Intermittent hypoxia causes a form of serotonin-dependent synaptic plasticity in the spinal cord known as phrenic long-term facilitation (pLTF). Here we show that increased synthesis of brain-derived neurotrophic factor (BDNF) in the spinal cord is necessary and sufficient for pLTF in adult rats. We found that intermittent hypoxia elicited serotonin-dependent increases in BDNF synthesis in ventral spinal segments containing the phrenic nucleus, and the magnitude of these BDNF increases correlated with pLTF magnitude. We used RNA interference (RNAi) to interfere with BDNF expression, and tyrosine kinase receptor inhibition to block BDNF signaling. These disruptions blocked pLTF, whereas intrathecal injection of BDNF elicited an effect similar to pLTF. Our findings demonstrate new roles and regulatory mechanisms for BDNF in the spinal cord and suggest new therapeutic strategies for treating breathing disorders such as respiratory insufficiency after spinal injury. These experiments also illustrate the potential use of RNAi to investigate functional consequences of gene expression in the mammalian nervous system in vivo.

Intermittent hypoxia elicits long-term facilitation (LTF), a persistent augmentation (hours) of respiratory motor output. Considerable recent progress has been made toward an understanding of the mechanisms and manifestations of this potentially important model of respiratory plasticity. LTF is elicited by intermittent but not sustained hypoxia, indicating profound pattern sensitivity in its underlying mechanism. During intermittent hypoxia, episodic spinal serotonin receptor activation initiates cell signaling events, increasing spinal protein synthesis. One associated protein is brain-derived neurotrophic factor, a neurotrophin implicated in several forms of synaptic plasticity. Our working hypothesis is that increased brain-derived neurotrophic factor enhances glutamatergic synaptic currents in phrenic motoneurons, increasing their responsiveness to bulbospinal inspiratory inputs. LTF is heterogeneous among respiratory outputs, differs among experimental preparations, and is influenced by age, gender, and genetics. Furthermore, LTF is enhanced following chronic intermittent hypoxia, indicating a degree of metaplasticity. Although the physiological relevance of LTF remains unclear, it may reflect a general mechanism whereby intermittent serotonin receptor activation elicits respiratory plasticity, adapting system performance to the ever-changing requirements of life.

Pompe disease is a severe form of muscular dystrophy due to glycogen accumulation in all tissues, especially striated muscle. Disease severity is directly related to the deficiency of acid ␣-glucosidase (GAA), which degrades glycogen in the lysosome. Respiratory dysfunction is a hallmark of the disease, muscle weakness has been viewed as the underlying cause, and the possibility of an associated neural contribution has not been evaluated previously. Therefore, we examined behavioral and neurophysiological aspects of breathing in 2 animal models of Pompe disease-the Gaa ؊/؊ mouse and a transgenic line (MTP) expressing GAA only in skeletal muscle, as well as a detailed analysis of the CNS in a Pompe disease patient. Glycogen content was elevated in the Gaa ؊/؊ mouse cervical spinal cord. Retrograde labeling of phrenic motoneurons showed significantly greater soma size in Gaa ؊/؊ mice vs. isogenic controls, and glycogen was observed in Gaa ؊/؊ phrenic motoneurons. Ventilation, assessed via plethysmography, was attenuated during quiet breathing and hypercapnic challenge in Gaa ؊/؊ mice (6 to >21 months of age) vs. controls. We confirmed that MTP mice had normal diaphragmatic contractile properties; however, MTP mice had ventilation similar to the Gaa ؊/؊ mice during quiet breathing. Neurophysiological recordings indicated that efferent phrenic nerve inspiratory burst amplitudes were substantially lower in Gaa ؊/؊ and MTP mice vs. controls. In human samples, we demonstrated similar pathology in the cervical spinal cord and greater accumulation of glycogen in spinal cord compared with brain. We conclude that neural output to the diaphragm is deficient in Gaa ؊/؊ mice, and therapies targeting muscle alone may be ineffective in Pompe disease.glycogenosis ͉ motor neuron ͉ muscular dystrophy ͉ myopathy

While monosynaptic bulbospinal projections to phrenic motoneurons have been extensively described, little is known about the organization of phrenic premotor neurons in the adult rat spinal cord. As interneurons may play an important role in normal breathing and recovery following spinal cord injury, the present study has used anterograde and transneuronal retrograde tracing to study their distribution and synaptic relations. Exclusive unilateral, first-order labeling of the phrenic motoneuron pool with pseudorabies virus demonstrated a substantial number of second-order, bilaterally-distributed cervical interneurons predominantly in the dorsal horn and around the central canal. Combined transneuronal and anterograde tracing revealed ventral respiratory column projections to pre-phrenic interneurons suggesting some propriospinal relays exist between medullary neurons and the phrenic nucleus. Dual-labeling studies with pseudorabies virus recombinants also showed pre-phrenic interneurons integrated with either contralateral phrenic or intercostal motoneuron pools. The stability of interneuronal pseudorabies virus labeling patterns following lateral cervical hemisection was then addressed. Except for fewer infected contralateral interneurons at the level of the central canal, the number and distribution of phrenicassociated interneurons was not significantly altered two weeks post-hemisection (i.e. when the earliest post-injury recovery of phrenic activity has been reported). These results demonstrate a heterogeneous population of phrenic-related interneurons. Their connectivity and relative stability after cervical hemisection raises speculation for potentially diverse roles in modulating phrenic function normally and post-injury.

