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.

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% O(2)). 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% O(2)). 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.

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.

5-HT3 receptor-dependent modulation of respiratory burst frequency, regularity, and episodicity in isolated adult turtle brainstems.

To determine the role of central serotonin 5-HT(3) receptors in respiratory motor control, respiratory motor bursts were recorded from hypoglossal (XII) nerve rootlets on isolated adult turtle brainstems during bath-application of 5-HT(3) receptor agonists and antagonists. mCPBG and PBG (5-HT(3) receptor agonists) acutely increased XII burst frequency and regularity, and decreased bursts/episode. Tropisetron and MDL72222 (5-HT(3) antagonists) increased bursts/episode, suggesting endogenous 5-HT(3) receptor activation modulates burst timing in vitro. Tropisetron blocked all mCPBG effects, and the PBG-induced reduction in bursts/episode. Tropisetron application following mCPBG application did not reverse the long-lasting (2h) mCPBG-induced decrease in bursts/episode. We conclude that endogenous 5-HT(3) receptor activation regulates respiratory frequency, regularity, and episodicity in turtles and may induce a form of respiratory plasticity with the long-lasting changes in respiratory regularity.

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.

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 GABA(A) 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 microM). Rhythmic activity persisted during combined bicuculline and strychnine application (50 microM each) with increased amplitude and frequency, decreased burst duration, and a rapid onset-decrementing burst pattern. The bicuculline-strychnine rhythm frequency decreased during mu-opioid receptor activation or decreased bath P(C)(O(2)). 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.