David D. Fuller

BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia

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.

Long term facilitation of phrenic motor output

Episodic hypoxia or electrical stimulation of carotid chemoafferent neurons elicits a sustained, serotonin-dependent augmentation of respiratory motor output known as long term facilitation (LTF). The primary objectives of this paper are to provide an updated review of the literature pertaining to LTF, to investigate the influence of selected variables on LTF via meta-analysis of a large data set from LTF experiments on anesthetized rats, and to propose an updated mechanism of LTF. LTF has been demonstrated in anesthetized and awake experimental preparations, and can be evoked in some human subjects during sleep. The mechanism underlying LTF requires episodic chemoafferent stimulation, and is not elicited by similar cumulative durations of sustained hypoxia. Meta-analysis of phrenic nerve responses following episodic hypoxia in 63 experiments on anesthetized rats (conducted by four investigators over a period of several years) indicates that phrenic LTF magnitude correlates with peak phrenic responses during hypoxia and hypercapnia, but not with the level of hypoxia during episodic exposures. Potential mechanisms underlying these relationships are discussed, and currently available data are synthesized into an updated mechanistic model of LTF. In this model, we propose that LTF arises predominantly from episodic activation of serotonergic receptors on phrenic motoneurons, activating intracellular kinases and, thus, phosphorylating and potentiating ionic currents associated with the glutamate receptors that mediate respiratory drive.

Effect of co-activation of tongue protrudor and retractor muscles on tongue movements and pharyngeal airflow mechanics in the rat

1 The purpose of these experiments was to examine the mechanisms by which either co‐activation 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 (VI,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 VI,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 VI,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 VI,max at all stimulation frequencies were significantly correlated during co‐activation of protrudor and retractor muscles (r2= 0.63, P < 0.05), but not during independent protrudor muscle stimulation (r2= 0.09). 4 These findings indicate that either co‐activation of protrudor and retractor muscles or independent activation of protrudor muscles can improve upper airway flow mechanics, although the underlying mechanisms are different. We suggest that co‐activation decreases pharyngeal collapsibility but does not dilate the pharyngeal airway. In contrast, unopposed tongue protrusion dilates the oropharynx, but has a minimal effect on pharyngeal airway collapsibility.

Co-activation of tongue protrudor and retractor muscles during chemoreceptor stimulation in the rat

1 Our primary purpose was to test the hypothesis that the tongue protrudor (genioglossus, GG) and retractor (styloglossus, SG and hyoglossus, HG) muscles are co‐activated when respiratory drive increases, and that co‐activation will cause retraction of the tongue. This was addressed by performing two series of experiments using a supine, anaesthetized, tracheotomized rat in which tongue muscle force and the neural drive to the protrudor and retractor muscles could be measured during spontaneous breathing. In the first series of experiments, respiratory drive was increased progressively by occluding the tracheal cannula for thirty respiratory cycles; in the second series of experiments, the animals were subjected to hyperoxic hypercapnia and poikilocapnic hypoxia. 2 Airway occlusion for thirty breaths caused progressive, quantitatively similar increases in efferent motor nerve activity to protrudor and retractor tongue muscles. Net tongue muscle force was always consistent with tongue retraction during occlusion, and peak force rose in parallel with the neural activites. When airway occlusion was repeated following section of the lateral XIIth nerve branch (denervation of retractor muscles) the tongue either protruded (15/21 animals; 10 ± 2 mN at the 30th occluded breath) or retracted weakly (6/21 animals; 6 ± 2 mN at 30th occluded breath). 3 To ensure that our findings were not the result of damage to the muscle nerves, occlusion experiments were also done in eight animals in which GG EMG activity was recorded instead of nerve activities. Changes in peak integrated GG electryomyogram (EMG) activity and peak retraction force during occlusion were highly correlated (r2= 0.86, slope = 1.05). 4 In separate experiments in fourteen rats, we found that hyperoxic hypercapnia and poikilocapnic hypoxia also result in parallel increases in the respiratory‐related EMG activity of the GG and HG muscles. Also, as in the occlusion experiments, augmentations of protrudor and retractor muscle EMG activities were associated with parallel changes in tongue retraction force. 5 These studies in anaesthetized rats demonstrate that tracheal occlusion and independent stimulation of central or peripheral chemoreceptors results in inspiratory‐related co‐activation of the protrudor and retractor muscles, and proportional changes in tongue retraction force. These observations also demonstrate that recording GG EMG activity in isolation could lead to erroneous conclusions about respiratory‐related movements of the tongue.

