Enhancement of phrenic long-term facilitation following repetitive acute intermittent hypoxia is blocked by the glycolytic inhibitor 2-deoxyglucose.

Moderate acute intermittent hypoxia (mAIH) elicits a form of respiratory motor plasticity known as phrenic long-term facilitation (pLTF). Preconditioning with modest protocols of chronic intermittent hypoxia enhances pLTF, demonstrating pLTF metaplasticity. Since "low-dose" protocols of repetitive acute intermittent hypoxia (rAIH) show promise as a therapeutic modality to restore respiratory (and nonrespiratory) motor function in clinical disorders with compromised breathing, we tested 1) whether preconditioning with a mild rAIH protocol enhances pLTF and hypoglossal (XII) LTF and 2) whether the enhancement is regulated by glycolytic flux. In anesthetized, paralyzed, and ventilated adult male Lewis rats, mAIH (three 5-min episodes of 10% O2) elicited pLTF (pLTF at 60 min post-mAIH: 49 ± 5% baseline). rAIH preconditioning (ten 5-min episodes of 11% O2/day with 5-min normoxic intervals, 3 times per week, for 4 wk) significantly enhanced pLTF (100 ± 16% baseline). XII LTF was unaffected by rAIH. When glycolytic flux was inhibited by 2-deoxy-d-glucose (2-DG) administered via drinking water (~80 mg·kg-1·day-1), pLTF returned to normal levels (58 ± 8% baseline); 2-DG had no effect on pLTF in normoxia-pretreated rats (59 ± 7% baseline). In ventral cervical (C4/5) spinal homogenates, rAIH increased inducible nitric oxide synthase mRNA vs. normoxic controls, an effect blocked by 2-DG. However, there were no detectable effects of rAIH or 2-DG on several molecules associated with phrenic motor plasticity, including serotonin 2A, serotonin 7, brain-derived neurotrophic factor, tropomyosin receptor kinase B, or VEGF mRNA. We conclude that modest, but prolonged, rAIH elicits pLTF metaplasticity and that a drug known to inhibit glycolytic flux (2-DG) blocks pLTF enhancement.

Unexpected benefits of intermittent hypoxia: enhanced respiratory and nonrespiratory motor function.

Intermittent hypoxia (IH) is most often thought of for its role in morbidity associated with sleep-disordered breathing, including central nervous system pathology. However, recent evidence suggests that the nervous system fights back in an attempt to minimize pathology by increasing the expression of growth/trophic factors that confer neuroprotection and neuroplasticity. For example, even modest ("low dose") IH elicits respiratory motor plasticity, increasing the strength of respiratory contractions and breathing. These low IH doses upregulate hypoxia-sensitive growth/trophic factors within respiratory motoneurons but do not elicit detectable pathologies such as hippocampal cell death, neuroinflammation, or systemic hypertension. Recent advances have been made toward understanding cellular mechanisms giving rise to IH-induced respiratory plasticity, and attempts have been made to harness the benefits of low-dose IH to treat respiratory insufficiency after cervical spinal injury. Our recent realization that IH also upregulates growth/trophic factors in nonrespiratory motoneurons and improves limb (or leg) function after incomplete chronic spinal injuries suggests that IH-induced plasticity is a general feature of motor systems. Collectively, available evidence suggests that low-dose IH may represent a safe and effective treatment to restore lost motor function in diverse clinical disorders that impair motor function.

Spinal 5-HT7 receptors induce phrenic motor facilitation via EPAC-mTORC1 signaling.

Spinal serotonin type 7 (5-HT7) receptors elicit complex effects on motor activity. Whereas 5-HT7 receptor activation gives rise to long-lasting phrenic motor facilitation (pMF), it also constrains 5-HT2 receptor-induced pMF via "cross-talk inhibition." We hypothesized that divergent cAMP-dependent signaling pathways give rise to these distinct 5-HT7 receptor actions. Specifically, we hypothesized that protein kinase A (PKA) mediates cross-talk inhibition of 5-HT2 receptor-induced pMF whereas 5-HT7 receptor-induced pMF results from exchange protein activated by cAMP (EPAC) signaling. Anesthetized, paralyzed, and ventilated rats receiving intrathecal (C4) 5-HT7 receptor agonist (AS-19) injections expressed pMF for >90 min, an effect abolished by pretreatment with a selective EPAC inhibitor (ESI-05) but not a selective PKA inhibitor (KT-5720). Furthermore, intrathecal injections of a selective EPAC activator (8-pCPT-2'-Me-cAMP) were sufficient to elicit pMF. Finally, spinal mammalian target of rapamycin complex-1 (mTORC1) inhibition via intrathecal rapamycin abolished 5-HT7 receptor- and EPAC-induced pMF, demonstrating that spinal 5-HT7 receptors elicit pMF by an EPAC-mTORC1 signaling pathway. Thus 5-HT7 receptors elicit and constrain spinal phrenic motor plasticity via distinct signaling mechanisms that diverge at cAMP (EPAC vs. PKA). Selective manipulation of these molecules may enable refined regulation of serotonin-dependent spinal motor plasticity for therapeutic advantage.

