Magnitude and Mechanism of Phrenic Long-term Facilitation Shift Between Daily Rest Versus Active Phase.

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, and interact via powerful cross-talk inhibition. Here, we demonstrate that the daily rest/active phase and the duration of hypoxic episodes within an AIH protocol have profound impact on the magnitude and mechanism of pLTF due to shifts in 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 midactive versus midrest phase, yet the mechanism driving pLTF shifts from serotonin-dominant (midrest) to adenosine-dominant (midactive). This mechanistic "flip" results from combined influences of hypoxia-evoked adenosine release and daily fluctuations in basal spinal adenosine. Since AIH evokes less adenosine with shorter (15, 1-min) hypoxic episodes, midrest pLTF is amplified due to diminished adenosine constraint on serotonin-driven plasticity; in contrast, elevated background adenosine during the midactive phase suppresses serotonin-dominant pLTF. These findings demonstrate the importance of the serotonin/adenosine balance in regulating the amplitude and mechanism of AIH-induced pLTF. Since AIH is emerging as a promising therapeutic modality to restore respiratory and nonrespiratory 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.

Dose-dependent phosphorylation of endogenous Tau by intermittent hypoxia in rat brain.

Intermittent hypoxia, or intermittent low oxygen interspersed with normal oxygen levels, has differential effects that depend on the "dose" of hypoxic episodes (duration, severity, number per day, and number of days). Whereas "low dose" daily acute intermittent hypoxia (dAIH) elicits neuroprotection and neuroplasticity, "high dose" chronic intermittent hypoxia (CIH) similar to that experienced during sleep apnea elicits neuropathology. Sleep apnea is comorbid in >50% of patients with Alzheimer's disease-a progressive, neurodegenerative disease associated with brain amyloid and chronic Tau dysregulation (pathology). Although patients with sleep apnea present with higher Tau levels, it is unknown if sleep apnea through attendant CIH contributes to onset of Tau pathology. We hypothesized CIH characteristic of moderate sleep apnea would increase dysregulation of phosphorylated Tau (phospho-Tau) species in Sprague-Dawley rat hippocampus and prefrontal cortex. Conversely, we hypothesized that dAIH, a promising neurotherapeutic, has minimal impact on Tau phosphorylation. We report a dose-dependent intermittent hypoxia effect, with region-specific increases in 1) phospho-Tau species associated with human Tauopathies in the soluble form and 2) accumulated phospho-Tau in the insoluble fraction. The latter observation was particularly evident with higher CIH intensities. This important and novel finding is consistent with the idea that sleep apnea and attendant CIH have the potential to accelerate the progression of Alzheimer's disease and/or other Tauopathies.NEW & NOTEWORTHY Sleep apnea is highly prevalent in people with Alzheimer's disease, suggesting the potential to accelerate disease onset and/or progression. These studies demonstrate that intermittent hypoxia (IH) induces dose-dependent, region-specific Tau phosphorylation, and are the first to indicate that higher IH "doses" elicit both endogenous, (rat) Tau hyperphosphorylation and accumulation in the hippocampus. These findings are essential for development and implementation of new treatment strategies that minimize sleep apnea and its adverse impact on neurodegenerative diseases.

Closed-Loop, Cervical, Epidural Stimulation Elicits Respiratory Neuroplasticity after Spinal Cord Injury in Freely Behaving Rats.

Over half of all spinal cord injuries (SCIs) are cervical, which can lead to paralysis and respiratory compromise, causing significant morbidity and mortality. Effective treatments to restore breathing after severe upper cervical injury are lacking; thus, it is imperative to develop therapies to address this. Epidural stimulation has successfully restored motor function after SCI for stepping, standing, reaching, grasping, and postural control. We hypothesized that closed-loop stimulation triggered via healthy hemidiaphragm EMG activity has the potential to elicit functional neuroplasticity in spinal respiratory pathways after cervical SCI (cSCI). To test this, we delivered closed-loop, electrical, epidural stimulation (CLES) at the level of the phrenic motor nucleus (C4) for 3 d after C2 hemisection (C2HS) in freely behaving rats. A 2 × 2 Latin Square experimental design incorporated two treatments, C2HS injury and CLES therapy resulting in four groups of adult, female Sprague Dawley rats: C2HS + CLES (n = 8), C2HS (n = 6), intact + CLES (n = 6), intact (n = 6). In stimulated groups, CLES was delivered for 12-20 h/d for 3 d. After C2HS, 3 d of CLES robustly facilitated the slope of stimulus-response curves of ipsilesional spinal motor evoked potentials (sMEPs) versus nonstimulated controls. To our knowledge, this is the first demonstration of CLES eliciting respiratory neuroplasticity after C2HS in freely behaving animals. These findings suggest CLES as a promising future therapy to address respiratory deficiency associated with cSCI.

Circadian clock genes and respiratory neuroplasticity genes oscillate in the phrenic motor system.