Respiratory-related control of extrinsic tongue muscle activity

The purpose of this brief report is to introduce new evidence showing that the protrudor and retractor muscles of the tongue are co-activated during inspiration in eupnea and hyperpnea in an anesthetized, tracheotomized rat model. We also review previous work on the respiratory related control of the tongue musculature, and briefly consider the clinical significance of this work. The important new findings are that: (1) Both hypoxia and hypercapnia cause parallel increases in drive to the tongue protrudor and retractor muscles (the genioglossus and hyoglossus muscles, respectively); (2) phasic volume feedback inhibits the peak inspiratory activity of both muscles; and (3) the tongue muscles consistently produce a retraction force when the genioglossus and hyoglossus are co-activated, in both animal and human subjects. This latter observation is consistent with previous work showing that the retractor muscles (hyoglossus and styloglossus) develop up to ten times more force than the genioglossus muscle. The possible mechanical consequences of tongue muscle co-activation are briefly considered.

Neurogenesis of cough, other airway defensive behaviors and breathing: A holarchical system?

Cough and breathing are generated by a common muscular system. However, these two behaviors differ significantly in their mechanical features and regulation. The current conceptualization of the neurogenic mechanism for these behaviors holds that the multifunctional respiratory pattern generator undergoes reconfiguration to produce cough. Our previous results indicate the presence of a functional cough gate mechanism that controls the excitability of this airway defensive behavior, but is not involved in the regulation of breathing. We propose that the neurogenesis of cough, breathing, and other nonbreathing behaviors is controlled by a larger network, of which the respiratory pattern generator is part. This network we term a holarchical system. This system is governed by functional control elements known as holons, which confer unique regulatory features to each behavior. The cough gate is an example of such a holon. Neurons that participate in a cough holon may include behavior selective elements. That is, neurons that are either specifically recruited during cough and/or tonically-active neurons with little or no modulation during breathing but with significant alterations in discharge during coughing. We also propose that the holarchical system is responsible for the orderly expression of different airway defensive behaviors such that each motor task is executed in a temporally and mechanically discrete manner. We further propose that a holon controlling one airway defensive behavior can regulate the excitability of, and cooperate with, holons unique to other behaviors. As such, co-expression of multiple rhythmic behaviors such as cough and swallow can occur without compromising airway defense.

Cervical prephrenic interneurons in the normal and lesioned spinal cord of the adult rat

Although 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. Because 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 prephrenic interneurons, suggesting that some propriospinal relays exist between medullary neurons and the phrenic nucleus. Dual‐labeling studies with pseudorabies virus recombinants also showed prephrenic 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 phrenic‐associated interneurons was not significantly altered 2 weeks posthemisection (i.e., the point at which the earliest postinjury 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 raise speculation for potentially diverse roles in modulating phrenic function normally and postinjury. J. Comp. Neurol. 511:692–709, 2008.

Neural deficits contribute to respiratory insufficiency in Pompe disease

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 α-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.

Spinal circuitry and respiratory recovery following spinal cord injury

Numerous studies have demonstrated anatomical and functional neuroplasticity following spinal cord injury. One of the more notable examples is return of ipsilateral phrenic motoneuron and diaphragm activity which can be induced under terminal neurophysiological conditions after high cervical hemisection in the rat. More recently it has been shown that a protracted, spontaneous recovery also occurs in this model. While a candidate neural substrate has been identified for the former, the neuroanatomical basis underlying spontaneous recovery has not been explored. Demonstrations of spinal respiratory interneurons in other species suggest such cells may play a role; however, the presence of interneurons in the adult rat phrenic circuit - the primary animal model of respiratory plasticity - has not been extensively investigated. Emerging neuroanatomical and electrophysiological results raise the possibility of a more complex neural network underlying spontaneous recovery of phrenic function and compensatory respiratory neuroplasticity after C2 hemisection than has been previously considered.

Respiratory plasticity: differential actions of continuous and episodic hypoxia and hypercapnia.

The objectives of this paper are: (1) to review advances in our understanding of the mechanisms of respiratory plasticity elicited by episodic versus continuous hypoxia in short to intermediate time domains (min to h); and (2) to present new data suggesting that different patterns of hypercapnia also elicit distinct forms of respiratory plasticity. Episodic, but not continuous hypoxia elicits long-term facilitation (LTF) of respiratory motor output. Phrenic LTF is a serotonin-dependent central neural mechanism that requires: (a) activation of spinal serotonin receptors; and (b) spinal protein synthesis. Continuous and episodic hypercapnia also elicit different mechanisms of plasticity. Continuous, severe hypercapnia (25 min of approximately 10% inspired CO(2)) elicits long-term depression (LTD) of phrenic motor output (-33+/-8% at 60 min post-hypercapnia) in anesthetized rats. In contrast, 3,5 min hypercapnic episodes do not elicit LTD (9+/-17% at 60 min). We hypothesize that the response of respiratory motoneurons to serotonergic and noradrenergic modulation may contribute to pattern sensitivity to hypoxia and hypercapnia.