Ralph F Fregosi

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• Boyd, B., Hoyer-Kimura, C., Wollman, L., & Fregosi, R. F. (2022). A homemade device for simultaneous measurement of pulmonary ventilation and metabolic rate in neonatal rodents. Respiratory physiology & neurobiology, 103858.
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  Various in vitro neonatal rodent models have been developed to study the control of breathing, but translation of the information requires a behavioral assay, which has led to the widespread use of plethysmography to measure breathing in awake neonatal rodents. Best practice requires correcting changes in ventilation to the corresponding change in metabolic rate, which is the main driver of pulmonary ventilation. Obtaining measures of both simultaneously is ideal, though technically difficult. Here we describe a simple, inexpensive home-made dual chamber approach for simultaneous measurement of pulmonary ventilation and metabolic rate. We found that the dual chamber provides values for pulmonary ventilation and metabolic rate that compare favorably with existing approaches.
• Buls Wollman, L., & Fregosi, R. F. (2022). Chronic, Episodic Nicotine Alters Hypoglossal Motor Neuron Function at a Critical Developmental Time Point in Neonatal Rats. eNeuro, 8(4).
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  Developmental nicotine exposure (DNE), alters brainstem neurons that control breathing, including hypoglossal motor neurons (XIIMNs), which innervate the tongue. Here, we tested the hypothesis that chronic, episodic DNE (eDNE), which mimics nicotine replacement therapies such as e-cigarettes or nicotine gum, alters the function of nicotinic acetylcholine receptors (nAChRs), XIIMN intrinsic properties, and tongue muscle function similar to what we have observed with a chronic, sustained exposure model. We delivered nicotine to pregnant Sprague Dawley rats through drinking water and studied pups of either sex in two age groups: postnatal day (P)1-P5 and P10-P12, which encompasses a critical period in brain development. At P1-P5, eDNE was associated with delayed recovery of nAChRs from desensitization; however, there were no changes in the magnitude of desensitization, XIIMN intrinsic properties, or tongue muscle function By P10-P12, eDNE XIIMNs had lower peak firing frequencies in response to depolarizing current injection, larger delayed rectifier potassium currents, and continued to exhibit delayed nAChR recovery. Moreover, this age group exhibited a blunted and delayed tongue muscle response to nasal occlusion , indicating that changes to XIIMN intrinsic properties is an important mechanism behind this effect, as it is not produced by altered nAChR function alone. Together, these results show that eDNE alters XIIMNs and tongue muscle function during a critical period in brain development and that the specific effects of chronic nicotine exposure may be pattern dependent.
• Wollman, L., Hill, A., Hasse, B., Young, C., Hernandez-De La Pena, G., Levine, R. B., & Fregosi, R. F. (2022). Influence of developmental nicotine exposure on serotonergic control of breathing-related motor output. Developmental neurobiology.
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  Serotonin plays an important role in the development of brainstem circuits that control breathing. Here, we test the hypothesis that developmental nicotine exposure (DNE) alters the breathing-related motor response to serotonin (5HT). Pregnant rats were exposed to nicotine or saline, and brainstem-spinal cord preparations from 1- to 5-day-old pups were studied in a split-bath configuration, allowing drugs to be applied selectively to the medulla or spinal cord. The activity of the fourth cervical ventral nerve roots (C4VR), which contain axons of phrenic motoneurons, was recorded. We applied 5HT alone or together with antagonists of 5HT1A, 5HT2A, or 5HT7 receptor subtypes. In control preparations, 5HT applied to the medulla consistently reduced C4VR frequency and this reduction could not be blocked by any of the three antagonists. In DNE preparations, medullary 5HT caused a large and sustained frequency increase (10 min), followed by a sustained decrease. Notably, the transient increase in frequency could be blocked by the independent addition of any of the antagonists. Experiments with subtype-specific agonists suggest that the 5HT7 subtype may contribute to the increased frequency response in the DNE preparations. Changes in C4VR burst amplitude in response to brainstem 5HT were uninfluenced by DNE. Addition of 5HT to the caudal chamber modestly increased phasic and greatly increased tonic C4VR activity, but there were no effects of DNE. The data show that DNE alters serotonergic signaling within brainstem circuits that control respiratory frequency but does not functionally alter serotonin signaling in the phrenic motoneuron pool.
• Dempsey, J. A., Fregosi, R. F., & Booth, F. W. (2021). A tribute to Charles M. "Tip" Tipton (1927-2021). Journal of applied physiology (Bethesda, Md. : 1985), 131(1), 192-193.
• Buls Wollman, L., Clarke, J., DeLucia, C. M., Levine, R. B., & Fregosi, R. F. (2020). Developmental Nicotine Exposure Alters Synaptic Input to Hypoglossal Motoneurons and Is Associated with Altered Function of Upper Airway Muscles. eNeuro, 6(6).
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  Nicotine exposure during the fetal and neonatal periods [developmental nicotine exposure (DNE)] is associated with ineffective upper airway protective reflexes in infants. This could be explained by desensitized chemoreceptors and/or mechanoreceptors, diminished neuromuscular transmission or altered synaptic transmission among central neurons, as each of these systems depend in part on cholinergic signaling through nicotinic AChRs (nAChRs). Here, we showed that DNE blunts the response of the genioglossus (GG) muscle to nasal airway occlusion in lightly anesthetized rat pups. The GG muscle helps keep the upper airway open and is innervated by hypoglossal motoneurons (XIIMNs). Experiments using the phrenic nerve-diaphragm preparation showed that DNE does not alter transmission across the neuromuscular junction. Accordingly, we used whole cell recordings from XIIMNs in brainstem slices to examine the influence of DNE on glutamatergic synaptic transmission under baseline conditions and in response to an acute nicotine challenge. DNE did not alter excitatory transmission under baseline conditions. Analysis of cumulative probability distributions revealed that acute nicotine challenge of P1-P2 preparations resulted in an increase in the frequency of nicotine-induced glutamatergic inputs to XIIMNs in both control and DNE. By contrast, P3-P5 DNE pups showed a decrease, rather than an increase in frequency. We suggest that this, together with previous studies showing that DNE is associated with a compensatory increase in inhibitory synaptic input to XIIMNs, leads to an excitatory-inhibitory imbalance. This imbalance may contribute to the blunting of airway protective reflexes observed in nicotine exposed animals and human infants.
• Pilarski, J. Q., Leiter, J. C., & Fregosi, R. F. (2019). Muscles of Breathing: Development, Function, and Patterns of Activation. Comprehensive Physiology, 9(3), 1025-1080.
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  This review is a comprehensive description of all muscles that assist lung inflation or deflation in any way. The developmental origin, anatomical orientation, mechanical action, innervation, and pattern of activation are described for each respiratory muscle fulfilling this broad definition. In addition, the circumstances in which each muscle is called upon to assist ventilation are discussed. The number of "respiratory" muscles is large, and the coordination of respiratory muscles with "nonrespiratory" muscles and in nonrespiratory activities is complex-commensurate with the diversity of activities that humans pursue, including sleep (8.27). The capacity for speech and adoption of the bipedal posture in human evolution has resulted in patterns of respiratory muscle activation that differ significantly from most other animals. A disproportionate number of respiratory muscles affect the nose, mouth, pharynx, and larynx, reflecting the vital importance of coordinated muscle activity to control upper airway patency during both wakefulness and sleep. The upright posture has freed the hands from locomotor functions, but the evolutionary history and ontogeny of forelimb muscles pervades the patterns of activation and the forces generated by these muscles during breathing. The distinction between respiratory and nonrespiratory muscles is artificial, as many "nonrespiratory" muscles can augment breathing under conditions of high ventilator demand. Understanding the ontogeny, innervation, activation patterns, and functions of respiratory muscles is clinically useful, particularly in sleep medicine. Detailed explorations of how the nervous system controls the multiple muscles required for successful completion of respiratory behaviors will continue to be a fruitful area of investigation. © 2019 American Physiological Society. Compr Physiol 9:1025-1080, 2019.
• Wealing, J. C., Cholanian, M., Flanigan, E. G., Levine, R. B., & Fregosi, R. F. (2019). Diverse physiological properties of hypoglossal motoneurons innervating intrinsic and extrinsic tongue muscles. Journal of neurophysiology, 122(5), 2054-2060.
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  The mammalian tongue contains eight muscles that collaborate to ensure that suckling, swallowing, and other critical functions are robust and reliable. Seven of the eight tongue muscles are innervated by hypoglossal motoneurons (XIIMNs). A somatotopic organization of the XII motor nucleus, defined in part by the mechanical action of a neuron's target muscle, has been described, but whether or not XIIMNs within a compartment are functionally specialized is unsettled. We hypothesize that developing XIIMNs are assigned unique functional properties that reflect the challenges that their target muscle faces upon the transition from in utero to terrestrial life. To address this, we studied XIIMNs that innervate intrinsic and extrinsic tongue muscles, because intrinsic muscles play a more prominent role in suckling than the extrinsic muscles. We injected dextran-rhodamine into the intrinsic longitudinal muscles (IL) and the extrinsic genioglossus, and physiologically characterized the labeled XIIMNs. Consistent with earlier work, IL XIIMNs ( = 150) were located more dorsally within the nucleus, and GG XIIMNs ( = 55) more ventrally. Whole cell recordings showed that resting membrane potential was, on average, 9 mV more depolarized in IL than in GG XIIMNs ( = 0.0019), and the firing threshold in response to current injection was lower in IL (-31 ± 23 pA) than in GG XIIMNs (225 ± 39 pA; < 0.0001). We also found that the appearance of net outward currents in GG XIIMNs occurred at more hyperpolarized membrane potentials than IL XIIMNs, consistent with lower excitability in GG XIIMNs. These observations document muscle-specific functional specializations among XIIMNs. The hypoglossal motor nucleus contains motoneurons responsible for innervating one of seven different muscles with notably different biomechanics and patterns of use. Whether or not motoneurons innervating the different muscles also have unique functional properties (e.g., spiking behavior, synaptic physiology) is poorly understood. In this work we show that neonatal hypoglossal motoneurons innervating muscles that shape the tongue (intrinsic longitudinal muscles) have different electrical properties than those innervating the genioglossus, which controls tongue position.
• Wollman, L. B., Levine, R. B., & Fregosi, R. F. (2018). Developmental nicotine exposure alters glycinergic neurotransmission to hypoglossal motoneurons in neonatal rats. Journal of neurophysiology, 120(3), 1135-1142.