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Episodic hypoxia evokes a sustained augmentation of respiratory motor output known as long-term facilitation (LTF). Phrenic LTF is prevented by pretreatment with the 5-hydroxytryptamine (5-HT) receptor antagonist ketanserin. We tested the hypothesis that 5-HT receptor activation is necessary for the induction but not maintenance of phrenic LTF. Peak integrated phrenic nerve activity (integralPhr) was monitored for 1 h after three 5-min episodes of isocapnic hypoxia (arterial PO(2) = 40 +/- 2 Torr; 5-min hyperoxic intervals) in four groups of anesthetized, vagotomized, paralyzed, and ventilated Sprague-Dawley rats [1) control (n = 11), 2) ketanserin pretreatment (2 mg/kg iv; n = 7), and ketanserin treatment 0 and 45 min after episodic hypoxia (n = 7 each)]. Ketanserin transiently decreased integralPhr, but it returned to baseline levels within 10 min. One hour after episodic hypoxia, integralPhr was significantly elevated from baseline in control and in the 0- and 45-min posthypoxia ketanserin groups. Conversely, ketanserin pretreatment abolished phrenic LTF. We conclude that 5-HT receptor activation is necessary to initiate (during hypoxia) but not maintain (following hypoxia) phrenic LTF.

Electromyographic recordings in animal models indicate that tongue protrudor (genioglossus, GG) and retractor muscles (styloglossus, SG; hyoglossus, HG) are co-activated during inspiration (Yasui et al. 1993, Fregosi & Fuller, 1997. Moreover, recent experiments indicate that respiratory-related co-activation of protrudor and retractor muscles results in retraction of the tongue (Fregosi & Fuller, 1997;Fuller et al. 1998). The observation that tongue muscle co-activation causes retraction of the tongue implies that respiratory-related tongue-motor activity narrows, rather than dilates, the oropharynx. However, two groups of investigators have shown that co-activation of the protrudor and retractor muscles evoked by XIIth nerve stimulation results in increased inspiratory flow rates in obstructive sleep apnoea (OSA) patients, in spite of clear tongue retraction (Eisele et al. 1997;De Backer et al. 1998). Protrusion of the tongue, evoked by either medial XIIth nerve branch stimulation (Eisele et al. 1997) or direct GG muscle stimulation (Schwartz et al. 1996) is also associated with increased inspiratory flow rates in human subjects. These observations suggest that either co-activation of tongue protrudor and retractor muscles, or independent protrudor muscle activation will improve pharyngeal flow mechanics. This is in spite of the fact that the tongue retracts 1. The purpose of these experiments was to examine the mechanisms by which either coactivation or independent activation of tongue protrudor and retractor muscles influence upper airway flow mechanics. We studied the influence of selective hypoglossal (XIIth) nerve stimulation on tongue movements and flow mechanics in anaesthetized rats that were prepared with an isolated upper airway. In this preparation, both nasal and oral flow pathways are available. 2. Inspiratory flow limitation was achieved by rapidly lowering hypopharyngeal pressure (Php) with a vacuum pump, and the maximal rate of flow (ýI,max) and the nasopharyngeal pressure associated with flow limitation (Pcrit) were measured. These experimental trials were repeated while nerve branches innervating tongue protrudor (genioglossus; medial XIIth nerve branch) and retractor (hyoglossus and styloglossus; lateral XIIth nerve branch) muscles were stimulated either simultaneously or independently at frequencies ranging from 20-100 Hz. Co-activating the protrudor and retractor muscles produced tongue retraction, whereas independently activating the genioglossus resulted in tongue protrusion. 3. Co-activation of tongue protrudor and retractor muscles increased ýI,max (peak increase 44%, P < 0·05), made Pcrit more negative (peak decrease of 44%, P < 0·05), and did not change upstream nasopharyngeal resistance (Rn). Independent protrudor muscle stimulation increased ýI,max (peak increase 61%, P < 0·05), did not change Pcrit, and decreased Rn (peak decrease of 41%, P < 0·05). Independent retractor muscle stimulation did not significantly alter flow mechanics. Changes in Pcrit and ýI,max at all stimulation fr...

Following C2 spinal hemisection (C2HS) in adult rats, ipsilateral phrenic motoneuron (PhMN) recovery occurs through a time-dependent activation of latent, crossed-spinal collaterals (i.e., spontaneous crossed phrenic phenomenon; sCPP) from contralateral bulbospinal axons. Ventilation is maintained during quiet breathing after C2HS, but the ability to increase ventilation during a respiratory stimulation (e.g. hypercapnia) is impaired. We hypothesized that long-term expression of the sCPP would correspond to a progressive normalization in ventilatory patterns during respiratory challenge. Breathing was assessed via plethsymography in unanesthetized animals and phrenic motor output was measured in urethane-anesthetized, paralyzed and vagotomized rats. At 2-week post-C2HS, minute ventilation (VE) was maintained during baseline (room air) conditions as expected but was substantially blunted during hypercapnic challenge (68±3% of VE in uninjured, weight-matched rats). However, by 12 weeks the spinal-lesioned rats achieved a hypercapnic VE response that was 85±7% of control (p = 0.017 vs. 2 wks). These rats also exhibited augmented breaths (AB's) or "sighs" more frequently (p<0.05) than controls; however, total AB volume was significantly less than control at 2-and 12-week post-injury (69±4% and 80±5%, p<0.05, respectively). We also noted that phrenic neurograms demonstrated a consistent delay in onset of the ipsilateral vs. contralateral inspiratory phrenic burst at 2-12-week post-injury. Finally, the ipsilateral phrenic response to respiratory challenge (hypoxia) was greater, though not normalized, at 4-12-vs. 2-week post-injury. We conclude that recovery of ventilation deficits occurs over 2-12-week post-C2HS; however, intrinsic neuroplasticity remains insufficient to concurrently restore a normal level of ipsilateral phrenic output.