Spinal 5-HT7 receptor activation induces long-lasting phrenic motor facilitation.

Acute intermittent hypoxia elicits a form of serotonin-dependent respiratory plasticity known as phrenic long term facilitation (pLTF). Episodic spinal serotonin-2 (5-HT2) receptor activation on or near phrenic motor neurons is necessary for pLTF. A hallmark of pLTF is the requirement for serotonin-dependent synthesis of brain-derived neurotrophic factor (BDNF), and activation of its high affinity receptor, TrkB. Activation of spinal Gs protein-coupled adenosine 2A receptors (GsPCRs) elicits a unique form of long-lasting phrenic motor facilitation (PMF), but via unique mechanisms (BDNF independent TrkB trans-activation).We hypothesized that other GsPCRs elicit PMF, specifically serotonin-7 (5-HT7) receptors, which are expressed in phrenic motor neurons. Cervical spinal (C4) injections of a selective 5-HT7 receptor agonist, AS-19 (10 µM, 5 µl; 3 × 5 min), in anaesthetized, vagotomized and ventilated male Sprague-Dawley rats elicited long-lasting PMF (>120 min), an effect prevented by pretreatment with a 5-HT7 receptor antagonist (SB 269970; 5mM, 7 µl).GsPCR activation 'trans-activates'TrkB by increasing synthesis of an immature TrkB isoform. Spinal injection of a TrkB inhibitor (k252a) and siRNAs that prevent TrkB (but not BDNF) mRNA translation both blocked 5-HT7 agonist-induced PMF, confirming a requirement for TrkB synthesis and activity. k252a affected late PMF (≥ 90 min) only. Spinal inhibition of the PI3K/AKT pathway blocked 5-HT7 agonist-induced PMF, whereas MEK/ERK inhibition delayed, but did not block, PMF. An understanding of signalling mechanisms giving rise to PMF may guide development of novel therapeutic strategies to treat ventilatory control disorders associated with respiratory insufficiency, such as spinal injury and motor neuron disease.

Spinal adenosine A2(A) receptor inhibition enhances phrenic long term facilitation following acute intermittent hypoxia.

Phrenic long term facilitation (pLTF) is a form of respiratory plasticity induced by acute intermittent hypoxia. pLTF requires spinal serotonin receptor activation, new BDNF synthesis and TrkB receptor activation. Spinal adenosine 2A (A(2A)) receptor activation also elicits phrenic motor facilitation, but by a distinct mechanism involving new TrkB synthesis. Because extracellular adenosine increases during hypoxia, we hypothesized that A(2A) receptor activation contributes to acute intermittent hypoxia (AIH)-induced pLTF. A selective A(2A) receptor antagonist (MSX-3, 8 microg kg(-1), 12 microl) was administered intrathecally (C4) to anaesthetized, vagotomized and ventilated male Sprague-Dawley rats before AIH (three 5 min episodes, 11% O(2)). Contrary to our hypothesis, pLTF was greater in MSX-3 versus vehicle (aCSF) treated rats (97 +/- 6% vs. 49 +/- 4% at 60 min post-AIH, respectively; P < 0.05). MSX-3 and aCSF treated rats did not exhibit facilitation without AIH (time controls; 7 +/- 5% and 9 +/- 9%, respectively; P > 0.05). A second A(2A) receptor antagonist (ZM2412385, 7 microg kg(11), 7 microl) enhanced pLTF (85 +/- 11%, P < 0.05), but an adenosine A(1) receptor antagonist (DPCPX, 3 microg kg(-1), 10 microl) had no effect (51% +/- 8%, P > 0.05), indicating specific A(2A) receptor effects. Intrathecal methysergide (306 microg kg(-1), 15 microl) blocked AIH-induced pLTF in both MSX-3 and aCSF treated rats, confirming that enhanced pLTF is serotonin dependent. Intravenous MSX-3 (140 microg kg(-1), 1 ml) enhanced both phrenic (104 +/- 7% vs. 57 +/- 5%, P < 0.05) and hypoglossal LTF (46 +/- 13% vs. 28 +/- 10%; P < 0.05). In conclusion, A(2A) receptors constrain the expression of serotonin-dependent phrenic and hypoglossal LTF following AIH. A(2A) receptor antagonists (such as caffeine) may exert beneficial therapeutic effects by enhancing the capacity for AIH-induced respiratory plasticity.

