Cervical spinal cord injury creates lasting respiratory deficits which can require mechanical ventilation long-term. We have shown that closed-loop epidural stimulation (CL-ES) elicits respiratory plasticity in the form of increased phrenic network excitability (Malone et. al., E Neuro, Vol 9, 0426-21.2021, 2022); however, the ability of this treatment to create functional benefits for breathing function per se after injury has not been demonstrated. Here, we demonstrate in C2 hemisected anesthetized rats, a 20-minute bout of CL-ES administered at current amplitudes below the motor threshold restores paralyzed hemidiaphragm activity in-phase with breathing while potentiating contralesional activity. While this acute bout of stimulation did not elicit the increased network excitability seen in our chronic model, a subset of stimulated animals continued spontaneous ipsilesional diaphragm activity for several seconds after stopping stimulation. These results support the use of CL-ES as a therapeutic to rescue breathing after high cervical spinal cord injury, with the potential to lead to lasting recovery and device independence.

Chronic hypoxia (CH) from birth attenuates the acute hypoxic ventilatory response (HVR) in rats and other mammals, but CH is often reported to augment the HVR in adult mammals. To test the hypothesis that this transition - from blunting to augmenting the HVR - occurs in the third or fourth postnatal week in rats, juvenile and adult rats were exposed to normobaric CH (12% O2) for 7 days and the HVR was assessed by whole-body plethysmography. No transition was observed, however, and the acute HVR was reduced by 61 - 85% across all ages studied. The failure to observe an augmented HVR in adult rats could not be explained by the substrain of Sprague Dawley rats used, the duration of the CH exposure, the order in which test gases were presented, the level of hypoxia used for CH and to assess the HVR, or the effects of CH on the metabolic response to hypoxia and the hypercapnic ventilatory response. A literature survey revealed several distinct patterns of ventilatory acclimatization to hypoxia (VAH) in adult rats, with most studies (77%) revealing a decrease or no change in the acute HVR after CH. In conclusion, the effects of CH on respiratory control are qualitatively similar across age groups, at least within the populations of Sprague Dawley rats used in the present study, and there does not appear to be one \"typical\" pattern for VAH in adult rats.

Intrapleural injections of cholera toxin B conjugated to saporin (CTB-SAP) selectively eliminates respiratory (e.g., phrenic) motor neurons, and mimics motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. Additionally, microglial density increases in the phrenic motor nucleus following CTB-SAP. This CTB-SAP rodent model allows us to study the impact of motor neuron death on the output of surviving phrenic motor neurons, and the underlying mechanisms that contribute to enhancing or constraining their output at 7 days (d) or 28d post-CTB-SAP injection. 7d CTB-SAP rats elicit enhanced phrenic long-term facilitation (pLTF) through the Gs-pathway (inflammation-resistant in naïve rats), while pLTF is elicited though the Gq-pathway (inflammation-sensitive in naïve rats) in control and 28d CTB-SAP rats. In 7d and 28d male CTB-SAP rats and controls, we evaluated the effect of cyclooxygenase-1/2 enzymes on pLTF by delivery of the nonsteroidal anti-inflammatory drug, ketoprofen (IP), and we hypothesized that pLTF would be unaffected by ketoprofen in 7d CTB-SAP rats, but pLTF would be enhanced in 28d CTB-SAP rats. In anesthetized, paralyzed and ventilated rats, pLTF was surprisingly attenuated in 7d CTB-SAP rats and enhanced in 28d CTB-SAP rats (both p < 0.05) following ketoprofen delivery. Additionally in CTB-SAP rats: 1) microglia were more amoeboid in the phrenic motor nucleus; and 2) cervical spinal inflammatory-associated factor expression (TNF-α, BDNF, and IL-10) was increased vs. controls in the absence of ketoprofen (p < 0.05). Following ketoprofen delivery, TNF-α and IL-10 expression was decreased back to control levels, while BDNF expression was differentially affected over the course of motor neuron death in CTB-SAP rats. This study furthers our understanding of factors (e.g., cyclooxygenase-1/2-induced inflammation) that contribute to enhancing or constraining pLTF and its implications for breathing following respiratory motor neuron death.