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  We tested the hypothesis that nicotine exposure in utero and after birth [developmental nicotine exposure (DNE)] disrupts development of glycinergic synaptic transmission to hypoglossal motoneurons (XIIMNs). Glycinergic spontaneous and miniature inhibitory postsynaptic currents (sIPSC/mIPSC) were recorded from XIIMNs in brain stem slices from 1- to 5-day-old rat pups of either sex, under baseline conditions and following stimulation of nicotinic acetylcholine (ACh) receptors with nicotine (i.e., an acute nicotine challenge). Under baseline conditions, there were no significant effects of DNE on the amplitude or frequency of either sIPSCs or mIPSCs. In addition, DNE did not alter the magnitude of the whole cell current evoked by bath application of glycine, consistent with an absence of change in postsynaptic glycine-mediated conductance. An acute nicotine challenge (bath application of 0.5 μM nicotine) increased sIPSC frequency in the DNE cells, but not control cells. In contrast, nicotine challenge did not change mIPSC frequency in either control or DNE cells. In addition, there were no significant changes in the amplitude of either sIPSCs or mIPSCs in response to nicotine challenge. The increased frequency of sIPSCs in response to an acute nicotine challenge in DNE cells reflects an enhancement of action potential-mediated input from glycinergic interneurons to hypoglossal motoneurons. This could lead to more intense inhibition of hypoglossal motoneurons in response to exogenous nicotine or endogenous ACh. The former would occur with smoking or e-cigarette use while the latter occurs with changes in sleep state and with hypercapnia. NEW & NOTEWORTHY Here we show that perinatal nicotine exposure does not impact baseline glycinergic neurotransmission to hypoglossal motoneurons but enhances glycinergic inputs to hypoglossal motoneurons in response to activation of nicotinic acetylcholine (ACh) receptors with acute nicotine. Given that ACh is the endogenous ligand for nicotinic ACh receptors, the latter reveals a potential mechanism whereby perinatal nicotine exposure alters motor function under conditions where ACh release increases, such as the transition from non-rapid-eye movement to rapid-eye movement sleep, and during hypercapnia.
• Wollman, L. B., Levine, R. B., & Fregosi, R. F. (2018). Developmental plasticity of GABAergic neurotransmission to brainstem motoneurons. The Journal of physiology.
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  Nicotinic acetylcholine receptor (nAChR) signalling regulates neuronal differentiation and synaptogenesis. Here we test the hypothesis that developmental nicotine exposure (DNE) disrupts the development of GABAergic synaptic transmission to hypoglossal motoneurons (XIIMNs). GABAergic spontaneous and miniature inhibitory postsynaptic currents (sIPSC/mIPSC) were recorded from XIIMNs in brainstem slices from control and DNE rat pups of either sex, 1-5 days old, at baseline and following acute stimulation of nAChRs with nicotine. At baseline, sIPSCs were less frequent and smaller in DNE cells (consistent with decreased action potential mediated GABA release), and mIPSCs were more frequent (consistent with increased vesicular GABA release from presynaptic terminals). Acute nicotine challenge increased sIPSC frequency in both groups, though the increase was greater in DNE cells. Acute nicotine challenge did not change the frequency of mIPSCs in either group, though mIPSC amplitude increased significantly in DNE cells, but not control cells. Stimulation of postsynaptic GABAA receptors with muscimol caused a significantly greater chloride current in DNE cells than in control cells. The increased quantal release of GABA, coupled with the rise in the strength of postsynaptic inhibition may be homeostatic adjustments to the decreased action-potential mediated input from GABAergic interneurons. However, this will exaggerate synaptic inhibition under conditions where the release of GABA (e.g. hypoxia) or ACh (sleep-wake transitions) is increased. These findings reveal a mechanism that may explain why DNE is associated with deficits in the ability to respond appropriately to chemosensory stimuli or to changes in neuromodulation secondary to changes in central nervous system state. This article is protected by copyright. All rights reserved.
• Cholanian, M., Powell, G. L., Levine, R. B., & Fregosi, R. F. (2017). Influence of developmental nicotine exposure on glutamatergic neurotransmission in rhythmically active hypoglossal motoneurons. Experimental neurology, 287(Pt 2), 254-260.
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  Developmental nicotine exposure (DNE) is associated with increased risk of cardiorespiratory, intellectual, and behavioral abnormalities in neonates, and is a risk factor for apnea of prematurity, altered arousal responses and Sudden Infant Death Syndrome. Alterations in nicotinic acetylcholine receptor signaling (nAChRs) after DNE lead to changes in excitatory neurotransmission in neural networks that control breathing, including a heightened excitatory response to AMPA microinjection into the hypoglossal motor nucleus. Here, we report on experiments designed to probe possible postsynaptic and presynaptic mechanisms that may underlie this plasticity. Pregnant dams were exposed to nicotine or saline via an osmotic mini-pump implanted on the 5th day of gestation. We used whole-cell patch clamp electrophysiology to record from hypoglossal motoneurons (XIIMNs) in thick medullary slices from neonatal rat pups (N=26 control and 24 DNE cells). To enable the translation of our findings to breathing-related consequences of DNE, we only studied XIIMNs that were receiving rhythmic excitatory drive from the respiratory central pattern generator. Tetrodotoxin was used to isolate XIIMNs from presynaptic input, and their postsynaptic responses to bath application of l-glutamic acid (glutamate) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were studied under voltage clamp. DNE had no influence on inward current magnitude evoked by either glutamate or AMPA. However, in cells from DNE animals, bath application of AMPA was associated with a right shift in the amplitude distribution (P=0.0004), but no change in the inter-event interval distribution of miniature excitatory postsynaptic currents (mEPSCs). DNE had no influence on mEPSC amplitude or frequency evoked by glutamate application, or under (unstimulated) baseline conditions. Thus, in the presence of AMPA, DNE is associated with a small but significant increase in quantal size, but no change in the probability of glutamate release.
• Cholanian, M., Wealing, J., Levine, R. B., & Fregosi, R. F. (2017). Developmental nicotine exposure alters potassium currents in hypoglossal motoneurons of neonatal rat. Journal of neurophysiology, 117(4), 1544-1552.
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  We previously showed that nicotine exposure in utero and after birth via breast milk [developmental nicotine exposure (DNE)] is associated with many changes in the structure and function of hypoglossal motoneurons (XIIMNs), including a reduction in the size of the dendritic arbor and an increase in cell excitability. Interestingly, the elevated excitability was associated with a reduction in the expression of glutamate receptors on the cell body. Together, these observations are consistent with a homeostatic compensation aimed at restoring cell excitability. Compensation for increased cell excitability could also occur by changing potassium conductance, which plays a critical role in regulating resting potential, spike threshold, and repetitive spiking behavior. Here we test the hypothesis that the previously observed increase in the excitability of XIIMNs from DNE animals is associated with an increase in whole cell potassium currents. Potassium currents were measured in XIIMNs in brain stem slices derived from DNE and control rat pups ranging in age from 0 to 4 days by whole cell patch-clamp electrophysiology. All currents were measured after blockade of action potential-dependent synaptic transmission with tetrodotoxin. Compared with control cells, XIIMNs from DNE animals showed significantly larger transient and sustained potassium currents, but this was observed only under conditions of increased cell and network excitability, which we evoked by raising extracellular potassium from 3 to 9 mM. These observations suggest that the larger potassium currents in nicotine-exposed neurons are an important homeostatic compensation that prevents "runaway" excitability under stressful conditions, when neurons are receiving elevated excitatory synaptic input.NEW & NOTEWORTHY Developmental nicotine exposure is associated with increased cell excitability, which is often accompanied by compensatory changes aimed at normalizing excitability. Here we show that whole cell potassium currents are also increased in hypoglossal motoneurons from nicotine-exposed neonatal rats under conditions of increased cell and network excitability. This is consistent with a compensatory response aimed at preventing instability under conditions in which excitatory synaptic input is high and is compatible with the concept of homeostatic plasticity.
• Fregosi, R. F. (2017). Developmental nicotine exposure alters potassium currents in hypoglossal motoneurons of neonatal rat. Journal of Neurophysiology.
• Fregosi, R. F., & Wagner, P. D. (2017). Reply to Drs. Nuzzo and Barry. Journal of applied physiology (Bethesda, Md. : 1985), 123(5), 1421.
• Fregosi, R. F., & Wagner, P. D. (2016). When negative is positive. Journal of applied physiology (Bethesda, Md. : 1985), 121(3), 605.
• Jaiswal, S. J., Buls Wollman, L., Harrison, C. M., Pilarski, J. Q., & Fregosi, R. F. (2016). Developmental nicotine exposure enhances inhibitory synaptic transmission in motor neurons and interneurons critical for normal breathing. Developmental neurobiology, 76(3), 337-54.
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  Nicotine exposure in utero negatively affects neuronal growth, differentiation, and synaptogenesis. We used rhythmic brainstems slices and immunohistochemistry to determine how developmental nicotine exposure (DNE) alters inhibitory neurotransmission in two regions essential to normal breathing, the hypoglossal motor nucleus (XIIn), and preBötzinger complex (preBötC). We microinjected glycine or muscimol (GABAA agonist) into the XIIn or preBötC of rhythmic brainstem slices from neonatal rats while recording from XII nerve roots to obtain XII motoneuron population activity. Injection of glycine or muscimol into the XIIn reduced XII nerve burst amplitude, while injection into the preBötC altered nerve burst frequency. These responses were exaggerated in preparations from DNE animals. Quantitative immunohistochemistry revealed a significantly higher GABAA receptor density on XII motoneurons from DNE pups. There were no differences in GABAA receptor density in the preBötC, and there were no differences in glycine receptor expression in either region. Nicotine, in the absence of other chemicals in tobacco smoke, alters normal development of brainstem circuits that are critical for normal breathing. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 337-354, 2016.
• Powell, G. L., Gaddy, J., Xu, F., Fregosi, R. F., & Levine, R. B. (2016). Developmental Nicotine Exposure disrupts dendritic arborization patterns of hypoglossal motoneurons in the neonatal rat. Developmental neurobiology.
