Julia E. R. Wilkerson

Repetitive Intermittent Hypoxia Induces Respiratory and Somatic Motor Recovery after Chronic Cervical Spinal Injury

Spinal injury disrupts connections between the brain and spinal cord, causing life-long paralysis. Most spinal injuries are incomplete, leaving spared neural pathways to motor neurons that initiate and coordinate movement. One therapeutic strategy to induce functional motor recovery is to harness plasticity in these spared neural pathways. Chronic intermittent hypoxia (CIH) (72 episodes per night, 7 nights) increases synaptic strength in crossed spinal synaptic pathways to phrenic motoneurons below a C2 spinal hemisection. However, CIH also causes morbidity (e.g., high blood pressure, hippocampal apoptosis), rendering it unsuitable as a therapeutic approach to chronic spinal injury. Less severe protocols of repetitive acute intermittent hypoxia may elicit plasticity without associated morbidity. Here we demonstrate that daily acute intermittent hypoxia (dAIH; 10 episodes per day, 7 d) induces motor plasticity in respiratory and nonrespiratory motor behaviors without evidence for associated morbidity. dAIH induces plasticity in spared, spinal pathways to respiratory and nonrespiratory motor neurons, improving respiratory and nonrespiratory (forelimb) motor function in rats with chronic cervical injuries. Functional improvements were persistent and were mirrored by neurochemical changes in proteins that contribute to respiratory motor plasticity after intermittent hypoxia (BDNF and TrkB) within both respiratory and nonrespiratory motor nuclei. Collectively, these studies demonstrate that repetitive acute intermittent hypoxia may be an effective and non-invasive means of improving function in multiple motor systems after chronic spinal injury.

Daily intermittent hypoxia augments spinal BDNF levels, ERK phosphorylation and respiratory long-term facilitation

Acute intermittent hypoxia (AIH) elicits a form of respiratory plasticity known as long-term facilitation (LTF). We hypothesized that: 1) daily AIH (dAIH) preconditioning enhances phrenic and hypoglossal (XII) LTF in a rat strain with low constitutive LTF expression; 2) dAIH induces brain-derived neurotrophic factor (BDNF), a critical protein for phrenic LTF (pLTF) in the cervical spinal cord; and 3) dAIH increases post-AIH extracellular regulated kinase (ERK) activation. Phrenic and XII motor output were monitored in anesthetized dAIH- or sham-treated Brown Norway rats with and without acute AIH. pLTF was observed in both sham (18+/-9% baseline; 60 min post-hypoxia; p<0.05; n=18) and dAIH treated rats (37+/-8%; p<0.05; n=14), but these values were not significantly different (p=0.13). XII LTF was not observed in sham-treated rats (4+/-5%), but was revealed in dAIH pretreated rats (48+/-18%; p<0.05). dAIH preconditioning increased basal ventral cervical BDNF protein levels (24+/-8%; p<0.05), but had no significant effect on ERK phosphorylation. AIH increased BDNF in sham (25+/-8%; p<0.05), but not dAIH-pretreated rats (-7+/-4%), and had complex effects on ERK phosphorylation (ERK2 increased in shams whereas ERK1 increased in dAIH-treated rats). Thus, dAIH elicits metaplasticity in LTF, revealing XII LTF in a rat strain with no constitutive XII LTF expression. Increased BDNF synthesis may no longer be necessary for phrenic LTF following dAIH preconditioning since BDNF concentration is already elevated.

Reactive oxygen species and respiratory plasticity following intermittent hypoxia.

The neural network controlling breathing exhibits plasticity in response to environmental or physiological challenges. For example, while hypoxia initiates rapid and robust increases in respiratory motor output to defend against hypoxemia, it also triggers persistent changes, or plasticity, in chemosensory neurons and integrative pathways that transmit brainstem respiratory activity to respiratory motor neurons. Frequently studied models of hypoxia-induced respiratory plasticity include: (1) carotid chemosensory plasticity and metaplasticity induced by chronic intermittent hypoxia (CIH), and (2) acute intermittent hypoxia (AIH) induced phrenic long-term facilitation (pLTF) in naïve and CIH preconditioned rats. These forms of plasticity share some mechanistic elements, although they differ in anatomical location and the requirement for CIH preconditioning. Both forms of plasticity require serotonin receptor activation and formation of reactive oxygen species (ROS). While the cellular sources and targets of ROS are not well known, recent evidence suggests that ROS modify the balance of protein phosphatase and kinase activities, shifting the balance towards net phosphorylation and favoring cellular reactions that induce and/or maintain plasticity. Here, we review possible sources of ROS, and the impact of ROS on phosphorylation events relevant to respiratory plasticity.

