanisms giving rise to iPMF differ in their requirement for RA and protein synthesis depending on the pattern of reduced respiratory neural activity. NIH HL 105511

Intermittent reductions in respiratory neural activity elicit spinal TNF-α-independent, atypical PKC-dependent inactivity-induced phrenic motor facilitation.

In many neural networks, mechanisms of compensatory plasticity respond to prolonged reductions in neural activity by increasing cellular excitability or synaptic strength. In the respiratory control system, a prolonged reduction in synaptic inputs to the phrenic motor pool elicits a TNF-α- and atypical PKC-dependent form of spinal plasticity known as inactivity-induced phrenic motor facilitation (iPMF). Although iPMF may be elicited by a prolonged reduction in respiratory neural activity, iPMF is more efficiently induced when reduced respiratory neural activity (neural apnea) occurs intermittently. Mechanisms giving rise to iPMF following intermittent neural apnea are unknown. The purpose of this study was to test the hypothesis that iPMF following intermittent reductions in respiratory neural activity requires spinal TNF-α and aPKC. Phrenic motor output was recorded in anesthetized and ventilated rats exposed to brief intermittent (5, ∼1.25 min), brief sustained (∼6.25 min), or prolonged sustained (30 min) neural apnea. iPMF was elicited following brief intermittent and prolonged sustained neural apnea, but not following brief sustained neural apnea. Unlike iPMF following prolonged neural apnea, spinal TNF-α was not required to initiate iPMF during intermittent neural apnea; however, aPKC was still required for its stabilization. These results suggest that different patterns of respiratory neural activity induce iPMF through distinct cellular mechanisms but ultimately converge on a similar downstream pathway. Understanding the diverse cellular mechanisms that give rise to inactivity-induced respiratory plasticity may lead to development of novel therapeutic strategies to treat devastating respiratory control disorders when endogenous compensatory mechanisms fail.

Spinal NMDA receptor activation constrains inactivity-induced phrenic motor facilitation in Charles River Sprague-Dawley rats.

Reduced spinal synaptic inputs to phrenic motor neurons elicit a unique form of spinal plasticity known as inactivity-induced phrenic motor facilitation (iPMF). iPMF requires tumor necrosis factor-α (TNF-α) and atypical protein kinase C (aPKC) activity within spinal segments containing the phrenic motor nucleus to stabilize early, transient increases in phrenic burst amplitude into long-lasting iPMF. Here we tested the hypothesis that spinal N-methyl-d-aspartate receptor (NMDAR) activation constrains long-lasting iPMF in some rat substrains. Phrenic motor output was recorded in anesthetized, ventilated Harlan (HSD) and Charles River (CRSD) Sprague-Dawley rats exposed to a 30-min central neural apnea. HSD rats expressed a robust, long-lasting (>60 min) increase in phrenic burst amplitude (i.e., long-lasting iPMF) when respiratory neural activity was restored. By contrast, CRSD rats expressed an attenuated, transient (∼15 min) iPMF. Spinal NMDAR inhibition with DL-2-amino-5-phosphonopentanoic acid (APV) before neural apnea or shortly (4 min) prior to the resumption of respiratory neural activity revealed long-lasting iPMF in CRSD rats that was phenotypically similar to that in HSD rats. By contrast, APV did not alter iPMF expression in HSD rats. Spinal TNF-α or aPKC inhibition impaired long-lasting iPMF enabled by NMDAR inhibition in CRSD rats, suggesting that similar mechanisms give rise to long-lasting iPMF in CRSD rats with NMDAR inhibition as those giving rise to long-lasting iPMF in HSD rats. These results suggest that NMDAR activation can impose constraints on TNF-α-induced aPKC activation after neural apnea, impairing stabilization of transient iPMF into long-lasting iPMF. These data may have important implications for understanding differential responses to reduced respiratory neural activity in a heterogeneous human population.