Episodic spinal serotonin receptor activation elicits long-lasting phrenic motor facilitation by an NADPH oxidase-dependent mechanism.

Phrenic long-term facilitation (pLTF) is a serotonin (5-HT)-dependent augmentation of phrenic motor output induced by acute intermittent hypoxia (AIH). AIH-induced pLTF requires spinal NADPH oxidase activity and reactive oxygen species (ROS) formation. Since 5-HT receptor activation stimulates NADPH oxidase activity in some cell types, we tested the hypothesis that episodic spinal 5-HT receptor activation (without AIH) is sufficient to elicit an NADPH oxidase-dependent facilitation of phrenic motor output (pMF). In anaesthetised, artificially ventilated adult male rats, episodic intrathecal 5-HT injections (3 x 6 microl injections at 5 min intervals) into the cerebrospinal fluid (CSF) near cervical spinal segments containing the phrenic motor nucleus elicited a progressive increase in integrated phrenic nerve burst amplitude (i.e. pMF) lasting at least 60 min post-5-HT administration. Hypoglossal (XII) nerve activity was unaffected, suggesting that effective doses of 5-HT did not reach the brainstem. A single 5-HT injection was without effect. 5-HT-induced pMF was dose dependent, but exhibited a bell-shaped dose-response curve. Activation of different 5-HT receptor subtypes, specifically 5-HT(2) versus 5-HT(7) receptors, may underlie the bell-shaped dose-response curve via a mechanism of 'cross-talk' inhibition. Pre-treatment with NADPH oxidase inhibitors, apocynin or diphenylenodium (DPI), blocked 5-HT induced pMF. Thus, episodic spinal 5-HT receptor activation is sufficient to elicit pMF by an NADPH oxidase-dependent mechanism, suggesting common mechanisms of ROS formation with AIH-induced pLTF. An understanding of the mechanisms giving rise to AIH-induced pLTF and 5-HT induced pMF may inspire novel therapeutic strategies for respiratory insufficiency in diverse conditions, such as sleep apnoea, cervical spinal injury or amyotrophic lateral sclerosis.

NADPH oxidase activity is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation.

Phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) is a form of spinal, serotonin-dependent synaptic plasticity that requires reactive oxygen species (ROS) formation. We tested the hypothesis that spinal NADPH oxidase activity is a necessary source of ROS for pLTF. Sixty minutes post-AIH (three 5-min episodes of 11% O(2), 5 min intervals), integrated phrenic and hypoglossal (XII) nerve burst amplitudes were increased from baseline, indicative of phrenic and XII LTF. Intrathecal injections (approximately C(4)) of apocynin or diphenyleneiodonium chloride (DPI), two structurally and functionally distinct inhibitors of the NADPH oxidase complex, attenuated phrenic, but not XII, LTF. Immunoblots from soluble (cytosolic) and particulate (membrane) fractions of ventral C(4) spinal segments revealed predominantly membrane localization of the NADPH oxidase catalytic subunit, gp91(phox), whereas membrane and cytosolic expression were both observed for the regulatory subunits, p47(phox) and RAC1. Immunohistochemical analysis of fixed tissues revealed these same subunits in presumptive phrenic motoneurons of the C(4) ventral horn, but not in neighbouring astrocytes or microglia. Collectively, these data demonstrate that NADPH oxidase subunits localized within presumptive phrenic motoneurons are a major source of ROS necessary for AIH-induced pLTF. Thus, NADPH oxidase activity is a key regulator of spinal synaptic plasticity, and may be a useful pharmaceutical target in developing therapeutic strategies for respiratory insufficiency in patients with, for example, cervical spinal injury.

Optical imaging of Cerenkov light generation from positron-emitting radiotracers.