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  Maternal smoking or use of other products containing nicotine during pregnancy can have significant adverse consequences for respiratory function in neonates. We have shown, in previous studies, that developmental nicotine exposure (DNE) in a model system compromises the normal function of respiratory circuits within the brainstem. The effects of DNE include alterations in the excitability and synaptic interactions of the hypoglossal motoneurons, which innervate muscles of the tongue. This study was undertaken to test the hypothesis that these functional consequences of DNE are accompanied by changes in the dendritic morphology of hypoglossal motoneurons. Hypoglossal motoneurons in brain stem slices were filled with neurobiotin during whole-cell patch clamp recordings and subjected to histological processing to reveal dendrites. Morphometric analysis, including the Sholl method, revealed significant effects of DNE on dendritic branching patterns. In particular, whereas within the first 5 postnatal days there was significant growth of the higher-order dendritic branches of motoneurons from control animals, the growth was compromised in motoneurons from neonates that were subjected to DNE. This article is protected by copyright. All rights reserved.
• Wollman, L. B., Haggerty, J., Pilarski, J. Q., Levine, R. B., & Fregosi, R. F. (2016). Developmental nicotine exposure alters cholinergic control of respiratory frequency in neonatal rats. Developmental neurobiology.
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  Prenatal nicotine exposure with continued exposure through breast milk over the first week of life (developmental nicotine exposure, DNE) alters the development of brainstem circuits that control breathing. Here, we test the hypothesis that DNE alters the respiratory motor response to endogenous and exogenous acetylcholine (ACh) in neonatal rats. We used the brainstem-spinal cord preparation in the split-bath configuration, and applied drugs to the brainstem compartment while measuring the frequency and amplitude of the fourth cervical ventral nerve roots (C4VR), which contain the axons of phrenic motoneurons. We applied ACh alone; the nicotinic acetylcholine receptor (nAChR) antagonist curare, either alone or in the presence of ACh; and the muscarinic acetylcholine receptor (mAChR) antagonist atropine, either alone or in the presence of ACh. The main findings include: 1) atropine reduced frequency similarly in controls and DNE animals, while curare caused modest slowing in controls but no consistent change in DNE animals; 2) DNE greatly attenuated the increase in C4VR frequency mediated by exogenous ACh; 4) stimulation of nAChRs with ACh in the presence of atropine increased frequency markedly in controls, but not DNE animals; 5) stimulation of mAChRs with ACh in the presence of curare caused a modest increase in frequency, with no treatment group differences. DNE blunts the response of the respiratory central pattern generator to exogenous ACh, consistent with reduced availability of functionally competent nAChRs; DNE did not alter the muscarinic control of respiratory motor output. This article is protected by copyright. All rights reserved.
• Ferng, J., & Fregosi, R. F. (2015). Influence of developmental nicotine exposure on the ventilatory and metabolic response to hyperthermia. The Journal of physiology, 593(23), 5201-13.
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  Both developmental nicotine exposure and hyperthermia are associated with breathing abnormalities in neonates. Whether or not gestational nicotine exposure interacts with heat stress to alter the normal ventilatory response to hyperthermia is unclear. With moderate thermal stress, nicotine-exposed rat pups breathed less and had longer apnoeas than control pups, although this was true at both normal and high temperatures, suggesting an effect of gestational nicotine exposure that is independent of body temperature. With severe thermal stress, nicotine-exposed pups failed to increase pulmonary ventilation, and had a delayed recovery from hyperthermia-induced gasping The results show that gestational nicotine exposure has subtle but physiologically significant effects on the ventilatory response to severe hyperthermia, which could impact upon both gas exchange and temperature regulation in infants with fever, excessive swaddling or exposure to hot environmental temperatures.
• Fregosi, R. F., Pilarski, J. Q., & Leiter, J. L. (2018). "The muscles of breathing: Development, Function and Patterns of Activation". Comprehensive Physiology.
• Patel-Khurana, N., & Fregosi, R. F. (2015). Motor unit number in a small facial muscle, dilator naris. Experimental brain research, 233(10), 2897-902.
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  A loss of functioning motor units underlies many neuromuscular disorders. The facial nerve innervates the muscles of facial expression, including nasal muscles, which also play an important role in the regulation of airflow resistance. It is difficult to accurately assess motor unit number in the facial muscles, because the muscles are difficult to activate in isolation. Here, we apply the manual McComas method to estimate the number of motor units in a nasal dilator muscle. EMG of the dilator naris was recorded during graded stimulation of the zygomatic branch of the facial nerve in 26 subjects (12 males and 14 females), aged 20-41 years. Each subject was studied twice, on separate days, to estimate method reproducibility. As a check on our use of the McComas method, we also estimated motor unit number in the first dorsal interosseus muscle (FDI) of six subjects, as the muscle is also small and has been studied with the McComas method. Reproducibility was evaluated with a rigorous statistical approach, the Bland-Altman procedure. We estimate that dilator naris is composed of 75 ± 15.6 (SD) motor units, compared to 144 ± 35.5 in FDI. The coefficient of variation for test-retest reproducibility of dilator naris motor unit estimates was 29.6 %, similar to separate-day reproducibility reported for other muscles. Recording and stimulation were done with surface electrodes, and the recordings were of high quality and reproducible. This simple technique could be applied clinically to track motor neuron loss and to monitor facial nerve integrity.
• Uribe, J. M., Stump, C. S., Tipton, C. M., & Fregosi, R. F. (1992). Influence of exercise training on the oxidative capacity of rat abdominal muscles. Respiration physiology, 88(1-2), 171-80.
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  Our purpose was to determine if endurance exercise training would increase the oxidative capacity of the abdominal expiratory muscles of the rat. Accordingly, 9 male rats were subjected to an endurance training protocol (1 h/day, 6 days/week, 9 weeks) and 9 litter-mates served as controls. Citrate synthase (CS) activity was used as an index of oxidative capacity, and was determined in the following muscles: soleus, plantaris, costal diaphragm, crural diaphragm, and in all four abdominal muscles: rectus abdominis, transversus abdominis, external oblique, and internal oblique. Compared to their non-trained litter-mates, the trained rats had higher peak whole body oxygen consumption rates (+ 16%) and CS activities in plantaris (+34%) and soleus (+36%) muscles. Thus, the training program caused substantial systemic and locomotor muscle adaptations. The CS activity of costal diaphragm was 20% greater in the trained animals, but no difference was observed in crural diaphragm. The CS activity in the abdominal muscles was less than one-half of that in locomotor and diaphragm muscles, and there were no significant changes with training except in the rectus abdominis where a 26% increase was observed. The increase in rectus abdominis CS activity may reflect its role in postural support and/or locomotion, as none of the primary expiratory pumping muscles adapted to the training protocol. The relatively low levels of CS activity in the abdominal muscles suggests that they are not recruited frequently at rest, and the lack of an increase with training indicates that these muscles do not contribute significantly to the increased ventilatory activity accompanying exercise in the rat.
• Wakefield, H. E., Fregosi, R. F., & Fuglevand, A. J. (2015). Current Injection And Receptor-Mediated Excitation Produce Similar Maximal Firing Rates In Hypoglossal Motor Neurons. Journal of neurophysiology, jn.00848.2015.
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  The maximum firing rates of motor neurons (MNs) activated in response to synaptic drive appear to be much lower than that elicited by current injection. It could be that the decrease in input resistance associated with increased synaptic activity (but not current injection) might blunt overall changes in membrane depolarization and thereby limit spike-frequency output. To test this idea, we recorded, in the same cells, maximal firing responses to current injection and to synaptic activation. We prepared 300 μm medullary slices in neonatal rats that contained hypoglossal motor neurons (HMNs) and used whole cell patch clamp electrophysiology to record their maximum firing rates in response to triangular-ramp current injections and to glutamate receptor-mediated excitation. Brief pressure pulses of high concentration glutamate led to significant depolarization, high firing rates, and temporary cessation of spiking due to spike inactivation. In the same cells, we applied current clamp protocols that approximated the time-course of membrane potential change associated with glutamate application and with peak current levels large enough to cause spike inactivation. Mean (SD) maximum firing rates obtained in response to glutamate application were nearly identical to those obtained in response to ramp current injection (glutamate 47.1 ± 12.0 impulses/s, current injection 47.5 ± 11.2 impulses/s) even though input resistance was 40% less during glutamate application compared to current injection. Therefore, these data suggest that the reduction in input resistance associated with receptor-mediated excitation does not by itself limit the maximal firing rate responses in MNs.
• Fregosi, R. F., & Ludlow, C. L. (2014). Activation of upper airway muscles during breathing and swallowing. Journal of applied physiology (Bethesda, Md. : 1985), 116(3), 291-301.
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  The upper airway is a complex muscular tube that is used by the respiratory and digestive systems. The upper airway is invested with several small and anatomically peculiar muscles. The muscle fiber orientations and their nervous innervation are both extremely complex, and how the activity of the muscles is initiated and adjusted during complex behaviors is poorly understood. The bulk of the evidence suggests that the entire assembly of tongue and laryngeal muscles operate together but differently during breathing and swallowing, like a ballet rather than a solo performance. Here we review the functional anatomy of the tongue and laryngeal muscles, and their neural innervation. We also consider how muscular activity is altered as respiratory drive changes, and briefly address upper airway muscle control during swallowing.
• Powell, G. L., Levine, R. B., Frazier, A. M., & Fregosi, R. F. (2014). Influence of developmental nicotine exposure on spike timing precision & reliability in hypoglossal motoneurons. Journal of neurophysiology, jn.00838.2014.
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  Smoothly graded muscle contractions depend in part on the precision and reliability of motoneuron action potential generation. Whether or not a motoneuron generates spikes precisely and reliably depends on both its intrinsic membrane properties and the nature of the synaptic input that it receives. Factors that perturb neuronal intrinsic properties and/or synaptic drive may compromise the temporal precision and the reliability of action potential generation. We have previously shown that developmental nicotine exposure (DNE) alters intrinsic properties and synaptic transmission in hypoglossal motoneurons (XIIMNs). Here we show that the effects of DNE also include alterations in spike-timing precision and reliability, and spike-frequency adaptation, in response to sinusoidal current injection. Current-clamp experiments in brainstem slices from neonatal rats show that DNE lowers the threshold for spike generation, but increases the variability of spike timing mechanisms. DNE is also associated with an increase in spike-frequency adaptation and reductions in both peak and steady-state firing rate in response to brief, square wave current injections. Taken together, our data indicate that DNE causes significant alterations in the input-output efficiency of XIIMNs. These alterations may play a role in the increased frequency of obstructive apneas and altered suckling strength and coordination observed in nicotine exposed neonatal humans.
• Fregosi, R., Jaiswal, S. J., Pilarski, J. Q., Harrison, C. M., & Fregosi, R. F. (2013). Developmental nicotine exposure alters AMPA neurotransmission in the hypoglossal motor nucleus and pre-Botzinger complex of neonatal rats. The Journal of neuroscience : the official journal of the Society for Neuroscience, 33(6).