Inactivity‐induced phrenic motor facilitation does not require new protein synthesis (713.8)

Reduced respiratory neural activity elicits a TNFα‐ and atypical PKC‐dependent form of spinal plasticity in phrenic motor output termed inactivity‐induced phrenic motor facilitation (iPMF), a long‐lasting increase in phrenic nerve burst amplitude apparent once respiratory neural activity is restored. Many forms of neuroplasticity require protein synthesis for long‐term expression, including the most widely studied form of spinal respiratory plasticity, phrenic long‐term facilitation (pLTF; Baker‐Herman and Mitchell, 2002) elicited following acute intermittent hypoxia. We hypothesized that iPMF would also require de novo protein synthesis. Briefly, Sprague Dawley rats were urethane anesthetized, vagotomized, paralyzed, and prepared for electrophysiological recording. Rats received intrathecal injection (C4) of vehicle (aCSF; n=5) or the protein synthesis inhibitor emetine (n=6; 1ug/kg; Baker‐Herman and Mitchell, 2002) ~15 min prior to a prolonged (30min) period of neural apnea. As expected, in aCSF treated rats, phrenic nerve amplitude was significantly increased compared to baseline 5 min following reversal of neural apnea (53±11, %baseline) and was maintained for >60 min (54±16, %baseline), indicating long‐lasting iPMF. Contrary to our hypothesis, rats receiving emetine also expressed long‐lasting iPMF such that phrenic nerve burst amplitude was significantly increased from baseline at both 5 (41±8, %baseline) and 60 min (42±12,%baseline) following neural apnea (p<0.05). Importantly, phrenic nerve burst amplitude in rats receiving intrathecal emetine was not significantly different from rats receiving aCSF at any time point following neural apnea (p>0.05). Together, these data suggest that iPMF is a form of long‐lasting plasticity that does not require new protein synthesis.Grant Funding Source: Supported by NIH HL105511

Decreased spinal synaptic inputs to phrenic motor neurons elicit localized inactivity-induced phrenic motor facilitation

Phrenic motor neurons receive rhythmic synaptic inputs throughout life. Since even brief disruption in phrenic neural activity is detrimental to life, on-going neural activity may play a key role in shaping phrenic motor output. To test the hypothesis that spinal mechanisms sense and respond to reduced phrenic activity, anesthetized, ventilated rats received micro-injections of procaine in the C2 ventrolateral funiculus (VLF) to transiently (~30min) block axon conduction in bulbospinal axons from medullary respiratory neurons that innervate one phrenic motor pool; during procaine injections, contralateral phrenic neural activity was maintained. Once axon conduction resumed, a prolonged increase in phrenic burst amplitude was observed in the ipsilateral phrenic nerve, demonstrating inactivity-induced phrenic motor facilitation (iPMF). Inhibition of tumor necrosis factor alpha (TNFα) and atypical PKC (aPKC) activity in spinal segments containing the phrenic motor nucleus impaired ipsilateral iPMF, suggesting a key role for spinal TNFα and aPKC in iPMF following unilateral axon conduction block. A small phrenic burst amplitude facilitation was also observed contralateral to axon conduction block, indicating crossed spinal phrenic motor facilitation (csPMF). csPMF was independent of spinal TNFα and aPKC. Ipsilateral iPMF and csPMF following unilateral withdrawal of phrenic synaptic inputs were associated with proportional increases in phrenic responses to chemoreceptor stimulation (hypercapnia), suggesting iPMF and csPMF increase phrenic dynamic range. These data suggest that local, spinal mechanisms sense and respond to reduced synaptic inputs to phrenic motor neurons. We hypothesize that iPMF and csPMF may represent compensatory mechanisms that assure adequate motor output is maintained in a physiological system in which prolonged inactivity ends life.

Spinal TNFα is necessary for inactivity‐induced phrenic motor facilitation

A prolonged reduction in central neural respiratory activity elicits a form of plasticity known as inactivity-induced phrenic motor facilitation (iPMF), a 'rebound' increase in phrenic burst amplitude apparent once respiratory neural activity is restored. iPMF requires atypical protein kinase C (aPKC) activity within spinal segments containing the phrenic motor nucleus to stabilize an early transient increase in phrenic burst amplitude and to form long-lasting iPMF following reduced respiratory neural activity. Upstream signal(s) leading to spinal aPKC activation are unknown. We tested the hypothesis that spinal tumour necrosis factor-α (TNFα) is necessary for iPMF via an aPKC-dependent mechanism. Anaesthetized, ventilated rats were exposed to a 30 min neural apnoea; upon resumption of respiratory neural activity, a prolonged increase in phrenic burst amplitude (42 ± 9% baseline; P < 0.05) was apparent, indicating long-lasting iPMF. Pretreatment with recombinant human soluble TNF receptor 1 (sTNFR1) in the intrathecal space at the level of the phrenic motor nucleus prior to neural apnoea blocked long-lasting iPMF (2 ± 8% baseline; P > 0.05). Intrathecal TNFα without neural apnoea was sufficient to elicit long-lasting phrenic motor facilitation (pMF; 62 ± 7% baseline; P < 0.05). Similar to iPMF, TNFα-induced pMF required spinal aPKC activity, as intrathecal delivery of a ζ-pseudosubstrate inhibitory peptide (PKCζ-PS) 35 min following intrathecal TNFα arrested TNFα-induced pMF (28 ± 8% baseline; P < 0.05). These data demonstrate that: (1) spinal TNFα is necessary for iPMF; and (2) spinal TNFα is sufficient to elicit pMF via a similar aPKC-dependent mechanism. These data are consistent with the hypothesis that reduced respiratory neural activity elicits iPMF via a TNFα-dependent increase in spinal aPKC activity.