Okadaic Acid-Sensitive Protein Phosphatases Constrain Phrenic Long-Term Facilitation after Sustained Hypoxia

Phrenic long-term facilitation (pLTF) is a serotonin-dependent form of pattern-sensitive respiratory plasticity induced by intermittent hypoxia (IH), but not sustained hypoxia (SH). The mechanism(s) underlying pLTF pattern sensitivity are unknown. SH and IH may differentially regulate serine/threonine protein phosphatase activity, thereby inhibiting relevant protein phosphatases uniquely during IH and conferring pattern sensitivity to pLTF. We hypothesized that spinal protein phosphatase inhibition would relieve this braking action of protein phosphatases, thereby revealing pLTF after SH. Anesthetized rats received intrathecal (C4) okadaic acid (25 nm) before SH (25 min, 11% O2). Unlike (vehicle) control rats, SH induced a significant pLTF in okadaic acid-treated rats that was indistinguishable from rats exposed to IH (three 5 min episodes, 11% O2). IH and SH with okadaic acid may elicit pLTF by similar, serotonin-dependent mechanisms, because intravenous methysergide blocks pLTF in rats receiving IH or okadaic acid plus SH. Okadaic acid did not alter IH-induced pLTF. In summary, pattern sensitivity in pLTF may reflect differential regulation of okadaic acid-sensitive serine/threonine phosphatases; presumably, these phosphatases are less active during/after IH versus SH. The specific okadaic acid-sensitive phosphatase(s) constraining pLTF and their spatiotemporal dynamics during and/or after IH and SH remain to be determined.

Differential expression of respiratory long-term facilitation among inbred rat strains

We tested the hypotheses that: (1) long-term facilitation (LTF) following acute intermittent hypoxia (AIH) varies among three inbred rat strains: Fischer 344 (F344), Brown Norway (BN) and Lewis rats and (2) ventral cervical spinal levels of genes important for phrenic LTF (pLTF) vary in association with pLTF magnitude. Lewis and F344, but not BN rats exhibited significant increases in phrenic and hypoglossal burst amplitude 60min post-AIH that were significantly greater than control experiments without AIH, indicating strain differences in phrenic (98%, 56% and 20%, respectively) and hypoglossal LTF (66%, 77% and 5%, respectively). Ventral spinal 5-HT(2A) receptor mRNA and protein levels were higher in F344 and Lewis versus BN, suggesting that higher 5-HT(2A) receptor levels are associated with greater pLTF. More complex relationships were found for 5-HT(7), BDNF and TrkB mRNA. BN had higher 5-HT(7) and TrkB mRNA versus F344; BN and Lewis had higher BDNF mRNA levels versus F344. Genetic variations in serotonergic function may underlie strain differences in AIH-induced pLTF.

Role of synaptic inhibition in turtle respiratory rhythm generation

In vitro brainstem and brainstem‐spinal cord preparations were used to determine the role of synaptic inhibition in respiratory rhythm generation in adult turtles. Bath application of bicuculline (a GABAA receptor antagonist) to brainstems increased hypoglossal burst frequency and amplitude, with peak discharge shifted towards the burst onset. Strychnine (a glycine receptor antagonist) increased amplitude and frequency, and decreased burst duration, but only at relatively high concentrations (10‐100 μM). Rhythmic activity persisted during combined bicuculline and strychnine application (50 μM each) with increased amplitude and frequency, decreased burst duration, and a rapid onset‐decrementing burst pattern. The bicuculline‐strychnine rhythm frequency decreased during μ‐opioid receptor activation or decreased bath PCO2. Synaptic inhibition blockade in the brainstem of brainstem‐spinal cord preparations increased burst amplitude in spinal expiratory (pectoralis) nerves and nearly abolished spinal inspiratory activity (serratus nerves), suggesting that medullary expiratory motoneurons were mainly active. Under conditions of synaptic inhibition blockade in vitro, the turtle respiratory network was able to produce a rhythm that was sensitive to characteristic respiratory stimuli, perhaps via an expiratory (rather than inspiratory) pacemaker‐driven mechanism. Thus, these data indicate that the adult turtle respiratory rhythm generator has the potential to operate in a pacemaker‐driven manner.

