Nicole L. Nichols

Hypoxia‐induced phrenic long‐term facilitation: emergent properties

As in other neural systems, plasticity is a hallmark of the neural system controlling breathing. One spinal mechanism of respiratory plasticity is phrenic long‐term facilitation (pLTF) following acute intermittent hypoxia. Although cellular mechanisms giving rise to pLTF occur within the phrenic motor nucleus, different signaling cascades elicit pLTF under different conditions. These cascades, referred to as Q and S pathways to phrenic motor facilitation (pMF), interact via cross‐talk inhibition. Whereas the Q pathway dominates pLTF after mild to moderate hypoxic episodes, the S pathway dominates after severe hypoxic episodes. The biological significance of multiple pathways to pMF is unknown. This review will discuss the possibility that interactions between pathways confer emergent properties to pLTF, including pattern sensitivity and metaplasticity. Understanding these mechanisms and their interactions may enable us to optimize intermittent hypoxia‐induced plasticity as a treatment for patients that suffer from ventilatory impairment or other motor deficits.

Severe acute intermittent hypoxia elicits phrenic long-term facilitation by a novel adenosine-dependent mechanism

Acute intermittent hypoxia [AIH; 3, 5-min episodes; 35-45 mmHg arterial PO(2) (Pa(O(2)))] elicits serotonin-dependent phrenic long-term facilitation (pLTF), a form of phrenic motor facilitation (pMF) initiated by G(q) protein-coupled metabotropic 5-HT(2) receptors. An alternate pathway to pMF is induced by G(s) protein-coupled metabotropic receptors, including adenosine A(2A) receptors. AIH-induced pLTF is dominated by the serotonin-dependent pathway and is actually restrained via inhibition from the adenosine-dependent pathway. Here, we hypothesized that severe AIH shifts pLTF from a serotonin-dependent to an adenosine-dependent form of pMF. pLTF induced by severe (25-30 mmHg Pa(O(2))) and moderate (45-55 mmHg Pa(O(2))) AIH were compared in anesthetized rats, with and without intrathecal (C4) spinal A(2A) (MSX-3, 130 ng/kg, 12 μl) or 5-HT receptor antagonist (methysergide, 300 μg/kg, 15 μl) injections. During severe, but not moderate AIH, progressive augmentation of the phrenic response during hypoxic episodes was observed. Severe AIH (78% ± 8% 90 min post-AIH, n = 6) elicited greater pLTF vs. moderate AIH (41% ± 12%, n = 8; P < 0.05). MSX-3 (28% ± 6%; n = 6; P < 0.05) attenuated pLTF following severe AIH, but enhanced pLTF following moderate AIH (86% ± 26%; n = 8; P < 0.05). Methysergide abolished pLTF after moderate AIH (12% ± 5%; n = 6; P = 0.035), but had no effect after severe AIH (66 ± 13%; n = 5; P > 0.05). Thus severe AIH shifts pLTF from a serotonin-dependent to an adenosine-dependent mechanism; the adenosinergic pathway inhibits the serotonergic pathway following moderate AIH. Here we demonstrate a novel adenosine-dependent pathway to pLTF following severe AIH. Shifts in the mechanisms of respiratory plasticity provide the ventilatory control system greater flexibility as challenges that differ in severity are confronted.

Intermittent Hypoxia and Stem Cell Implants Preserve Breathing Capacity in a Rodent Model of Amyotrophic Lateral Sclerosis

RATIONALE Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1(G93A) rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure. OBJECTIVES To preserve or restore phrenic nerve activity in SOD1(G93A) rats at disease end stage. METHODS SOD1(G93A) rats were injected with human neural progenitor cells (hNPCs) bracketing the phrenic motor nucleus before disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end stage. MEASUREMENTS AND MAIN RESULTS The capacity to generate phrenic motor output in anesthetized rats at disease end stage was: (1) transiently restored by a single presentation of AIH; and (2) preserved ipsilateral to hNPC transplants made before disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival. CONCLUSIONS AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in patients with ALS.