Radiotracers labeled with high-energy positron emitters, such as those commonly used for positron emission tomography studies, emit visible light immediately following decay in a medium. This phenomenon, not previously described for these imaging tracers, is consistent with Cerenkov radiation and has several potential applications, especially for in vivo molecular imaging studies. Herein we detail a new molecular imaging tool, Cerenkov Luminescence Imaging, the experiments conducted that support our interpretation of the source of the signal, and proof-of-concept in vivo studies that set the foundation for future application of this new method.

Respiratory long-term facilitation following intermittent hypoxia requires reactive oxygen species formation.

Acute intermittent hypoxia (AIH) elicits a form of respiratory plasticity known as long-term facilitation (LTF). LTF is a progressive and sustained increase in respiratory motor output as expressed in phrenic and hypoglossal (XII) nerve activity. Since reactive oxygen species (ROS) play important roles in several forms of neuroplasticity, and ROS production is increased by intermittent hypoxia, we tested the hypothesis that ROS are necessary for phrenic and XII LTF following AIH. Urethane-anesthetized, paralyzed, vagotomized and pump-ventilated Sprague-Dawley rats were exposed to AIH (11% O2, 3, 5 min episodes, 5 min intervals), and both phrenic and XII nerve activity were monitored for 60 min post-AIH. Although phrenic and XII LTF were observed in control rats, i.v. manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP), a superoxide anion scavenger, attenuated both phrenic and XII LTF in a dose dependent manner. Localized application of MnTMPyP (5.5 mM; 10 microl) to the intrathecal space of the cervical spinal cord (C4) abolished phrenic, but not XII LTF. Thus, ROS are necessary for AIH-induced respiratory LTF, and the relevant ROS appear to be localized near respiratory motor nuclei since cervical MnTMPyP injections impaired phrenic (and not XII) LTF. Phrenic LTF is a novel form of ROS-dependent neuroplasticity since its ROS-dependence resides in the spinal cord.

Sustained hypoxia-induced proliferation of carotid body type I cells in rats.

Sustained hypoxia (SH) has been shown to cause profound morphological and cellular changes in carotid body (CB). However, results regarding whether SH causes CB type I cell proliferation are conflicting. By using bromodeoxyuridine, a uridine analog that is stably incorporated into cells undergoing DNA synthesis, we have found that SH causes the type I cell proliferation in the CB; the proliferation occurs mainly during the first 1-3 days of hypoxic exposure. Moreover, the new cells survive for at least 1 mo after the return to normoxia. Also, SH does not cause any cell death in CB as examined by the terminal deoxynucleotidyl transferase-mediated dUTP-X nick-end labeling assay. Taken together, our results suggest that SH stimulates CB type I cell proliferation, which may produce long-lasting changes in CB morphology and function.

Spinal serotonin receptor activation modulates the exercise ventilatory response with increased dead space in goats.

Small increases in respiratory dead space (VD) augment the exercise ventilatory response by a serotonin-dependent mechanism known as short-term modulation (STM). We tested the hypotheses that the relevant serotonin receptors for STM are in the spinal cord, and are of the 5-HT2-receptor subtype. After preparing adult female goats with a mid-thoracic (T6-T8) subarachnoid catheter, ventilation and arterial blood gases were measured at rest and during treadmill exercise (4.8 km/h; 5% grade) with and without an increased VD (0.2-0.3 L). Measurements were made before and after spinal or intravenous administration of a broad-spectrum serotonin receptor antagonist (methysergide, 1-2mg total) and a selective 5-HT2-receptor antagonist (ketanserin, 5-12 mg total). Although spinal methysergide had no effect on the exercise ventilatory response in control conditions, the augmented response with increased VD was impaired, allowing Pa(CO)(2) to increase from rest to exercise. Spinal methysergide diminished both mean inspiratory flow and frequency responses to exercise with increased VD. Spinal ketanserin impaired Pa(CO)(2) regulation with increased VD, although its ventilatory effects were less clear. Intrathecal dye injections indicated CSF drug distribution was caudal to the upper cervical spinal cord and intravenous drugs at the same total dose did not affect STM. We conclude that spinal 5-HT2 receptors modulate the exercise ventilatory response with increased VD in goats.

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.

Daily acute intermittent hypoxia improves breathing function with acute and chronic spinal injury via distinct mechanisms.