Respiratory plasticity following intermittent hypoxia: roles of protein phosphatases and reactive oxygen species

Plasticity is an important property of the respiratory control system. One of the best-studied models of respiratory plasticity is pLTF (phrenic long-term facilitation). pLTF is a progressive increase in phrenic motor output lasting several hours following acute exposure to intermittent hypoxia. Similar to many other forms of neuroplasticity, pLTF is pattern-sensitive; it is induced by intermittent, but not sustained hypoxia of similar cumulative duration. Our understanding of the cellular/synaptic mechanisms underlying pLTF has increased considerably in recent years. Here, we review accumulating evidence suggesting that the pattern-sensitivity of pLTF arises substantially from differential reactive oxygen species formation and subsequent protein phosphatase inhibition during intermittent compared with sustained hypoxia in or near phrenic motor neurons. A detailed understanding of the cellular/synaptic mechanisms of pLTF may provide the rationale for new pharmacological approaches in the treatment of severe ventilatory control disorders, such as obstructive sleep apnoea and respiratory insufficiency either following spinal cord injury or during neurodegenerative diseases such as amyotrophic lateral sclerosis.

Time-dependent changes in spontaneous respiratory activity in turtle brainstems in vitro.

Our goal was to determine whether time-dependent changes in respiratory motor output in vitro could be minimized by altering bath solution composition. Adult turtle brainstems were bathed in standard solution, nutrient-rich Dulbeccos Eagle media (100 or 25% concentration), or standard solution with phenylbiguanide (PBG, 5-HT3 agonist which increases respiratory drive). Except for a 63% frequency increase in PBG solution, hypoglossal bursts were unaltered within 100 min of observation. Respiratory activity was abolished within 7 h in 100% Dulbeccos compared with a mean of 24-31 h in other test solutions. At 12 h, burst frequency decreased faster in standard solution and 25% Dulbeccos (-0.28+/-0.07 and -0.13+/-0.05 bursts/h, respectively) compared with PBG solution (-0.09+/-0.04 bursts/h); amplitude declined at approximately 2%/h in all solutions. The tendency for episodic discharge decreased gradually in standard solution, but was eliminated in 25% Dulbeccos and PBG solution. Certain bath solutions may minimize time-dependent frequency reductions but may also cause breathing pattern changes.

Intermittent but not sustained moderate hypoxia elicits long-term facilitation of hypoglossal motor output

Phrenic long-term facilitation (pLTF) is a form of serotonin-dependent respiratory motor plasticity induced by moderate acute intermittent hypoxia (AIH), but not by moderate acute sustained hypoxia (ASH) of similar cumulative duration. Thus, moderate AIH-induced pLTF is sensitive to the pattern of hypoxia. On the other hand, pLTF induced by severe AIH protocols is neither pattern sensitive nor serotonin dependent (it converts to an adenosine-dependent mechanism). Although moderate AIH also induces hypoglossal LTF (hLTF), no data are available concerning its sensitivity/insensitivity to the pattern of hypoxia. Since hLTF following moderate hypoxia is serotonin-dependent, we hypothesized that hLTF is pattern-sensitive, similar to serotonin-dependent pLTF. Integrated hypoglossal nerve activity was recorded in urethane-anesthetized, vagotomized, paralyzed, and ventilated rats exposed to isocapnic AIH (3, 5min episodes of 11% O2) or ASH (a single 25min episode of 11% O2). Similar to previous studies of pLTF, hypoglossal motor output was elevated for more than 1h following AIH (50±20%, p<0.01), but not ASH (-6±9%, p>0.05). Frequency LTF was not observed following either hypoxic exposure. Thus, in agreement with our hypothesis, hypoglossal LTF following moderate AIH is pattern-sensitive, similar to phrenic LTF.