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  Developmental nicotine exposure (DNE) impacts central respiratory control in neonates born to smoking mothers. We previously showed that DNE enhances the respiratory motor response to bath application of AMPA to the brainstem, although it was unclear which brainstem respiratory neurons mediated these effects (Pilarski and Fregosi, 2009). Here we examine how DNE influences AMPA-type glutamatergic neurotransmission in the pre-Bötzinger complex (pre-BötC) and the hypoglossal motor nucleus (XIIMN), which are neuronal populations located in the medulla that are necessary for normal breathing. Using rhythmic brainstem slices from neonatal rats, we microinjected AMPA into the pre-BötC or the XIIMN while recording from XII nerve rootlets (XIIn) as an index of respiratory motor output. DNE increased the duration of tonic activity and reduced rhythmic burst amplitude after AMPA microinjection into the XIIMN. Also, DNE led to an increase in respiratory burst frequency after AMPA injection into the pre-BötC. Whole-cell patch-clamp recordings of XII motoneurons showed that DNE increased motoneuron excitability but did not change inward currents. Immunohistochemical studies indicate that DNE reduced the expression of glutamate receptor subunits 2 and 3 (GluR2/3) in the XIIMN and the pre-BötC. Our data show that DNE alters AMPAergic synaptic transmission in both the XIIMN and pre-BötC, although the mechanism by which this occurs is unclear. We suggest that the DNE-induced reduction in GluR2/3 may represent an attempt to compensate for increased cell excitability, consistent with mechanisms underlying homeostatic plasticity.
• Fregosi, R., Powell, G. L., Rice, A., Bennett-Cross, S. J., & Fregosi, R. F. (2013). Respiration-related discharge of hyoglossus muscle motor units in the rat. Journal of neurophysiology.
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  Although respiratory muscle motor units have been studied during natural breathing, simultaneous measures of muscle force have never been obtained. Tongue retractor muscles, such as the hyoglossus (HG), play an important role in swallowing, licking, chewing, breathing, and in humans, speech. The HG is phasically recruited during the inspiratory phase of the respiratory cycle. Moreover, in urethane anesthetized rats the drive to the HG waxes and wanes spontaneously, providing a unique opportunity to study motor unit firing patterns as the muscle is driven naturally by the central pattern generator for breathing. We recorded tongue retraction force, the whole HG muscle EMG and the activity of 38 HG motor units in spontaneously breathing anesthetized rats under low force and high force conditions. Activity in all cases was confined to the inspiratory phase of the respiratory cycle. Changes in the EMG were correlated significantly with corresponding changes in force, with the change in EMG able to predict 53-68% of the force variation. Mean and peak motor unit firing rates were greater under high force conditions, though the magnitude of discharge rate modulation varied widely across the population. Changes in mean and peak firing rates were significantly correlated with the corresponding changes in force, but the correlations were weak (r2 = 0.27 and 0.25, respectively). These data indicate that during spontaneous breathing recruitment of hyoglossus motor units plays a critical role in the control of muscle force, with firing rate modulation playing an important but lesser role.
• Pilarski, J. Q., Wakefield, H. E., Fuglevand, A. J., Levine, R. B., & Fregosi, R. F. (2012). Increased nicotinic receptor desensitization in hypoglossal motor neurons following chronic developmental nicotine exposure. Journal of neurophysiology, 107(1), 257-64.
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  Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed on hypoglossal motor neurons (XII MNs) that innervate muscles of the tongue. Activation of XII MN nAChRs evokes depolarizing currents, which are important for regulating the size and stiffness of the upper airway. Although data show that chronic developmental nicotine exposure (DNE) blunts cholinergic neurotransmission in the XII motor nucleus, it is unclear how nAChRs are involved. Therefore, XII MN nAChR desensitization and recovery were examined in tissues from DNE or control pups using a medullary slice preparation and tight-seal whole cell patch-clamp recordings. nAChR-mediated inward currents were evoked by brief pressure pulses of nicotine or the α4β2 nAChR agonist RJR-2403. We found that, regardless of treatment, activatable nAChRs underwent desensitization, but, following DNE, nAChRs exhibited increased desensitization and delayed recovery. Similar results were produced using RJR-2403, showing that DNE influences primarily the α4β2 nAChR subtype. These results show that while some nAChRs preserve their responsiveness to acute nicotine following DNE, they more readily desensitize and recover more slowly from the desensitized state. These data provide new evidence that chronic DNE modulates XII MN nAChR function, and suggests an explanation for the association between DNE and the incidence of central and obstructive apneas.
• Fregosi, R. F., Bailey, E. F., & Fuller, D. D. (2011). Respiratory muscles and motoneurons. Respiratory physiology & neurobiology, 179(1), 1-2.
• Fregosi, R., & Fregosi, R. F. (2011). Influence of tongue muscle contraction and transmural pressure on nasopharyngeal geometry in the rat. Journal of applied physiology (Bethesda, Md. : 1985), 111(3).
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  The mammalian pharynx is a hollow muscular tube that participates in ingestion and respiration, and its size, shape, and stiffness can be altered by contraction of skeletal muscles that lie inside or outside of its walls. MRI was used to determine the interaction between pharyngeal pressure and selective stimulation of extrinsic tongue muscles on the shape of the rat nasopharynx. Pressure (-9, -6, -3, 3, 6, and 9 cmH₂O) was applied randomly to the isolated pharyngeal airway of anesthetized rats that were positioned in a 4.7-T MRI scanner. The anterior-posterior (AP) and lateral diameters of the nasopharynx were measured in eight axial slices at each level of pressure, with and without bilateral hypoglossal nerve stimulation (0.1-ms pulse, 1/3 maximal force, 80 Hz). The rat nasopharynx is nearly circular, and positive pharyngeal pressure caused similar expansion of AP and lateral diameters; as a result, airway shape (ratio of lateral to AP diameter) remained constant. Negative pressure did not change AP or lateral diameter significantly, suggesting that a negative pressure reflex activated the tongue or other pharyngeal muscles. Stimulation of tongue protrudor muscles alone or coactivation of protrudor and retractor muscles caused greater AP than lateral expansion, making the nasopharynx slightly more elliptical, with the long axis in the AP direction. These effects tended to be more pronounced at negative pharyngeal pressures and greater in the caudal than rostral nasopharynx. These data show that stimulation of rodent tongue muscles can adjust pharyngeal shape, extending previous work showing that tongue muscle contraction alters pharyngeal compliance and volume, and provide physiological insight that can be applied to the treatment of obstructive sleep apnea.
• Fregosi, R., & Fregosi, R. F. (2011). Respiratory related control of hypoglossal motoneurons--knowing what we do not know. Respiratory physiology & neurobiology, 179(1).
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  Because tongue position and stiffness help insure that the pharyngeal airspace is sufficiently open during breathing, the respiration-related behavior of the tongue muscles has been studied in detail, particularly during the last two decades. Although eight different muscles act upon the mammal tongue, we know very little about the respiration-related control of the majority of these, and almost nothing about how they work together as a complex electro-mechanical system. Other significant gaps include how hypoglossal motoneuron axons find their appropriate muscle target during development, whether the biophysical properties of hypoglossal motoneurons driving different muscles are the same, and how afferent information from cardiorespiratory reflex systems is transmitted from major brainstem integrating centers to the hypoglossal motoneuron pool. This brief review outlines some of these issues, with the hope that this will spur research in the field, ultimately leading to an improved understanding of the respiration-related control of the mammalian tongue musculature.
• Fregosi, R., Pilarski, J. Q., Wakefield, H. E., Fuglevand, A. J., Levine, R. B., & Fregosi, R. F. (2011). Developmental nicotine exposure alters neurotransmission and excitability in hypoglossal motoneurons. Journal of neurophysiology, 105(1).
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  Hypoglossal motoneurons (XII MNs) control muscles of the mammalian tongue and are rhythmically active during breathing. Acetylcholine (ACh) modulates XII MN activity by promoting the release of glutamate from neurons that express nicotinic ACh receptors (nAChRs). Chronic nicotine exposure alters nAChRs on neurons throughout the brain, including brain stem respiratory neurons. Here we test the hypothesis that developmental nicotine exposure (DNE) reduces excitatory synaptic input to XII MNs. Voltage-clamp experiments in rhythmically active medullary slices showed that the frequency of excitatory postsynaptic currents (EPSCs) onto XII MNs from DNE animals is reduced by 61% (DNE = 1.7 ± 0.4 events/s; control = 4.4 ± 0.6 events/s; P < 0.002). We also examine the intrinsic excitability of XII MNs to test whether cells from DNE animals have altered membrane properties. Current-clamp experiments showed XII MNs from DNE animals had higher intrinsic excitability, as evaluated by measuring their response to injected current. DNE cells had high-input resistances (DNE = 131.9 ± 13.7 MΩ, control = 78.6 ± 9.7 MΩ, P < 0.008), began firing at lower current levels (DNE = 144 ± 22 pA, control = 351 ± 45 pA, P < 0.003), and exhibited higher frequency-current gain values (DNE = 0.087 ± 0.012 Hz/pA, control = 0.050 ± 0.004 Hz/pA, P < 0.02). Taken together, our data show previously unreported effects of DNE on XII MN function and may also help to explain the association between DNE and the incidence of central and obstructive apneas.
• Fregosi, R., Rice, A., Fuglevand, A. J., Laine, C. M., & Fregosi, R. F. (2011). Synchronization of presynaptic input to motor units of tongue, inspiratory intercostal, and diaphragm muscles. Journal of neurophysiology, 105(5).
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  The respiratory central pattern generator distributes rhythmic excitatory input to phrenic, intercostal, and hypoglossal premotor neurons. The degree to which this input shapes motor neuron activity can vary across respiratory muscles and motor neuron pools. We evaluated the extent to which respiratory drive synchronizes the activation of motor unit pairs in tongue (genioglossus, hyoglossus) and chest-wall (diaphragm, external intercostals) muscles using coherence analysis. This is a frequency domain technique, which characterizes the frequency and relative strength of neural inputs that are common to each of the recorded motor units. We also examined coherence across the two tongue muscles, as our previous work shows that, despite being antagonists, they are strongly coactivated during the inspiratory phase, suggesting that excitatory input from the premotor neurons is distributed broadly throughout the hypoglossal motoneuron pool. All motor unit pairs showed highly correlated activity in the low-frequency range (1-8 Hz), reflecting the fundamental respiratory frequency and its harmonics. Coherence of motor unit pairs recorded either within or across the tongue muscles was similar, consistent with broadly distributed premotor input to the hypoglossal motoneuron pool. Interestingly, motor units from diaphragm and external intercostal muscles showed significantly higher coherence across the 10-20-Hz bandwidth than tongue-muscle units. We propose that the lower coherence in tongue-muscle motor units over this range reflects a larger constellation of presynaptic inputs, which collectively lead to a reduction in the coherence between hypoglossal motoneurons in this frequency band. This, in turn, may reflect the relative simplicity of the respiratory drive to the diaphragm and intercostal muscles, compared with the greater diversity of functions fulfilled by muscles of the tongue.