Intrinsic Chemosensitivity of Individual Nucleus Tractus Solitarius (NTS) and Locus Coeruleus (LC) Neurons from Neonatal Rats

Chemosensitive (CS) neurons are found in discrete brainstem regions, but whether the CS response of these neurons is due to intrinsic chemosensitivity of individual neurons or is mediated by changes in chemical and/or electrical synaptic input is largely unknown. We studied the effect of synaptic blockade (11.4 mM Mg2+/0.2mM Ca2+) solution (SNB) and a gap junction uncoupling agent carbenoxolone (CAR--100 microM) on the response of neurons from two CS brainstem regions, the NTS and the LC. In NTS neurons, SNB decreased spontaneous firing rate (FR). We calculated the magnitude of the FR response to hypercapnic acidosis (HA; 15% CO2) using the Chemosensitivity Index (CI). The percentage of NTS neurons activated and CI were the same in the absence and presence of SNB. Blocking gap junctions with CAR did not significantly alter spontaneous FR. CAR did not alter the CI in NTS neurons and resulted in a small decrease in the percentage of activated neurons, which was most evident in NTS neurons from rats younger than postnatal day 10. In LC neurons, SNB resulted in an increase in spontaneous FR. As with NTS neurons, SNB did not alter the percentage of activated neurons or the CI in LC neurons. CAR resulted in a small increase in spontaneous FR in LC neurons. In contrast, CAR had a marked effect on the response of LC neurons to HA: a reduced percentage of CS LC neurons and decreased CI. In summary, both NTS and LC neurons appear to contain intrinsically CS neurons. CS neurons from the two regions receive different tonic input in slices (excitatory for NTS and inhibitory for LC); however, blocking chemical synaptic input does not affect the CS response in either region. In NTS neurons, gap junction coupling plays a small role in the CS response, but gap junctions play a major role in the chemosensitivity of many LC neurons.

Development of chemosensitivity in neurons from the nucleus tractus solitarii (NTS) of neonatal rats

We studied the development of chemosensitivity during the neonatal period in rat nucleus tractus solitarii (NTS) neurons. We determined the percentage of neurons activated by hypercapnia (15% CO(2)) and assessed the magnitude of the response by calculating the chemosensitivity index (CI). There were no differences in the percentage of neurons that were inhibited (9%) or activated (44.8%) by hypercapnia or in the magnitude of the activated response (CI 164+/-4.9%) in NTS neurons from neonatal rats of all ages. To assess the degree of intrinsic chemosensitivity in these neurons we used chemical synaptic block medium and the gap junction blocker carbenoxolone. Chemical synaptic block medium slightly decreased basal firing rate but did not affect the percentage of NTS neurons that responded to hypercapnia at any neonatal age. However, in neonates aged <P10, but not in older neonates, chemical synaptic block medium increased CI. Carbenoxolone did not significantly alter the number of NTS neurons activated by hypercapnia in neonatal rats of any age. In summary, the response of NTS neurons from neonatal rats appears to be intrinsic and largely unchanged throughout early development. In young neonates (<P10) chemical synaptic input reduces the magnitude of the firing rate response to hypercapnia, but otherwise neither chemical synaptic input nor gap junctions significantly alter the percentage of NTS neurons that respond to hypercapnia or the magnitude of that response.

Characterization of the chemosensitive response of individual solitary complex neurons from adult rats

We studied the CO(2)/H(+)-chemosensitive responses of individual solitary complex (SC) neurons from adult rats by simultaneously measuring the intracellular pH (pH(i)) and electrical responses to hypercapnic acidosis (HA). SC neurons were recorded using the blind whole cell patch-clamp technique and loading the soma with the pH-sensitive dye pyranine through the patch pipette. We found that SC neurons from adult rats have a lower steady-state pH(i) than SC neurons from neonatal rats. In the presence of chemical and electrical synaptic blockade, adult SC neurons have firing rate responses to HA (percentage of neurons activated or inhibited and the magnitude of response as determined by the chemosensitivity index) that are similar to SC neurons from neonatal rats. They also have a typical response to isohydric hypercapnia, including decreased DeltapH(i), followed by pH(i) recovery, and increased firing rate. Thus, the chemosensitive response of SC neurons from adults is similar to the chemosensitive response of SC neurons from neonatal rats. Because our findings for adults are similar to previously reported values for neurons from neonatal rats, we conclude that intrinsic chemosensitivity is established early in development for SC neurons and is maintained throughout adulthood.

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.