Daily acute intermittent hypoxia (dAIH) elicits respiratory plasticity, enhancing respiratory motor output and restoring breathing capacity after incomplete cervical spinal injuries (cSCI). We hypothesized that dAIH-induced functional recovery of breathing capacity would occur after both acute (2 weeks) and chronic (8 weeks) cSCI, but through distinct cellular mechanisms. Specifically, we hypothesized that dAIH-induced breathing recovery would occur through serotonin-independent mechanisms 2wks post C2 cervical hemisection (C2Hs), versus serotonin-dependent mechanisms 8wks post C2Hs. In two independent studies, dAIH or sham (normoxia) was initiated 1 week (Study 1) or 7 weeks (Study 2) post-C2Hs to test our hypothesis. Rats were pre-treated with intra-peritoneal vehicle or methysergide, a broad-spectrum serotonin receptor antagonist, to determine the role of serotonin signaling in dAIH-induced functional recovery. Our data support the hypothesis that dAIH-induced recovery of breathing capacity transitions from a serotonin-independent mechanism with acute C2Hs to a serotonin-dependent mechanism with chronic C2Hs. An understanding of shifting mechanisms giving rise to dAIH-induced respiratory motor plasticity is vital for clinical translation of dAIH as a therapeutic modality.

Divergent cAMP signaling differentially regulates serotonin-induced spinal motor plasticity.

Spinal metabotropic serotonin receptors encode transient experiences into long-lasting changes in motor behavior (i.e. motor plasticity). While interactions between serotonin receptor subtypes are known to regulate plasticity, the significance of molecular divergence in downstream G protein coupled receptor signaling is not well understood. Here we tested the hypothesis that distinct cAMP dependent signaling pathways differentially regulate serotonin-induced phrenic motor facilitation (pMF); a well-studied model of spinal motor plasticity. Specifically, we studied the capacity of cAMP-dependent protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) to regulate 5-HT2A receptor-induced pMF within adult male rats. Although spinal PKA, EPAC and 5-HT2A each elicit pMF when activated alone, concurrent PKA and 5-HT2A activation interact via mutual inhibition thereby blocking pMF expression. Conversely, concurrent EPAC and 5-HT2A activation enhance pMF expression reflecting additive contributions from both mechanisms. Thus, we demonstrate that distinct downstream cAMP signaling pathways enable differential regulation of 5-HT2A-induced pMF. Conditional activation of independent signaling mechanisms may explain experience amendable changes in plasticity expression (i.e. metaplasticity), an emerging concept thought to enable flexible motor control within the adult central nervous system.

Enhanced recovery of breathing capacity from combined adenosine 2A receptor inhibition and daily acute intermittent hypoxia after chronic cervical spinal injury.

Daily acute intermittent hypoxia (dAIH) improves breathing capacity after C2 spinal hemisection (C2HS) in rats. Since C2HS disrupts spinal serotonergic innervation below the injury, adenosine-dependent mechanisms underlie dAIH-induced functional recovery 2weeks post-injury. We hypothesized that dAIH-induced functional recovery converts from an adenosine-dependent to a serotonin-dependent, adenosine-constrained mechanism with chronic injury. Eight weeks post-C2HS, rats began dAIH (10, 5-min episodes, 10.5% O2; 5-min intervals; 7days) followed by AIH 3× per week (3×wAIH) for 8 additional weeks with/without systemic A2A receptor inhibition (KW6002) on each AIH exposure day. Tidal volume (VT) and bilateral diaphragm (Dia) and T2 external intercostal motor activity were assessed in unanesthetized rats breathing air and during maximum chemoreflex stimulation (MCS: 7% CO2, 10.5% O2). Nine weeks post-C2HS, dAIH increased VT versus time controls (p<0.05), an effect enhanced by KW6002 (p<0.05). dAIH increased bilateral Dia activity (p<0.05), and KW6002 enhanced this effect in contralateral (p<0.05) and ipsilateral Dia activity (p<0.001), but not T2 inspiratory activity. Functional benefits of combined AIH plus systemic A2A receptor inhibition were maintained for 4weeks. Thus, in rats with chronic injuries: 1) dAIH improves VT and bilateral diaphragm activity; 2) VT recovery is enhanced by A2A receptor inhibition; and 3) functional recovery with A2A receptor inhibition and AIH "reminders" last 4weeks. Combined dAIH and A2A receptor inhibition may be a simple, safe, and effective strategy to accelerate/enhance functional recovery of breathing capacity in patients with respiratory impairment from chronic spinal injury.