• Fregosi, R., Huang, Y., Brown, A. R., Cross, S. J., Cruz, J., Rice, A., Jaiswal, S., & Fregosi, R. F. (2010). Influence of prenatal nicotine exposure on development of the ventilatory response to hypoxia and hypercapnia in neonatal rats. Journal of applied physiology (Bethesda, Md. : 1985), 109(1).
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  In a recent study (Huang YH et al. Respir Physiol Neurobiol 143: 1-8, 2004), we showed that prenatal nicotine exposure (PNE) increased the frequency of spontaneous apneic events on the first 2 days of life in unanesthetized neonatal rats. Here we test the hypothesis that PNE blunts chemoreceptor reflexes. Ventilatory responses to three levels each of hypoxia (inspired O(2) fraction: 16, 12, and 10%) and hypercapnia (3, 6, and 9% inspired CO(2) fraction, all in 50% O(2), balance N(2)), and one level each of combined hypoxia-hypercapnia (H/H; 12% inspired O(2) fraction/5% inspired CO(2) fraction) and hyperoxia (50% O(2), 50% N(2)) were recorded with head-out plethysmography in neonatal rats exposed to either nicotine (N = 12) or physiological saline (N = 12) in the prenatal period. Recordings were made on postnatal day 1 (P1), P3, P6, P9, P12, and P18, in each animal. The change in ventilation in response to hypoxia was blunted in PNE animals on P1 and P3, but there were no other treatment effects. Hyperoxia significantly depressed ventilation in both groups from P3-P18, but there were no significant treatment effects. The ventilatory response to 3, 6, and 9% inspired CO(2) was significantly blunted in PNE animals at all ages studied, due exclusively to a blunted tidal volume response. PNE also blunted the ventilatory response to H/H at all ages, due primarily to blunting of the tidal volume response. PNE had no significant effect on body mass or metabolic rate, except that PNE animals had a slightly higher mass on P18 and a lower metabolic rate on P1. As shown by others, PNE has small and inconsistent effects on hypoxic ventilatory responses, but here we show that responses to hypercapnia and H/H are consistently blunted by PNE due to a diminished tidal volume response. The combination of reduced hypoxic and hypercapnic sensitivity over the first 3 days of life may define an especially vulnerable developmental period.
• Rice, A., Costy-Bennett, S., Goodwin, J. L., Quan, S. F., & Fregosi, R. F. (2010). Control of breathing in children with mild sleep apnoea: a 6-year follow-up study. The European respiratory journal, 35(1), 124-31.
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  We have previously shown that children (average age 9 yrs) with mildly elevated obstructive apnoea/hypopnoea indices (OAHI) retained CO(2) at rest. Here, we report the results of a 6-yr follow-up study on 14 children from that study. Minute ventilation (V'(E)) and end-tidal CO(2) partial pressure (P(ET,CO(2))) were measured during hypercapnic challenge. OAHI decreased from 7.5+/-4.7 events x h(-1) at age 9 yrs to 2.5+/-1.8 events x h(-1) at age 15 yrs (p
• Sandhu, M. S., Lee, K. Z., Fregosi, R. F., & Fuller, D. D. (2010). Phrenicotomy alters phrenic long-term facilitation following intermittent hypoxia in anesthetized rats. Journal of applied physiology (Bethesda, Md. : 1985), 109(2), 279-87.
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  Intermittent hypoxia (IH) can induce a persistent increase in neural drive to the respiratory muscles known as long-term facilitation (LTF). LTF of phrenic inspiratory activity is often studied in anesthetized animals after phrenicotomy (PhrX), with subsequent recordings being made from the proximal stump of the phrenic nerve. However, severing afferent and efferent axons in the phrenic nerve has the potential to alter the excitability of phrenic motoneurons, which has been hypothesized to be an important determinant of phrenic LTF. Here we test the hypothesis that acute PhrX influences immediate and long-term phrenic motor responses to hypoxia. Phrenic neurograms were recorded in anesthetized, ventilated, and vagotomized adult male rats with intact phrenic nerves or bilateral PhrX. Data were obtained before (i.e., baseline), during, and after three 5-min bouts of isocapnic hypoxia. Inspiratory burst amplitude during hypoxia (%baseline) was greater in PhrX than in phrenic nerve-intact rats (P < 0.001). Similarly, burst amplitude 55 min after IH was greater in PhrX than in phrenic nerve-intact rats (175 + or - 9 vs. 126 + or - 8% baseline, P < 0.001). In separate experiments, phrenic bursting was recorded before and after PhrX in the same animal. Afferent bursting that was clearly observable in phase with lung deflation was immediately abolished by PhrX. The PhrX procedure also induced a form of facilitation as inspiratory burst amplitude was increased at 30 min post-PhrX (P = 0.01 vs. pre-PhrX). We conclude that, after PhrX, axotomy of phrenic motoneurons and, possibly, removal of phrenic afferents result in increased phrenic motoneuron excitability and enhanced LTF following IH.
• Fregosi, R., Pilarski, J. Q., & Fregosi, R. F. (2009). Prenatal nicotine exposure alters medullary nicotinic and AMPA-mediated control of respiratory frequency in vitro. Respiratory physiology & neurobiology, 169(1).
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  Prenatal nicotine exposure (PNE) is correlated with breathing abnormalities in humans and other animals. Despite evidence that this relationship results from alterations in nicotinic acetylcholine receptors (nAChRs), the mechanisms are poorly understood. Here, we hypothesize that PNE blunts nAChR-mediated respiratory-related motor output. We also hypothesize that the PNE-induced changes in nAChRs leads to secondary alterations in glutamatergic neurotransmission. To test these hypotheses, we used an in vitro brainstem-spinal cord preparation and recorded C4 ventral root (C4 VR) nerve bursts from 0 to 4-day-old rats that were exposed to either nicotine (6mgkg(-1)day(-1)) or saline (control) in utero. Nicotine bitartrate, nAChR antagonists, NMDA and AMPA were applied to the brainstem compartment of a "split-bath" configuration, which physically separated the medulla from the spinal cord. Nicotine (0.2 or 0.5microM) increased peak C4 VR burst frequency by over 230% in control pups, but only 140% in PNE animals. The application of nAChR antagonists showed that these effects were mediated by the alpha4beta2 nAChR subtype with no effect on alpha7 nAChRs in either group. We also show that AMPA-mediated excitatory neurotransmission is enhanced by PNE, but NMDA-mediated neurotransmission is unaltered. These data and the work of others suggest that the PNE may functionally desensitize alpha4beta2 nAChRs located on the presynaptic terminals of glutamatergic neurons leading to less neurotransmitter release, which in turn up-regulates AMPA receptors on rhythm generating neurons.
• Fregosi, R., Schmitt, K., DelloRusso, C., & Fregosi, R. F. (2009). Force-EMG changes during sustained contractions of a human upper airway muscle. Journal of neurophysiology, 101(2).
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  Human upper airway and facial muscles support breathing, swallowing, speech, mastication, and facial expression, but their endurance performance in sustained contractions is poorly understood. The muscular fatigue typically associated with task failure during sustained contractions has both central and intramuscular causes, with the contribution of each believed to be task dependent. Previously we failed to show central fatigue in the nasal dilator muscles of subjects that performed intermittent maximal voluntary contractions (MVCs). Here we test the hypothesis that central mechanisms contribute to the fatigue of submaximal, sustained contractions in nasal dilator muscles. Nasal dilator muscle force and EMG activities were recorded in 11 subjects that performed submaximal contractions (20, 35, and 65% MVC) until force dropped to or=3 s, which we defined as task failure. MVC and twitch forces (the latter obtained by applying supramaximal shocks to the facial nerve) were recorded before the trial and at several time points over the first 10 min of recovery. The time to task failure was inversely related to contraction intensity. MVC force was depressed by roughly 30% at task failure in all three trials, but recovered within 2 min. Twitch force fell by 30-44% depending on contraction intensity and remained depressed after 10 min of recovery, consistent with low-frequency fatigue. Average EMG activity increased with time, but never exceeded 75% of the maximal, pretrial level despite task failure. EMG mean power frequency declined by 20-25% in all trials, suggesting reduced action potential conduction velocity at task failure. In contrast, the maximal evoked potential did not change significantly in any of the tasks, indicating that the EMG deficit at task failure was due largely to mechanisms proximal to the neuromuscular junction. Additional experiments using the interpolated twitch technique suggest that subjects can produce about 92% of the maximal evocable force with this muscle, which is not a large enough deficit to explain the entire shortfall in the EMG at task failure. These data show that the nervous system fails to fully activate the nasal dilator muscles during sustained, submaximal contractions; putative mechanisms are discussed.
• Fregosi, R. F., & Pilarski, J. Q. (2008). Prenatal nicotine exposure and development of nicotinic and fast amino acid-mediated neurotransmission in the control of breathing. Respiratory physiology & neurobiology, 164(1-2), 80-6.
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  There is mounting evidence that neonatal animals exposed to nicotine in the prenatal period exhibit a variety of anatomic and functional abnormalities that adversely affect their respiratory and cardiovascular control systems, but how nicotine causes these developmental alterations is unknown. The principle that guides our work is that PNE impairs the ability of nicotinic acetylcholine receptors (nAChRs) to modulate the pre-synaptic release of both inhibitory (particularly GABA) and excitatory (glutamate) neurotransmitters, leading to marked alterations in the density and/or function of receptors on the (post-synaptic) membrane of respiratory neurons. Such changes could lead to impaired ventilatory responses to sensory afferent stimulation, and altered breathing patterns, including central apneic events. In this brief review we summarize the work that lead to the development of this hypothesis, and introduce some new data that support and extend it.
• Fregosi, R., & Fregosi, R. F. (2008). Influence of tongue muscle contraction and dynamic airway pressure on velopharyngeal volume in the rat. Journal of applied physiology (Bethesda, Md. : 1985), 104(3).