Adrenergic α1 receptor activation is sufficient, but not necessary for phrenic long-term facilitation

Acute intermittent hypoxia (AIH; three 5-min hypoxic episodes) causes a form of phrenic motor facilitation (pMF) known as phrenic long-term facilitation (pLTF); pLTF is initiated by spinal activation of Gq protein-coupled 5-HT2 receptors. Because α1 adrenergic receptors are expressed in the phrenic motor nucleus and are also Gq protein-coupled, we hypothesized that α1 receptors are sufficient, but not necessary for AIH-induced pLTF. In anesthetized, paralyzed, and ventilated rats, episodic spinal application of the α1 receptor agonist phenylephrine (PE) elicited dose-dependent pMF (10 and 100 μM, P < 0.05; but not 1 μM). PE-induced pMF was blocked by the α1 receptor antagonist prazosin (1 mM; -20 ± 20% at 60 min, -5 ± 21% at 90 min; n = 6). Although α1 receptor activation is sufficient to induce pMF, it was not necessary for AIH-induced pLTF because intrathecal prazosin (1 mM) did not alter AIH-induced pLTF (56 ± 9% at 60 min, 78 ± 12% at 90 min; n = 9). Intravenous (iv) prazosin (150 μg/kg) appeared to reduce pLTF (21 ± 9% at 60 min, 26 ± 8% at 90 min), but this effect was not significant. Hypoglossal long-term facilitation was unaffected by intrathecal prazosin, but was blocked by iv prazosin (-4 ± 14% at 60 min, -13 ± 18% at 90 min), suggesting different LTF mechanisms in different motor neuron pools. In conclusion, Gq protein-coupled α1 adrenergic receptors evoke pMF, but they are not necessary for AIH-induced pLTF.

Chronic hypoxia suppresses the CO2 response of solitary complex (SC) neurons from rats

We studied the effect of chronic hypobaric hypoxia (CHx; 10-11% O(2)) on the response to hypercapnia (15% CO(2)) of individual solitary complex (SC) neurons from adult rats. We simultaneously measured the intracellular pH and firing rate responses to hypercapnia of SC neurons in superfused medullary slices from control and CHx-adapted adult rats using the blind whole cell patch clamp technique and fluorescence imaging microscopy. We found that CHx caused the percentage of SC neurons inhibited by hypercapnia to significantly increase from about 10% up to about 30%, but did not significantly alter the percentage of SC neurons activated by hypercapnia (50% in control vs. 35% in CHx). Further, the magnitudes of the responses of SC neurons from control rats (chemosensitivity index for activated neurons of 166+/-11% and for inhibited neurons of 45+/-15%) were the same in SC neurons from CHx-adapted rats. This plasticity induced in chemosensitive SC neurons by CHx appears to involve intrinsic changes in neuronal properties since they were the same in synaptic blockade medium.

Acute intermittent hypoxia induced phrenic long-term facilitation despite increased SOD1 expression in a rat model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by motor neuron death. Since most ALS patients succumb to ventilatory failure from loss of respiratory motor neurons, any effective ALS treatment must preserve and/or restore breathing capacity. In rats over-expressing mutated super-oxide dismutase-1 (SOD1(G93A)), the capacity to increase phrenic motor output is decreased at disease end-stage, suggesting imminent ventilatory failure. Acute intermittent hypoxia (AIH) induces phrenic long-term facilitation (pLTF), a form of spinal respiratory motor plasticity with potential to restore phrenic motor output in clinical disorders that compromise breathing. Since pLTF requires NADPH oxidase activity and reactive oxygen species (ROS) formation, it is blocked by NADPH oxidase inhibition and SOD mimetics in normal rats. Thus, we hypothesized that SOD1(G93A) (mutant; MT) rats do not express AIH-induced pLTF due to over-expression of active mutant superoxide dismutase-1. AIH-induced pLTF and hypoglossal (XII) LTF were assessed in young, pre-symptomatic and end-stage anesthetized MT rats and age-matched wild-type littermates. Contrary to predictions, pLTF and XII LTF were observed in MT rats at all ages; at end-stage, pLTF was actually enhanced. SOD1 levels were elevated in young and pre-symptomatic MT rats, yet superoxide accumulation in putative phrenic motor neurons (assessed with dihydroethidium) was unchanged; however, superoxide accumulation significantly decreased at end-stage. Thus, compensatory mechanisms appear to maintain ROS homoeostasis until late in disease progression, preserving AIH-induced respiratory plasticity. Following intrathecal injections of an NADPH oxidase inhibitor (apocynin; 600 μM; 12 μL), pLTF was abolished in pre-symptomatic, but not end-stage MT rats, demonstrating that pLTF is NADPH oxidase dependent in pre-symptomatic, but NADPH oxidase independent in end-stage MT rats. Mechanisms preserving/enhancing the capacity for pLTF in MT rats are not known.