Plasticity is a hallmark of the respiratory neural control system. Phrenic long-term facilitation (pLTF) is one form of respiratory plasticity characterized by persistent increases in phrenic nerve activity following acute intermittent hypoxia (AIH). Although there is evidence that key steps in the cellular pathway giving rise to pLTF are localized within phrenic motor neurons (PMNs), the impact of AIH on the strength of breathing-related synaptic inputs to PMNs remains unclear. Furthermore, the functional impact of AIH is enhanced by repeated/daily exposure to AIH (dAIH). Here, we explored the effects of AIH versus 2 wk of dAIH preconditioning on spontaneous and evoked phrenic responses in anesthetized, paralyzed, and mechanically ventilated rats. Evoked phrenic potentials were elicited by respiratory cycle-triggered lateral funiculus stimulation at the C2 spinal level delivered before and 60 min post-AIH (or the equivalent in time controls). Charge-balanced biphasic pulses (100 μs/phase) of progressively increasing intensity (100-700 μA) were delivered during the inspiratory and expiratory phases of the respiratory cycle. Although robust pLTF (∼60% from baseline) was observed after a single exposure to moderate AIH (3 × 5 min; 5-min intervals), there was no effect on evoked phrenic responses, contrary to our initial hypothesis. However, in rats preconditioned with dAIH, baseline phrenic nerve activity and evoked responses were increased, suggesting that repeated exposure to AIH enhances functional synaptic strength when assessed using this technique. The impact of daily AIH preconditioning on synaptic inputs to PMNs raises interesting questions that require further exploration.NEW & NOTEWORTHY Two weeks of daily acute intermittent hypoxia (dAIH) preconditioning enhanced stimulus-evoked phrenic responses to lateral funiculus stimulation (targeting respiratory bulbospinal projection to phrenic motor neurons). Furthermore, dAIH preconditioning enhanced baseline phrenic motor output responses to maximal chemoreflex activation in intact rats.

Exploring whether the genioglossus discharge in chronic intermittent hypoxia(CIH) - pretreated rats could be enhanced by intermittent electrical stimulation combined with acute intermittent hypoxia(AIH).
Rats were pretreated with CIH for 4 weeks and then were randomly divided into 6 groups: time control, intermittent electric stimulation, AIH, intermittent electric stimulation + AIH, continuous electric stimulation and continuous hypoxia exposure. The genioglossus discharges were recorded and compared before and after stimulation. Normoxic-treated rats were grouped and treated with the same stimulation protocols.
Intermittent electrical stimulation or AIH temporarily increased the activity of the genioglossus discharge, in which the degree of the increase was significantly higher in CIH-pretreated rats than in normoxic rats.After intermittent electrical stimulation, AIH evoked a sustained elevation of genioglossus discharge activities in CIH-pretreated rats, in which the degree of the increase was significantly higher than in rats induced by a single intermittent electric stimulation.
Intermittent electrical stimulation combined with AIH strengthens the genioglossus plasticity in CIH-pretreated rats.

After spinal cord injury (SCI) respiratory complications are a leading cause of morbidity and mortality. Acute intermittent hypoxia (AIH) triggers spinal respiratory motor plasticity in rodent models, and repetitive AIH may have the potential to restore breathing capacity in those with SCI. As an initial approach to provide proof of principle for such effects, we tested single-session AIH effects on breathing function in adults with chronic SCI. 17 adults (13 males; 34.1 ± 14.5 years old; 13 motor complete SCI; >6 months post injury) completed two randomly ordered sessions, AIH versus sham. AIH consisted of 15, 1-min episodes (hypoxia: 10.3% O2; sham: 21% O2) interspersed with room air breathing (1.5 min, 21% oxygen); no attempt was made to regulate arterial CO2 levels. Blood oxygen saturation (SpO2), maximal inspiratory and expiratory pressures (MIP; MEP), forced vital capacity (FVC), and mouth occlusion pressure within 0.1 s (P0.1) were assessed. Outcomes were compared using nonparametric Wilcoxon\'s tests, or a 2 × 2 ANOVA. Baseline SpO2 was 97.2 ± 1.3% and was unchanged during sham experiments. During hypoxic episodes, SpO2 decreased to 84.7 ± 0.9%, and returned to baseline levels during normoxic intervals. Outcomes were unchanged from baseline post-sham. Greater increases in MIP were evident post AIH vs. sham (median values; +10.8 cmH2O vs. -2.6 cmH2O respectively, 95% confidence interval (-18.7) - (-4.3), p = .006) with a moderate Cohen\'s effect size (0.68). P0.1, MEP and FVC did not change post-AIH. A single AIH session increased maximal inspiratory pressure generation, but not other breathing functions in adults with SCI. Reasons may include greater spared innervation to inspiratory versus expiratory muscles or differences in the capacity for AIH-induced plasticity in inspiratory motor neuron pools. Based on our findings, the therapeutic potential of AIH on breathing capacity in people with SCI warrants further investigation.