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  The mammalian pharynx is a collapsible tube that narrows during inspiration as transmural pressure becomes negative. The velopharynx (VP), which lies posterior to the soft palate, is considered to be one of the most collapsible pharyngeal regions. I tested the hypothesis that negative transmural pressure would narrow the VP, and that electrical stimulation of extrinsic tongue muscles would reverse this effect. Pressure (-6, -3, 3, and 6 cmH2O) was applied to the isolated pharyngeal airway of anesthetized rats that were positioned in a 4.7-T MRI scanner. The volume of eight axial slices encompassing the length of the VP was computed at each level of pressure, with and without bilateral hypoglossal nerve stimulation (0.1-ms pulse, one-third maximum force, 80 Hz). Negative pressure narrowed the VP, and either whole hypoglossal nerve stimulation (coactivation of protrudor and retractor muscles) or medial nerve branch stimulation (independent activation of tongue protrudor muscles) reversed this effect, with the greatest impact in the caudal one-third of the VP. The dilating effects of medial branch stimulation were slightly larger than whole nerve stimulation. Positive pressure dilated the VP, but tongue muscle contraction did not cause further dilation under these conditions. I conclude that the narrowest and most collapsible segment of the rat pharynx is in the caudal VP, posterior to the tip of the soft palate. Either coactivation of protrudor and retractor muscles or independent contraction of protrudor muscles caused dilation of this region, but the latter was slightly more effective.
• Fregosi, R., Luo, Z., McMullen, N. T., Costy-Bennett, S., & Fregosi, R. F. (2007). Prenatal nicotine exposure alters glycinergic and GABAergic control of respiratory frequency in the neonatal rat brainstem-spinal cord preparation. Respiratory physiology & neurobiology, 157(2-3).
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  Bath application of GABA-A receptor agonists in neonatal rat brainstem-spinal cord preparations (BSSC) reduces respiratory frequency, an effect that is enhanced by prenatal nicotine exposure. Here we test the hypothesis that these effects can be reproduced by microinjection of GABAergic and glycinergic agonists into the pre-Botzinger complex region (PBC). We recorded the activity of phrenic motor axons from the fourth cervical ventral root in 1-3 days old BSSC that were exposed to either nicotine (6 mg/(kg day)) or saline prenatally. Microinjection of glycine or muscimol into the PBC caused abrupt, reversible apnea in all experiments. Apnea duration with glycine averaged 50.3+/-5 s in saline-exposed (N=12), and 95.7+/-9.9 s in nicotine-exposed (N=12) neonates (P
• Fregosi, R., Van Zutphen, C., Janssen, P., Hassan, M., Cabrera, R., Bailey, E. F., & Fregosi, R. F. (2007). Regional velopharyngeal compliance in the rat: influence of tongue muscle contraction. NMR in biomedicine, 20(7).
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  The velopharynx is the most collapsible segment of the upper airway in patients with obstructive sleep apnea. However, we do not know if velopharyngeal compliance is uniform throughout its length, or if compliance is modified by contraction of upper airway muscles. We tested the hypothesis that rostral and caudal velopharyngeal (VP) compliance differs, and that tongue muscle contraction reduces compliance. High-resolution MR images of the VP were made at nasopharyngeal pressures ranging from -9 to 9 cmH(2)O in anesthetized rats. Images were obtained twice at each pressure, once with and once without bilateral hypoglossal nerve stimulation. The volume of the caudal and rostral VP was computed at each pressure. The caudal VP was significantly (P = 0.0058) more compliant than the rostral VP, but electrical stimulation of the tongue muscles did not change compliance. VP critical pressure (Pcrit; pressure at zero airway volume) averaged -25.2 and -12.1 cmH(2)O in the rostral and caudal VP, respectively (P < 0.0001). Coactivation of tongue protrudor and retractor muscles or contraction of protrudor muscles alone dilated the VP and made Pcrit more negative (P < 0.0001), but only in the caudal VP. In the rat, the caudal VP is more collapsible than the rostral VP, and either coactivation of tongue protrudor and retractor muscles or contraction of protrudor muscles alone makes this region more difficult to close. Thus, tongue muscle contraction protects the caudal VP, which appears to be a particularly vulnerable segment of the nasopharyngeal airway. With suitable modification, the methods described here, including tongue muscle stimulation at different pharyngeal pressures, may be appropriate for experiments in human subjects.
• Iizuka, M., & Fregosi, R. F. (2007). Influence of hypercapnic acidosis and hypoxia on abdominal expiratory nerve activity in the rat. Respiratory physiology & neurobiology, 157(2-3), 196-205.
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  We studied the influence of hypercapnic acidosis and hypoxia on the neural drive to abdominal muscles in anesthetized and decerebrate rats; this information is unavailable despite widespread use of the rat as an experimental model in respiratory physiology and neurobiology. To minimize confounding influences from receptors in the lungs and chest wall, the animals were vagotomized, paralyzed and mechanically ventilated, and electrical activity was recorded from abdominal muscle nerves. In anesthetized and decerebrate rats, both stimuli evoked steady, low amplitude expiratory discharge that persisted throughout the expiratory phase (E-all activity), but was inhibited during inspiration. We also observed late expiratory, high-amplitude bursts (E2 activity) superimposed on this steady activity, but only at the highest levels of respiratory drive. Hypoxia enhanced abdominal motor activity transiently, whereas hypercapnic acidosis caused a sustained increase in activity. Thus, both hypercapnic acidosis and hypoxia activate abdominal muscle motoneurons in the absence of phasic afferent inputs.
• Fregosi, R. F., Quan, S. F., Morgan, W. L., Goodwin, J. L., Cabrera, R., Shareif, I., Fridel, K. W., & Bootzin, R. R. (2006). Pharyngeal critical pressure in children with mild sleep-disordered breathing. Journal of applied physiology (Bethesda, Md. : 1985), 101(3), 734-9.
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  There is evidence that narrowing or collapse of the pharynx can contribute to obstructive sleep-disordered breathing (SDB) in adults and children. However, studies in children have focused on those with relatively severe SDB who generally were recruited from sleep clinics. It is unclear whether children with mild SDB who primarily have hypopneas, and not frank apnea, also have more collapsible airways. We estimated airway collapsibility in 10 control subjects (9.4 +/- 0.5 yr old; 1.9 +/- 0.2 hypopneas/h) and 7 children with mild SDB (10.6 +/- 0.5 yr old; 11.5 +/- 0.1 hypopneas/h) during stable, non-rapid eye movement sleep. None of the subjects had clinically significant enlargement of the tonsils or adenoids, nor had any undergone previous tonsillectomy or adenoidectomy. Airway collapsibility was measured by brief (2-breath duration) and sudden reductions in pharyngeal pressure by connecting the breathing mask to a negative pressure source. Negative pressure applications ranging from -1 to -20 cmH(2)O were randomly applied in each subject while respiratory airflow and mask pressure were measured. Flow-pressure curves were constructed for each subject, and the x-intercept gave the pressure at zero flow, the so-called critical pressure of the upper airway (Pcrit). Pcrit was significantly higher in children with SDB than in controls (-10.8 +/- 2.8 vs. -15.7 +/- 1.2 cmH(2)O; P < 0.05). There were no significant differences in the slopes of the pressure-flow relations or in baseline airflow resistance. These data support the concept that intrinsic pharyngeal collapsibility contributes to mild SDB in children.
• Fregosi, R., Bailey, E. F., & Fregosi, R. F. (2006). Modulation of upper airway muscle activities by bronchopulmonary afferents. Journal of applied physiology (Bethesda, Md. : 1985), 101(2).
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  Here we review the influence of bronchopulmonary receptors (slowly and rapidly adapting pulmonary stretch receptors, and pulmonary/bronchial C-fiber receptors) on respiratory-related motor output to upper airway muscles acting on the larynx, tongue, and hyoid arch. Review of the literature shows that all muscles in all three regions are profoundly inhibited by lung inflation, which excites slowly adapting pulmonary stretch receptors. This widespread coactivation includes the recruitment of muscles that have opposing mechanical actions, suggesting that the stiffness of upper airway muscles is highly regulated. A profound lack of information on the modulation of upper airway muscles by rapidly adapting receptors and bronchopulmonary C-fiber receptors prohibits formulation of a conclusive opinion as to their actions and underscores an urgent need for new studies in this area. The preponderance of the data support the view that discharge arising in slowly adapting pulmonary stretch receptors plays an important role in the initiation of the widespread and highly coordinated recruitment of laryngeal, tongue, and hyoid muscles during airway obstruction.
• Fregosi, R., Bailey, E. F., Huang, Y., & Fregosi, R. F. (2006). Anatomic consequences of intrinsic tongue muscle activation. Journal of applied physiology (Bethesda, Md. : 1985), 101(5).
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  We recently showed respiratory-related coactivation of both extrinsic and intrinsic tongue muscles in the rat. Here, we test the hypothesis that intrinsic tongue muscles contribute importantly to changes in velopharyngeal airway volume. Spontaneously breathing anesthetized rats were placed in a MRI scanner. A catheter was placed in the hypopharynx and connected to a pressure source. Axial and sagittal images of the velopharyngeal airway were obtained, and the volume of each image was computed at airway pressures ranging from +5.0 to -5.0 cm H2O. We obtained images in the hypoglossal intact animal (i.e., coactivation of intrinsic and extrinsic tongue muscles) and after selective denervation of the intrinsic tongue muscles, with and without electrical stimulation. Denervation of the intrinsic tongue muscles reduced velopharyngeal airway volume at atmospheric and positive airway pressures. Electrical stimulation of the intact hypoglossal nerve increased velopharyngeal airway volume; however, when stimulation was repeated after selective denervation of the intrinsic tongue muscles, the increase in velopharyngeal airway volume was significantly attenuated. These findings support our working hypothesis that intrinsic tongue muscles play a critical role in modulating upper airway patency.
• Fregosi, R. F., & Quan, S. F. (2005). MRI of pharyngeal airway in children with sleep-disordered breathing. Journal of applied physiology (Bethesda, Md. : 1985), 99(6), 2470; author reply 2470.
• Fregosi, R., Bailey, E. F., Janssen, P. L., & Fregosi, R. F. (2005). PO2-dependent changes in intrinsic and extrinsic tongue muscle activities in the rat. American journal of respiratory and critical care medicine, 171(12).