Inactivity‐induced phrenic motor facilitation (iPMF) requires atypical PKCζ activity within phrenic motor neurons

Reduced respiratory neural activity elicits a rebound increase in phrenic amplitude once respiratory neural activity is restored, a form of spinal plasticity termed inactivity‐induced phrenic motor facilitation (iPMF). We tested the hypothesis that the zeta isoform of atypical PKC (PKCζ) is necessary within phrenic motor neurons (PMNs) for iPMF. Rats received bilateral intrapleural injections of siRNA (Sieck et al., 2012; EB Abstract #1147.2) targeting PKCζ (30ul of 100pmol siRNA/side) once daily for 3 days to knock down PKCζ expression specifically within phrenic motor neurons. Control rats received a non‐targeting siRNA. The fourth day, rats were anesthetized and mechanically ventilated; the phrenic nerve was prepared for electrophysiological recordings. After baseline phrenic amplitude was established, respiratory neural activity was reversibly suppressed for 30min by hyperventilating until phrenic activity ceased. 60min following restoration of baseline conditions, rats treated with control injections expressed significant iPMF (82±19 %baseline, p&lt;0.05), whereas iPMF was significantly attenuated (13±10 %baseline, p&gt;;0.05) in siPKCζ treated rats. PKCζ knockdown was confirmed within ventral cervical spinal segments containing PMNs with western blot analysis (−31±3 %control; p&lt;0.05). These data indicate that expression of PKCζ specifically within PMNs is necessary for iPMF. NIH105511

Spinal Atypical Protein Kinase C Activity Is Necessary to Stabilize Inactivity-Induced Phrenic Motor Facilitation

The neural network controlling breathing must establish rhythmic motor output at a level adequate to sustain life. Reduced respiratory neural activity elicits a novel form of plasticity in circuits driving the diaphragm known as inactivity-induced phrenic motor facilitation (iPMF), a rebound increase in phrenic inspiratory output observed once respiratory neural drive is restored. The mechanisms underlying iPMF are unknown. Here, we demonstrate in anesthetized rats that spinal mechanisms give rise to iPMF and that iPMF consists of at least two mechanistically distinct phases: (1) an early, labile phase that requires atypical PKC (PKCζ and/or PKCι/λ) activity to transition to a (2) late, stable phase. Early (but not late) iPMF is associated with increased interactions between PKCζ/ι and the scaffolding protein ZIP (PKCζ-interacting protein)/p62 in spinal regions associated with the phrenic motor pool. Although PKCζ/ι activity is necessary for iPMF, spinal atypical PKC activity is not necessary for phrenic long-term facilitation (pLTF) following acute intermittent hypoxia, an activity-independent form of spinal respiratory plasticity. Thus, while iPMF and pLTF both manifest as prolonged increases in phrenic burst amplitude, they arise from distinct spinal cellular pathways. Our data are consistent with the hypotheses that (1) local mechanisms sense and respond to reduced respiratory-related activity in the phrenic motor pool and (2) inactivity-induced increases in phrenic inspiratory output require local PKCζ/ι activity to stabilize into a long-lasting iPMF. Although the physiological role of iPMF is unknown, we suspect that iPMF represents a compensatory mechanism, assuring adequate motor output in a physiological system in which prolonged inactivity ends life.