Serotonin-induced in vitro long-term facilitation exhibits differential pattern sensitivity in cervical and thoracic inspiratory motor output.

Intermittent hypoxia induces 5-HT-dependent, pattern-sensitive long-term facilitation (LTF) of spinal respiratory motor output. We used a split-bath in vitro neonatal rat brainstem-spinal cord preparation to test whether: 1) intermittent spinal 5-HT exposure (without hypoxia) is sufficient to induce LTF in phrenic and intercostal inspiratory motor outputs; 2) LTF magnitude is greater in intercostal versus phrenic activity; and 3) phrenic and intercostal motor output exhibits differential pattern sensitivity to 5-HT application. With a barrier at spinal segment C1, 5-HT (5 muM) was applied episodically (3 min 5-HT, 5 min wash, x3) to the spinal cord (C2-L1) while recording inspiratory bursts in cervical (C4 or C5) and thoracic (T5 or T6) ventral roots. Episodic 5-HT application increased cervical and thoracic burst amplitudes to 136+/-22% and 150+/-22% of baseline, respectively, at 120 min post-drug (P<0.01). Continuous 5-HT application (5 muM, 9 min) had no effect on cervical burst amplitude at 120 min post-drug, but increased thoracic burst amplitude to 142+/-11% of baseline at 120 min post-drug (P<0.001). Methysergide pretreatment abolished both cervical and thoracic 5-HT-induced LTF. Quantitative reverse transcriptase-polymerase chain reaction and immunocytochemistry revealed that 5-HT(2A) and 5-HT(7) receptor subtypes (receptors known to influence LTF expression in adult rats) are expressed in ventral cervical and thoracic spinal cord with no differences in expression levels due to spinal segment or age. Thus, 5-HT is sufficient to induce spinal LTF in neonatal rats and differences in pattern sensitivity suggest heterogeneity in underlying mechanisms.

Repetitive acute intermittent hypoxia increases growth/neurotrophic factor expression in non-respiratory motor neurons.

Repetitive acute intermittent hypoxia (rAIH) increases growth/trophic factor expression in respiratory motor neurons, thereby eliciting spinal respiratory motor plasticity and/or neuroprotection. Here we demonstrate that rAIH effects are not unique to respiratory motor neurons, but are also expressed in non-respiratory, spinal alpha motor neurons and upper motor neurons of the motor cortex. In specific, we used immunohistochemistry and immunofluorescence to assess growth/trophic factor protein expression in spinal sections from rats exposed to AIH three times per week for 10weeks (3×wAIH). 3×wAIH increased brain-derived neurotrophic factor (BDNF), its high-affinity receptor, tropomyosin receptor kinase B (TrkB), and phosphorylated TrkB (pTrkB) immunoreactivity in putative alpha motor neurons of spinal cervical 7 (C7) and lumbar 3 (L3) segments, as well as in upper motor neurons of the primary motor cortex (M1). 3×wAIH also increased immunoreactivity of vascular endothelial growth factor A (VEGFA), the high-affinity VEGFA receptor (VEGFR-2) and an important VEGF gene regulator, hypoxia-inducible factor-1α (HIF-1α). Thus, rAIH effects on growth/trophic factors are characteristic of non-respiratory as well as respiratory motor neurons. rAIH may be a useful tool in the treatment of disorders causing paralysis, such as spinal injury and motor neuron disease, as a pretreatment to enhance motor neuron survival during disease, or as preconditioning for cell-transplant therapies.

Activity-dependent plasticity of descending synaptic inputs to spinal motoneurons in an in vitro turtle brainstem-spinal cord preparation.