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  Historically, respiratory-related research in sleep apnea has focused exclusively on the extrinsic tongue muscles (i.e., genioglossus, hyoglossus, and styloglossus). Until recently, the respiratory control and function of intrinsic tongue muscles (i.e., inferior and superior longitudinalis, transverses, and verticalis), which comprise the bulk of the tongue, were unknown.
• Fregosi, R., John, J., Bailey, E. F., & Fregosi, R. F. (2005). Respiratory-related discharge of genioglossus muscle motor units. American journal of respiratory and critical care medicine, 172(10).
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  Little is known about the respiratory-related discharge properties of motor units driving any of the eight muscles that control the movement, shape, and stiffness of the mammalian tongue.
• Bailey, E. F., & Fregosi, R. F. (2004). Coordination of intrinsic and extrinsic tongue muscles during spontaneous breathing in the rat. Journal of applied physiology (Bethesda, Md. : 1985), 96(2), 440-9.
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  The muscular-hydrostat model of tongue function proposes a constant interaction of extrinsic (external bony attachment, insertion into base of tongue) and intrinsic (origin and insertion within the tongue) tongue muscles in all tongue movements (Kier WM and Smith KK. Zool J Linn Soc 83: 207-324, 1985). Yet, research that examines the respiratory-related effects of tongue function in mammals continues to focus almost exclusively on the respiratory control and function of the extrinsic tongue protrusor muscle, the genioglossus muscle. The respiratory control and function of the intrinsic tongue muscles are unknown. Our purpose was to determine whether intrinsic tongue muscles have a respiration-related activity pattern and whether intrinsic tongue muscles are coactivated with extrinsic tongue muscles in response to respiratory-related sensory stimuli. Esophageal pressure and electromyographic (EMG) activity of an extrinsic tongue muscle (hyoglossus), an intrinsic tongue muscle (superior longitudinal), and an external intercostal muscle were studied in anesthetized, tracheotomized, spontaneously breathing rats. Mean inspiratory EMG activity was compared at five levels of inspired CO2. Intrinsic tongue muscles were often quiescent during eupnea but active during hypercapnia, whereas extrinsic tongue muscles were active in both eupnea and hypercapnia. During hypercapnia, the activities of the airway muscles were largely coincident, although the onset of extrinsic muscle activity generally preceded the onset of intrinsic muscle activation. Our findings provide evidence, in an in vivo rodent preparation, of respiratory modulation of motoneurons supplying intrinsic tongue muscles. Distinctions noted between intrinsic and extrinsic activities could be due to differences in motoneuron properties or the central, respiration-related control of each motoneuron population.
• Fregosi, R. F., Luo, Z., & Iizuka, M. (2004). GABAA receptors mediate postnatal depression of respiratory frequency by barbiturates. Respiratory physiology & neurobiology, 140(3), 219-30.
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  We tested the hypothesis that barbiturates depress respiratory motor output by actions on the GABAA receptor. We examined the influence of pentobarbital sodium on nerve activity recorded from a fourth cervical (C4) ventral root (phrenic motoneuron output) in the in vitro brainstem-spinal cord preparation of neonatal rats aged 1-3 days. Bath application of pentobarbital slowed the respiratory rhythm but this effect could be reversed by drug washout or by simultaneous application of 8 microM bicuculline methiodide, a GABAA receptor antagonist. Pentobarbital up to a concentration of 80 microM (or 20 mg/l) did not change the magnitude of C4 nerve bursts. The GABAA receptor agonist muscimol evoked similar changes. The results support the hypothesis that respiratory depression by barbiturates is due to GABAA receptor-mediated inhibition, with the principal effects on rhythm generation. In the light of recent studies suggesting that GABAA receptors may be excitatory in the early neonatal period, we examined postnatal changes in the GABAergic slowing of respiratory rhythm. Stimulation of GABAA receptors slowed respiratory rhythm from the first postnatal day, with no change in efficacy over the first 3 days of life.
• Fregosi, R. F., Quan, S. F., Jackson, A. C., Kaemingk, K. L., Morgan, W. J., Goodwin, J. L., Reeder, J. C., Cabrera, R. K., & Antonio, E. (2004). Ventilatory drive and the apnea-hypopnea index in six-to-twelve year old children. BMC pulmonary medicine, 4, 4.
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  We tested the hypothesis that ventilatory drive in hypoxia and hypercapnia is inversely correlated with the number of hypopneas and obstructive apneas per hour of sleep (obstructive apnea hypopnea index, OAHI) in children.
• Fregosi, R., & Fregosi, R. F. (2004). Castrating the respiratory controller. The Journal of physiology, 561(Pt 2).
• Fregosi, R., Holm, P., Sattler, A., & Fregosi, R. F. (2004). Endurance training of respiratory muscles improves cycling performance in fit young cyclists. BMC physiology, 4.
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  Whether or not isolated endurance training of the respiratory muscles improves whole-body endurance exercise performance is controversial, with some studies reporting enhancements of 50% or more, and others reporting no change. Twenty fit (VO2 max 56.0 ml/kg/min), experienced cyclists were randomly assigned to three groups. The experimental group (n = 10) trained their respiratory muscles via 20, 45 min sessions of hyperpnea. The placebo group (n = 4) underwent "sham" training (20, 5 min sessions), and the control group (n = 6) did no training.
• Fregosi, R., Huang, Y., Brown, A. R., Costy-Bennett, S., Luo, Z., & Fregosi, R. F. (2004). Influence of prenatal nicotine exposure on postnatal development of breathing pattern. Respiratory physiology & neurobiology, 143(1).
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  To determine if prenatal nicotine exposure alters the postnatal development of the ventilatory pattern and the frequency and duration of apneas, we recorded respiratory airflow with head-out body plethysmography in awake neonates on postnatal days 1, 2, 6, 10, 14, and 18. Data from 12 nicotine-exposed animals were compared with data from 12 saline-exposed animals. Nicotine (6 mg/kg of nicotine tartrate per day) or saline exposure was induced by osmotic minipumps that were implanted subdermally on the fifth day of gestation in Sprague-Dawley Dams. Although both saline- and nicotine-exposed pups gained weight at the same rate throughout the studies, there were subtle differences in ventilatory indices between the two groups. Nicotine-exposed animals had a significantly higher breathing frequency on day 10, and a lower tidal volume on days 14 and 18. Although ventilation tended to be lower in the nicotine-exposed animals, the difference was not significant. There was a significantly higher frequency of apneas in the nicotine-exposed compared with the saline-exposed animals on postnatal days 1 and 2, but the apnea duration did not differ between the groups. No apneas were observed in any of the animals after the sixth postnatal day. Prenatal nicotine exposure is associated with a greater incidence of apneas on the first two postnatal days, and then an altered breathing pattern that manifests at a later stage of development.
• Fregosi, R., Luo, Z., Costy-Bennett, S., & Fregosi, R. F. (2004). Prenatal nicotine exposure increases the strength of GABA(A) receptor-mediated inhibition of respiratory rhythm in neonatal rats. The Journal of physiology, 561(Pt 2).
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  Infants born to mothers that smoke while pregnant have a relatively high incidence of central respiratory control abnormalities. Recent studies have shown that prenatal nicotine exposure increases GABA release and the frequency of GABAergic currents, leading to an up-regulation of GABA(A) receptors in central neurones. Activation of GABA(A) receptors inhibits ventilatory activity, with intense activation causing apnoea. These observations lead us to hypothesize that prenatal nicotine exposure alters GABAergic control of respiratory motor pattern in the early neonatal period. Osmotic minipumps were implanted in pregnant Sprague-Dawley rats on the fifth day of gestation, and filled with nicotine (6 mg kg(-1) day(-1), 2.5 microl h(-1)) or physiological saline (2.5 microl h(-1)). Brainstem-spinal cord preparations from 1- to 3-day-old neonates were studied under in vitro conditions. Electrical activity was recorded from the fourth cervical ventral root (C4 VR), which contains the axons of phrenic motoneurones. Bath application of GABA(A) receptor agonists muscimol (250 microM) or pentobarbital sodium (60 microM) to the brainstem led to consistent, reversible and significant reductions in C4 VR burst frequency. In saline-exposed animals, frequency (bursts min(-1)) fell from 6.8 +/- 0.4 to a nadir of 2.8 +/- 0.5 with muscimol, and from 6.5 +/- 0.3 to a nadir of 2.9 +/- 0.3 for pentobarbital; in nicotine-exposed animals, frequency fell from 6.3 +/- 0.4 to 1.0 +/- 0.4 with muscimol and from 6.4 +/- 0.2 to 1.7 +/- 0.4 with pentobarbital (P < 0.05 in all cases). The decrease in C4 VR frequency was significantly greater in nicotine-exposed compared to saline-exposed preparations with both muscimol and pentobarbital (P < 0.001 for both). There were no changes in the amplitude of C4 VR bursts under any condition. The GABA(A) receptor antagonist bicuculline methiodide (8 microM) did not change C4 VR frequency or amplitude in either group, although it was effective in reversing the effects of muscimol. These experiments demonstrate that prenatal nicotine exposure alters the GABAergic regulation of respiratory rhythm in a reduced preparation. The results may lead to a better understanding of the perturbed breathing pattern observed in neonates that are exposed to nicotine in utero.
• Goodwin, J. L., Kaemingk, K. L., Fregosi, R. F., Rosen, G. M., Morgan, W. J., Smith, T., & Quan, S. F. (2004). Parasomnias and sleep disordered breathing in Caucasian and Hispanic children - the Tucson children's assessment of sleep apnea study. BMC medicine, 2, 14.
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  Recent studies in children have demonstrated that frequent occurrence of parasomnias is related to increased sleep disruption, mental disorders, physical harm, sleep disordered breathing, and parental duress. Although there have been several cross-sectional and clinical studies of parasomnias in children, there have been no large, population-based studies using full polysomnography to examine the association between parasomnias and sleep disordered breathing. The Tucson Children's Assessment of Sleep Apnea study is a community-based cohort study designed to investigate the prevalence and correlates of objectively measured sleep disordered breathing (SDB) in pre-adolescent children six to 11 years of age. This paper characterizes the relationships between parasomnias and SDB with its associated symptoms in these children.
• Fregosi, R. F., Quan, S. F., Kaemingk, K. L., Morgan, W. J., Goodwin, J. L., Cabrera, R., & Gmitro, A. (2003). Sleep-disordered breathing, pharyngeal size and soft tissue anatomy in children. Journal of applied physiology (Bethesda, Md. : 1985), 95(5), 2030-8.