Moderate acute intermittent hypoxia (mAIH) elicits a progressive increase in phrenic motor output lasting hours post-mAIH, a form of respiratory motor plasticity known as phrenic long-term facilitation (pLTF). mAIH-induced pLTF is initiated by activation of spinally-projecting raphe serotonergic neurons during hypoxia and subsequent serotonin release near phrenic motor neurons. Since raphe serotonergic neurons are also sensitive to pH and CO2, the prevailing arterial CO2 pressure (PaCO2) may modulate their activity (and serotonin release) during hypoxic episodes. Thus, we hypothesized that changes in background PaCO2 directly influence the magnitude of mAIH-induced pLTF. mAIH-induced pLTF was evaluated in anesthetized, vagotomized, paralyzed and ventilated rats, with end-tidal CO2 (i.e., a PaCO2 surrogate) maintained at: (1) ≤39 mmHg (hypocapnia); (2) ∼41 mmHg (normocapnia); or (3) ≥48 mmHg (hypercapnia) throughout experimental protocols. Although baseline phrenic nerve activity tended to be lower in hypocapnia, short-term hypoxic phrenic response, i.e., burst amplitude (Δ = 5.1 ± 1.1 μV) and frequency responses (Δ = 21 ± 4 bpm), was greater than in normocapnic (Δ = 3.6 ± 0.6 μV and 8 ± 4, respectively) or hypercapnic rats (Δ = 2.0 ± 0.6 μV and -2 ± 2, respectively), followed by a progressive increase in phrenic burst amplitude (i.e., pLTF) for at least 60 min post mAIH. pLTF in the hypocapnic group (Δ = 4.9 ± 0.6 μV) was significantly greater than in normocapnic (Δ = 2.8 ± 0.7 μV) or hypercapnic rats (Δ = 1.7 ± 0.4 μV). In contrast, although hypercapnic rats also exhibited significant pLTF, it was attenuated versus hypocapnic rats. When pLTF was expressed as percent change from maximal chemoreflex stimulation, all pairwise comparisons were found to be statistically significant (p < 0.05). We conclude that elevated PaCO2 undermines mAIH-induced pLTF in anesthetized rats. These findings contrast with well-documented effects of PaCO2 on ventilatory LTF in awake humans.

Selective elimination of respiratory motor neurons using intrapleural injections of cholera toxin B fragment conjugated to saporin (CTB-SAP) mimics motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. This CTB-SAP model allows us to study the impact of motor neuron death on the output of surviving phrenic motor neurons. After 7(d) days of CTB-SAP, phrenic long-term facilitation (pLTF, a form of respiratory plasticity) is enhanced, but returns towards control levels at 28d. However, the mechanism responsible for this difference in magnitude of pLTF is unknown. In naïve rats, pLTF predominately requires 5-HT2 receptors, the new synthesis of BDNF, and MEK/ERK signaling; however, pLTF can alternatively be induced via A2A receptors, the new synthesis of TrkB, and PI3K/Akt signaling. Since A2A receptor-dependent pLTF is enhanced in naïve rats, we suggest that 7d CTB-SAP treated rats utilize the alternative mechanism for pLTF. Here, we tested the hypothesis that pLTF following CTB-SAP is: 1) TrkB and PI3K/Akt, not BDNF and MEK/ERK, dependent at 7d; and 2) BDNF and MEK/ERK, not TrkB and PI3K/Akt, dependent at 28d. Adult Sprague Dawley male rats were anesthetized, paralyzed, ventilated, and were exposed to acute intermittent hypoxia (AIH; 3, 5 min bouts of 10.5% O2) following bilateral, intrapleural injections at 7d and 28d of: 1) CTB-SAP (25 μg), or 2) un-conjugated CTB and SAP (control). Intrathecal C4 delivery included either: 1) small interfering RNA that targeted BDNF or TrkB mRNA; 2) UO126 (MEK/ERK inhibitor); or 3) PI828 (PI3K/Akt inhibitor). Our data suggest that pLTF in 7d CTB-SAP treated rats is elicited primarily through TrkB and PI3K/Akt-dependent mechanisms, whereas BDNF and MEK/ERK-dependent mechanisms induce pLTF in 28d CTB-SAP treated rats. This project increases our understanding of respiratory plasticity and its implications for breathing following motor neuron death.