Inactivity‐induced phrenic motor facilitation following intermittent neural inactivity is dependent on spinal atypical PKC

Prolonged reductions in respiratory neural activity in Harlan Sprague Dawley (HSD) rats elicit an atypical PKC (aPKC) dependent form of spinal plasticity called inactivity‐induced phrenic motor facilitation (iPMF), a rebound increase in phrenic burst amplitude that is apparent when respiratory neural activity is restored. In Charles River Sprague Dawley (CRSD) rats, iPMF is pattern sensitive and is only induced by intermittent, but not sustained neural apneas. We hypothesized that iPMF in CRSD requires spinal aPKC activity. Anesthetized and ventilated CRSD were hyperventilated to create five brief (~1 min) neural apneas, separated by 5 min. 10 min following intermittent neural apneas, rats received an aPKC inhibitor (ZIP) or scrambled ZIP in the intrathecal space over C4. As expected, significant iPMF was observed prior to intrathecal ZIP or scrambled ZIP (5 min post‐apnea: 56 + 8 and 57 + 12 %baseline; p&lt;0.01). Subsequent intrathecal ZIP significantly attenuated iPMF (p&lt;0.001), such that phrenic amplitude was no longer different from baseline at 60 min post‐intermittent apnea (6 + 7 %baseline; p&gt;0.05). By contrast, rats receiving scrambled ZIP continued to express significant iPMF (69+8 %baseline; p&lt;0.01). These data suggest that similar to iPMF following a prolonged apnea in HSD, increased phrenic burst amplitude following intermittent neural apnea in CRSD is dependent on spinal aPKC activity. NIH 105511

Local reductions in synaptic inputs to the phrenic motor pool elicits atypical Protein Kinase C (aPKC) and Tumor Necrosis Factor alpha (TNFá) dependent inactivity‐induced phrenic motor facilitation

A central neural apnea in ventilated rats elicits an aPKC and TNFα dependent form of plasticity called inactivity‐induced phrenic motor facilitation (iPMF), a rebound increase in phrenic burst amplitude that is apparent when respiratory neural activity is restored. We hypothesized that iPMF following local reductions in synaptic inputs to the phrenic pool occurs via similar mechanisms. Bilateral phrenic output was recorded in anesthetized and ventilated rats. Procaine or vehicle (~200 nl) was injected in the C2 ventrolateral funiculus (VLF) to reversibly reduce axon conduction (~25 min) on one side of the spinal cord. Procaine reduced ipsilateral (−82 + 5 %baseline, p&lt;0.05), but not contralateral (−22 + 16 %baseline, p&gt;0.05) phrenic amplitude. Upon axon conduction recovery, only ipsilateral phrenic amplitude was significantly increased (60 min post: ipsi: 83± 22 %baseline, p&lt;0.01; contra: 33 + 13 %baseline, p&gt;0.05); while no changes in ipsilateral or contralateral phrenic amplitude were observed in vehicle treated rats (10 + 6 and 6 + 8 %baseline; p&gt;0.05). Intrathecal aPKC inhibitor (ZIP) or TNFα scavenger (sTNFR1) blocked ipsilateral iPMF (60min: 24 + 11 and 16 + 7 %baseline, respectively, p&gt;0.05). These data suggest that iPMF induced by local reductions in descending synaptic inputs to the phrenic pool require similar cellular pathways as iPMF following global reductions in respiratory neural activity. NIH HL105511

Spinal atypical PKC activity is necessary for inactivity‐induced phrenic motor facilitation in Charles River Sprague Dawley rats with NMDA receptor inhibition

A central neural apnea elicits an atypical protein kinase C (aPKC) dependent increase in phrenic amplitude when respiratory neural activity is restored, a form of plasticity known as inactivity-induced phrenic motor facilitation (iPMF). Spinal NMDA receptor activation constrains iPMF in Charles River Sprague Dawley (CRSD) rats. We tested the hypothesis that aPKC is necessary for iPMF maintenance in CRSD with NMDA receptor inhibition. Anesthetized and ventilated CRSD received an intrathecal NMDA receptor antagonist, APV (200ng), over C4 spinal segments prior to a hypocapnia-induced neural apnea. When phrenic activity was restored, CRSD with NMDA receptor inhibition had increased phrenic amplitude (15 min post- hypocapnia: 57 +/− 19% baseline, p < 0.05), indicating iPMF. Subsequent delivery of an intrathecal aPKC inhibitor, ZIP (20ug), abolished iPMF, similar to CRSD without NMDA receptor inhibition (60 min post-hypocapnia: 11 +/− 22% and −4 +/− 5% baseline, respectively, p > 0.05). CRSD with NMDA receptor inhibition and no ZIP continued to show iPMF (60 min post-hypocapnia: 64 +/− 8% baseline, p < 0.05). These data suggest that spinal aPKC is necessary for iPMF maintenance in CRSD with NMDA receptor inhibition, similar to iPMF in other strains that lack NMDA receptor constraint. We hypothesize that genetic or epigenetic differences in spinal NMDA receptors underlie strain differences in iPMF expression.