Circadian rhythms are endogenous and entrainable daily patterns of physiology and behavior. Molecular mechanisms underlie circadian rhythms, characterized by an ~24-h pattern of gene expression of core clock genes. Although it has long been known that breathing exhibits circadian rhythms, little is known concerning clock gene expression in any element of the neuromuscular system controlling breathing. Furthermore, we know little concerning gene expression necessary for specific respiratory functions, such as phrenic motor plasticity. Thus, we tested the hypotheses that transcripts for clock genes (Bmal1, Clock, Per1, and Per2) and molecules necessary for phrenic motor plasticity (Htr2a, Htr2b, Bdnf, and Ntrk2) oscillate in regions critical for phrenic/diaphragm motor function via RT-PCR. Tissues were collected from male Sprague-Dawley rats entrained to a 12-h light-dark cycle at 4 zeitgeber times (ZT; n = 8 rats/group): ZT5, ZT11, ZT17, and ZT23; ZT0 = lights on. Here, we demonstrate that 1) circadian clock genes (Bmal1, Clock, Per1, and Per2) oscillate in regions critical for phrenic/diaphragm function, including the caudal medulla, ventral C3-C5 cervical spinal cord, and diaphragm; 2) the clock protein BMAL1 is localized within CtB-labeled phrenic motor neurons; 3) genes necessary for intermittent hypoxia-induced phrenic/diaphragm motor plasticity (Htr2b and Bdnf) oscillate in the caudal medulla and ventral C3-C5 spinal cord; and 4) there is higher intensity of immunofluorescent BDNF protein within phrenic motor neurons at ZT23 compared with ZT11 (n = 11 rats/group). These results suggest local circadian clocks exist in the phrenic motor system and confirm the potential for local circadian regulation of neuroplasticity and other elements of the neural network controlling breathing.

Attention to a threat-related feature does not interfere with concurrent attentive feature selection.

Visual features associated with a task and those that predict noxious events both prompt selectively heightened visuocortical responses. Conflicting views exist regarding how the competition between a task-related and a threat-related feature is resolved when they co-occur in time and space. Utilizing aversive classical Pavlovian conditioning, we investigated the visuocortical representation of two simultaneously presented, fully overlapping visual stimuli. Isoluminant red and green random dot kinematogram (RDK) stimuli were flickered at distinct tagging frequencies (8.57 Hz, 12 Hz) to elicit distinguishable steady-state visual evoked potentials (ssVEPs). Occasional coherent motion events prompted a motor response (task) or predicted a noxious noise (threat). These events occurred either in the green (task cue), the red (threat cue), or in both RDKs simultaneously. In the initial habituation phase, participants responded to coherent motion of the green RDK with a key press, but no loud noise was presented at any time. Here, selective amplification was seen for the task-relevant (green) RDK, and interference was observed when both RDKs simultaneously showed coherent motion. Upon pairing the threat cue with the noxious noise in the subsequent acquisition phase, the threat cue-evoked ssVEP (red RDK) was also amplified, but this amplification did not interact with amplification of the task cue or alter the behavioral or visuocortical interference effect observed during simultaneous coherent motion. Although competing feature conjunctions resulted in interference in the visual cortex, the acquisition of a bias toward an individual threat-related feature did not result in additional cost effects.

Compensatory plasticity in diaphragm and intercostal muscle utilization in a rat model of ALS.

In SOD1G93A transgenic rat model of ALS, breathing capacity is preserved until late in disease progression despite profound respiratory motor neuron (MN) cell death. To explore mechanisms preserving breathing capacity, we assessed inspiratory EMG activity in diaphragm and external intercostal T2 (EIC2) and T5 (EIC5) muscles in anesthetized SOD1G93A rats at disease end-stage (20% decrease in body mass). We hypothesized that despite significant phrenic motor neuron loss and decreased phrenic nerve activity, diaphragm electrical activity and trans-diaphragmatic pressure (Pdi) are maintained to sustain ventilation. We alternatively hypothesized that EIC activity is enhanced, compensating for impaired diaphragm function. Diaphragm, EIC2 and EIC5 muscle EMGs and Pdi were measured in urethane-anesthetized, spontaneously breathing female SOD1G93A rats versus wild-type littermates during normoxia (arterial PO2 ~90mmHg, PCO2 ~45mmHg), maximal chemoreceptor stimulation (MCS: 10.5% O2/7% CO2), spontaneous augmented breaths and sustained tracheal occlusion. Phrenic MNs were counted in C3-5; T2 and T5 ventrolateral MNs were counted. In end-stage SOD1G93A rats, 29% of phrenic MNs survived (vs. wild-type), yet integrated diaphragm EMG amplitude was normal. Nevertheless, maximal Pdi decreased ~30% vs. wild type (p<0.01) and increased esophageal to gastric pressure ratio (p<0.05), consistent with persistent diaphragm weakness. Despite major T2 and T5 MN death, integrated EIC2 (100% greater than wild type) and EIC5 (300%) EMG amplitudes were increased in mutant rats during normoxia (p<0.01), possibly compensating for decreased Pdi. Thus, despite significant phrenic MN loss, diaphragm EMG activity is maintained; in contrast, Pdi was not, suggesting diaphragm dysfunction. Presumably, increased EIC EMG activity compensated for persistent diaphragm weakness. These adjustments contribute to remarkable preservation of breathing ability despite major respiratory motor neuron death and diaphragm dysfunction.