The respiratory control network in the central nervous system undergoes critical developmental events early in life to ensure adequate breathing at birth. There are at least three \"critical windows\" in development of respiratory control networks: 1) in utero, 2) newborn (postnatal day 0-4 in rodents), and 3) neonatal (P10-13 in rodents, 2-4 months in humans). During these critical windows, developmental processes required for normal maturation of the respiratory control network occur, thereby increasing vulnerability of the network to insults, such as inflammation. Early life inflammation (induced by LPS, chronic intermittent hypoxia, sustained hypoxia, or neonatal maternal separation) acutely impairs respiratory rhythm generation, chemoreception and increases neonatal risk of mortality. These early life impairments are also greater in young males, suggesting sex-specific impairments in respiratory control. Further, neonatal inflammation has a lasting impact on respiratory control by impairing adult respiratory plasticity. This review focuses on how inflammation alters respiratory rhythm generation, chemoreception and plasticity during each of the three critical windows. We also highlight the need for additional mechanistic studies and increased investigation into how glia (such as microglia and astrocytes) play a role in impaired respiratory control after inflammation. Understanding how inflammation during critical windows of development disrupt respiratory control networks is essential for developing better treatments for vulnerable neonates and preventing adult ventilatory control disorders.

In rats, high ethanol doses during early postnatal life exert deleterious effects upon brain development that impact diverse social and cognitive abilities. This stage in development partially overlaps with the third human gestational trimester, commonly referred to as the brain growth spurt period. At this stage in development, human fetuses and rat neonates (postnatal days [PD] 3-9) exhibit relatively high respiratory rates that are affected by subteratogenic ethanol doses. Recent studies suggest conditioned breathing responses in the developing organism, given that there are explicit associations between exteroceptive stimuli and the state of ethanol intoxication. Furthermore, studies performed with near-term rat fetuses suggest heightened sensitivity to ethanol\'s motivational effects. The present study was meant to analyze the unconditioned effects of ethanol intoxication and the possible co-occurrence of learning mechanisms that can impact respiratory plasticity, and to analyze the preference for cues that signal the state of intoxication as well as the effects of the drug, related with motor stimulation. Neonatal rats were subjected to differential experiences with salient tactile cues explicitly paired or not paired with the effects of vehicle or ethanol (2.0 g/kg). A tactile discrimination procedure applied during PDs 3, 5, 7, and 9 allowed the identification of the emergence of ethanol-derived non-associative and associative learning processes that affect breathing plasticity, particularly when considering apneic disruptions. Ethanol was found to partially inhibit the disruptions that appeared to be intimately related with stressful circumstances defined by the experimental procedure. Tactile cues paired with the drug\'s effects were also observed to exert an inhibitory effect upon these breathing disruptions. The level of contingency between a given tactile cue and ethanol intoxication also resulted in significant changes in the probability of seeking this cue in a tactile preference test. In addition, the state of intoxication exerted motor-stimulating effects. When contrasting the data obtained via the analysis of the different dependent variables, it appears that most ethanol-derived changes are modulated by positive and/or negative (anti-anxiety) reinforcing effects of the drug. As a whole, the study indicates co-existence of ethanol-related functional changes in the developing organism that simultaneously affect respiratory plasticity and preference patterns elicited by stimuli that signal ethanol\'s motivational effects. These results emphasize the need to consider significant alterations due to minimal ethanol experiences that argue against \"safe\" levels of exposure in a critical stage in brain development.