Reduced respiratory neural activity elicits phrenic motor facilitation

We hypothesized that reduced respiratory neural activity elicits compensatory mechanisms of plasticity that enhance respiratory motor output. In urethane-anesthetized and ventilated rats, we reversibly reduced respiratory neural activity for 25-30 min using: hypocapnia (end tidal CO(2)=30 mmHg), isoflurane (~1%) or high frequency ventilation (HFV; ~100 breaths/min). In all cases, increased phrenic burst amplitude was observed following restoration of respiratory neural activity (hypocapnia: 92±22%; isoflurane: 65±22%; HFV: 54±13% baseline), which was significantly greater than time controls receiving the same surgery, but no interruptions in respiratory neural activity (3±5% baseline, p<0.05). Hypocapnia also elicited transient increases in respiratory burst frequency (9±2 versus 1±1bursts/min, p<0.05). Our results suggest that reduced respiratory neural activity elicits a unique form of plasticity in respiratory motor control which we refer to as inactivity-induced phrenic motor facilitation (iPMF). iPMF may prevent catastrophic decreases in respiratory motor output during ventilatory control disorders associated with abnormal respiratory activity.

Differential regulation of ventral spinal atypical PKC and ERK MAP kinase following hypocapnia‐induced respiratory neural inactivity

Hypocapnia‐induced respiratory neural inactivity elicits mechanisms of plasticity that enhance phrenic motor output once activity is restored, an effect known as inactivity‐induced phrenic motor facilitation (iPMF). Since atypical protein kinase C (aPKC) activity is necessary for iPMF, we tested the hypothesis that iPMF is associated with increased aPKC expression in spinal regions associated with the phrenic motor nucleus. Anesthetized and ventilated rats were exposed to 30 min of hypocapnia (5–10 mmHg below baseline) to reversibly stop respiratory neural activity. Following hypocapnia, ventral C3–C5 was fractionated into membrane‐enriched, cytoplasm‐enriched and nucleus‐enriched fractions. Total and phosphorylated aPKC increased in membrane‐enriched (47 +/− 10 and 39 +/− 11%, respectively, p&lt;0.05), but decreased in cytoplasm‐enriched fractions (−77 +/− 5 and −74 +/− 6%, respectively, p&lt;0.05) post‐hypocapnia. In contrast, there were no significant changes in membrane or cytoplasmic expression of phosphorylated ERK (−41 +/− 14 and −12 +/− 10%, respectively, p&gt;0.05), a kinase implicated in other forms of spinal plasticity. Phospho‐ERK was increased in nuclear fractions post‐hypocapnia (97 +/− 17%, p&lt;0.05), suggesting a possible role for ERK in longer‐lasting forms of plasticity elicited by neural inactivity. These data support a prominant role for aPKC in iPMF following hypocapnia (NIH HL69064).

Increased ventral spinal TNF‐alpha following hypocapnia‐induced respiratory neural inactivity

Respiratory‐related neural inactivity induced by hypocapnia elicits an atypical PKC‐dependent form of plasticity known as inactivity‐induced phrenic motor facilitation (iPMF). Since TNF‐alpha plays a prominent role in inactivity‐induced homeostatic plasticity in the cortex, we hypothesized that iPMF is associated with increased TNF‐alpha in spinal regions associated with the phrenic motor nucleus. Anesthetized and ventilated rats were exposed to 30 min of hypocapnia (5–10 mmHg below baseline) to reversibly deprive phrenic motor neurons of rhythmic respiratory activity. Sixty minutes after restoration of phrenic activity, ventral C3–C5 spinal segments were harvested and processed for TNF‐alpha concentration via ELISA. In rats exposed to hypocapnia, TNF‐alpha significantly increased in the ventral cervical spinal cord (44 +/− 10%; n = 3, p &lt; 0.05) versus time control rats not exposed to hypocapnia. Increased TNF‐alpha may be specific to iPMF since preliminary data suggest increased ventral spinal TNF‐alpha does not occur with phrenic motor plasticity induced by intermittent hypoxia (i.e., phrenic LTF). We are currently investigating the requirement for TNF‐alpha receptor activation for iPMF. These preliminary data suggest a prominent role for TNF‐alpha in phrenic motor plasticity following acute neural activity deprivation (NIH HL69064).