An in vitro brainstem-spinal cord preparation from adult turtles was used to test the hypothesis that descending synaptic inputs to multifunctional spinal motoneurons (i.e., involved in respiration and locomotion) express activity-dependent depression or potentiation. The tissue was placed in a chamber that allowed for separate superfusion of the brainstem, spinal segments C(2)-C(4), and C(5)-D(1). Action potential conduction between the brainstem and spinal segments C(5)-D(1) was blocked by superfusing C(2)-C(4) with Na(+)-free solution. With C(5)-D(1) at [K(+)] = 10 mM, electrical stimulation at C(5) every 2 min evoked potentials in intact pectoralis (expiratory, inward rotation of shoulder) and serratus (inspiratory, outward rotation of shoulder) nerves that were stable for at least 2 hr. Application of conditioning stimulation (900 pulses at 1 or 10 Hz) at C(5) decreased pectoralis evoked potential amplitudes by approximately 40% initially and by 20% after 90 min; serratus evoked potentials were unaltered. Conditioning stimulation (100 Hz, 900 pulses) transiently depressed pectoralis evoked potential amplitude by <20% but produced a delayed 72% increase in serratus evoked potential amplitude after approximately 80 min. Conditioning stimulation (10 Hz) at C(5) also reduced the amplitude of sensory afferent evoked potentials in pectoralis produced by stimulating ipsilateral dorsal roots at C(8). Thus, long-lasting changes in descending synaptic inputs to multifunctional spinal motoneurons were frequency-dependent and heterosynaptic. We hypothesize that activity-dependent plasticity may modulate descending synaptic drive to spinal motoneurons involved in both respiration and locomotion.

Cervical dorsal rhizotomy increases brain-derived neurotrophic factor and neurotrophin-3 expression in the ventral spinal cord.

Although neurotrophic factors have been implicated in several forms of neuroplasticity, little is known concerning their potential role in spinal plasticity. Cervical dorsal rhizotomy (CDR) enhances serotonin terminal density near (spinal) phrenic motoneurons and serotonin-dependent long-term facilitation of phrenic motor output (Kinkead et al., 1998). We tested the hypothesis that selected neurotrophic factors change in a manner consistent with an involvement in this model of spinal plasticity. Brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), glial cell line-derived neurotrophic factor (GDNF), and transforming growth factor-beta(1) (TGF-beta(1)) concentrations were measured (ELISA) in three regions of interest to respiratory control: (1) ventral cervical spinal segments associated with the phrenic motor nucleus (C3-C6), (2) ventral thoracic spinal segments associated with inspiratory intercostal motor output (T3-T6) and (3) the diaphragm. Tissues were harvested from rats 7 d after bilateral CDR and compared with sham-operated and unoperated control rats. CDR increased BDNF (110%; p = 0.002) and NT-3 (100%; p = 0.002) in the cervical and NT-3 in the thoracic spinal cord (98%; p = 0.009). GDNF and TGF-beta(1) were not altered by CDR in any tissue. Immunohistochemistry localized BDNF and NT-3 to motoneurons and interneurons of the ventral spinal cord. These studies provide novel, suggestive evidence that BDNF and NT-3, possibly through their trophic effects on serotonergic neurons and/or motoneurons, may underlie serotonin-dependent plasticity in (spinal) respiratory motor control after CDR.

Chronic intermittent hypoxia elicits serotonin-dependent plasticity in the central neural control of breathing.

We tested the hypothesis that chronic intermittent hypoxia (CIH) elicits plasticity in the central neural control of breathing via serotonin-dependent effects on the integration of carotid chemoafferent inputs. Adult rats were exposed to 1 week of nocturnal CIH (11-12% O(2)/air at 5 min intervals; 12 hr/night). CIH and untreated rats were then anesthetized, paralyzed, vagotomized, and artificially ventilated. Time-dependent hypoxic responses were assessed in the phrenic neurogram during and after three 5 min episodes of isocapnic hypoxia. Integrated phrenic amplitude (integralPhr) responses during hypoxia were greater after CIH at arterial oxygen pressures (PaO(2)) between 25 and 45 mmHg (p < 0.05), but not at higher PaO(2) levels. CIH did not affect hypoxic phrenic burst frequency responses, although the post-hypoxia frequency decline that is typical in rats was abolished. integralPhr and frequency responses to electrical stimulation of the carotid sinus nerve were enhanced by CIH (p < 0.05). Serotonin-dependent long-term facilitation (LTF) of integralPhr was enhanced after CIH at 15, 30, and 60 min after episodic hypoxia (p < 0.05). Pretreatment with the serotonin receptor antagonists methysergide (4 mg/kg, i.v.) and ketanserin (2 mg/kg, i.v.) reversed CIH-induced augmentation of the short-term hypoxic phrenic response and restored the post-hypoxia frequency decline in CIH rats. Whereas methysergide abolished CIH-enhanced phrenic LTF, the selective 5-HT(2) antagonist ketanserin only partially reversed this effect. The results suggest that CIH elicits unique forms of serotonin-dependent plasticity in the central neural control of breathing. Enhanced LTF after CIH may involve an upregulation of a non-5-HT(2) serotonin receptor subtype or subtypes.