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  We tested the hypothesis that pharyngeal geometry and soft tissue dimensions correlate with the severity of sleep-disordered breathing. Magnetic resonance images of the pharynx were obtained in 18 awake children, 7-12 yr of age, with obstructive apnea-hypopnea index (OAHI) values ranging from 1.81 to 24.2 events/h. Subjects were divided into low-OAHI (n = 9) and high-OAHI (n = 9) groups [2.8 +/- 0.7 and 13.5 +/- 4.9 (SD) P < 0.001]. The OAHI correlated positively with the size of the tonsils (r2 = 0.42, P = 0.024) and soft palate (r2 = 0.33, P = 0.049) and inversely with the volume of the oropharyx (r2 = 0.42, P = 0.038). The narrowest point in the pharyngeal airway was smaller in the high-compared with the low-OAHI group (4.4 +/- 1.2 vs. 6.0 +/- 1.3 mm; P = 0.024), and this point was in the retropalatal airway in all but two subjects. The airway cross-sectional area (CSA)-airway length relation showed that the high-OAHI group had a narrower retropapatal airway than the low-OAHI group, particularly in the retropalatal region where the soft palate, adenoids, and tonsils overlap (P = 0.001). The "retropalatal air space," which we defined as the ratio of the retropalatal airway CSA to the CSA of the soft palate, correlated inversely with the OAHI (r2 = 0.49, P = 0.001). We conclude that 7- to 12-yr-old children with a narrow retropalatal air space have significantly more apneas and hypopneas during sleep compared with children with relatively unobstructed retropalatal airways.
• Fregosi, R., Bailey, E. F., & Fregosi, R. F. (2003). Pressure-volume behaviour of the rat upper airway: effects of tongue muscle activation. The Journal of physiology, 548(Pt 2).
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  Our hypothesis was that the simultaneous activation of tongue protrudor and retractor muscles (co-activation) would constrict and stiffen the pharyngeal airway more than the independent activation of tongue protrudor muscles. Upper airway stiffness was determined by injecting known volumes of air into the sealed pharyngeal airway of the anaesthetized rat while measuring nasal pressure under control (no-stimulus) and stimulus conditions (volume paired with hypoglossal (XII) nerve stimulation). Stimulation of the whole XII nerves (co-activation) or the medial XII branches (protrudor activation) effected similar increases in total pharyngeal airway stiffness. Importantly, co-activation produced volume compression (airway narrowing) at large airway volumes (P < 0.05), but had no effect on airway dimension at low airway volumes. In comparison, protrudor activation resulted in significant volume expansion (airway dilatation) at low airway volumes and airway narrowing at high airway volumes (P < 0.05). In conclusion, both co-activation and independent protrudor muscle activation increase airway stiffness. However, their effects on airway size are complex and depend on the condition of the airway at the time of activation.
• Goodwin, J. L., Kaemingk, K. L., Fregosi, R. F., Rosen, G. M., Morgan, W. J., Sherrill, D. L., & Quan, S. F. (2003). Clinical outcomes associated with sleep-disordered breathing in Caucasian and Hispanic children--the Tucson Children's Assessment of Sleep Apnea study (TuCASA). Sleep, 26(5), 587-91.
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  This report describes clinical outcomes and threshold levels of respiratory disturbance index (RDI) associated with sleep-disordered breathing in children participating in the Tucson Children's Assessment of Sleep Apnea study.
• Kaemingk, K. L., Pasvogel, A. E., Goodwin, J. L., Mulvaney, S. A., Martinez, F., Enright, P. L., Rosen, G. M., Morgan, W. J., Fregosi, R. F., & Quan, S. F. (2003). Learning in children and sleep disordered breathing: findings of the Tucson Children's Assessment of Sleep Apnea (tuCASA) prospective cohort study. Journal of the International Neuropsychological Society : JINS, 9(7), 1016-26.
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  We examined the relationship between nocturnal respiratory disturbance and learning and compared learning in children with and without nocturnal respiratory disturbance. Subjects were 149 participants in a prospective cohort study examining sleep in children ages 6-12: The Tucson Children's Assessment of Sleep Apnea study (TuCASA). Sleep was assessed via home polysomnography. Intelligence, learning and memory, and academic achievement were assessed. Parents rated attention. Group comparisons were used to test the hypothesis that the group with an apnea/hypopnea index (AHI) of 5 or more (n = 77) would have weaker performance than the group with AHI less than 5 (n = 72). The group with AHI of 5 or more had weaker learning and memory though differences between groups decreased when arousals were taken into account. There was a greater percentage of Stage 1 sleep in the AHI 5 or more group, and Stage 1 percentage was negatively related to learning and memory in the sample (n = 149). There were negative relationships between AHI and immediate recall, Full Scale IQ, Performance IQ, and math achievement. Hypoxemia was associated with lower Performance IQ. Thus, findings suggest that nocturnal respiratory disturbance is associated with decreased learning in otherwise healthy children, that sleep fragmentation adversely impacts learning and memory, and that hypoxemia adversely influences nonverbal skills.
• Quan, S. F., Goodwin, J. L., Babar, S. I., Kaemingk, K. L., Enright, P. L., Rosen, G. M., Fregosi, R. F., & Morgan, W. J. (2003). Sleep architecture in normal Caucasian and Hispanic children aged 6-11 years recorded during unattended home polysomnography: experience from the Tucson Children's Assessment of Sleep Apnea Study (TuCASA). Sleep medicine, 4(1), 13-9.
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  To obtain normative sleep architecture data from unattended home polysomnography in Caucasian and Hispanic children aged 6-11 years.
• Fregosi, R., Abraham, K. A., Feingold, H., Fuller, D. D., Jenkins, M., Mateika, J. H., & Fregosi, R. F. (2002). Respiratory-related activation of human abdominal muscles during exercise. The Journal of physiology, 541(Pt 2).
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  We tested the hypothesis that abdominal muscles are active during the expiratory phase of the respiratory cycle during exercise. Electromyographic (EMG) activities of external oblique and rectus abdominis muscles were recorded during incremental exercise to exhaustion and during 30 min of constant work rate exercise at an intensity of 85 % of the peak oxygen consumption rate (V(O(2))). High amplitude intramuscular EMG activities of both abdominal muscles could be evoked with postural manoeuvres in all subjects. During cycling, respiratory-related activity of the external obliques was evoked in four of seven subjects, whereas rectus abdominis activity was observed in six of the seven subjects. We measured only the activity that was confined exclusively to the expiratory phase of the respiratory cycle. Expiratory activity of both muscles increased with exercise intensity, although peak values averaged only 10-20 or 20-40 % of the peak activity (obtained during maximal, voluntary expiratory efforts) for the external oblique and rectus abdominis muscles, respectively. To estimate how much of the recorded abdominal muscle activity was supporting leg movements during exercise, we compared the activity at the very end of incremental exercise to that recorded during the first five respiratory cycles after the abrupt cessation of exercise, when ventilation was still very high. Although external oblique activity was reduced after exercise stopped, clear expiratory activity remained. Rectus abdominis activity remained high after exercise cessation, showing a gradual decline that approximated the decline in ventilation. During constant work rate exercise, EMG activities increased to 40-50 and 5-10 % of peak in rectus and external oblique muscles, respectively, and then plateaued for the remainder of the bout in spite of a continual upward drift in (V(O(2))) and pulmonary ventilation. Linear regression analysis showed that the rise in respiratory-related expiratory muscle activity during progressive intensity exercise was significantly correlated with ventilation, although weakly. In constant work rate exercise, expiratory EMG activities increased, but the changes were highly variable and did not change as a function of exercise time, even though ventilation drifted significantly with time. These experiments suggest that abdominal muscles play a role in regulating the ventilatory response to progressive intensity bicycle exercise, although some of the observed activity may support postural adjustments or limb movements. The contribution of abdominal muscles to ventilation during constant work rate exercise is variable, and expiratory activity does not 'drift' significantly with time.
• Fregosi, R., DelloRusso, C., Khurana, N., Rankin, L., Sullivan, J., & Fregosi, R. F. (2002). Mechanisms of force failure during repetitive maximal efforts in a human upper airway muscle. Muscle & nerve, 26(1).
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  The upper airway respiratory muscles play an important role in the regulation of airway resistance, but surprisingly little is known about their contractile properties and endurance performance. We developed a technique that allows measurement of force and the electromyogram (EMG) of human nasal dilator muscles (NDMs). Endurance performance was quantified by measuring NDM "flaring" force and EMG activity as healthy human subjects performed 10 s maximal voluntary contractions (MVCs), separated by 10 s rest, until the area under the force curve fell to 50% MVC (the time limit of the fatigue task, Tlim), which was reached in 34.2 +/- 3.1 contractions (685.0 +/- 62.3 s). EMG activity was unchanged except at Tlim, where it averaged 78.7 +/- 3.6% of pretest activity (P < 0.01). M-wave amplitude did not change, suggesting that neuromuscular propagation was not impaired. MVC force increased to 80% of the pretest level within 10 min of recovery but twitch force failed to recover, suggesting low-frequency fatigue. The data suggest that a failure of the nervous system to excite muscle could explain at most only a small fraction of the NDM force loss during an intermittent fatigue task, and then only at Tlim. Thus, the majority of the force failure during this task is due to impairment of mechanisms that reside within the muscle fibers.
• Bailey, E. F., Jones, C. L., Reeder, J. C., Fuller, D. D., & Fregosi, R. F. (2001). Effect of pulmonary stretch receptor feedback and CO(2) on upper airway and respiratory pump muscle activity in the rat. The Journal of physiology, 532(Pt 2), 525-34.
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  1. Our purpose was to examine the effects of chemoreceptor stimulation and lung inflation on neural drive to tongue protrudor and retractor muscles in the rat. 2. Inspiratory flow, tidal volume, transpulmonary pressure, compliance and electromyographic (EMG) activity of genioglossus (GG), hyoglossus (HG) and inspiratory intercostal (IIC) muscles were studied in 11 anaesthetized, tracheotomized and spontaneously breathing rats. Mean EMG activity during inspiration was compared with mean EMG activity during an occluded inspiration, at each of five levels of inspired CO(2) (0, 3, 6, 9 and 12 %). 3. Lung inflation suppressed EMG activity in all muscles, with the effect on both tongue muscles exceeding that of the intercostal muscles. Static elevations of end-expiratory lung volume evoked by 2 cmH(2)O positive end-expiratory pressure (PEEP) had no effect on tongue muscle activity. 4. Despite increasing inspiratory flow, tidal volume and transpulmonary pressure, the inhibition of tongue muscle activity by lung inflation diminished as arterial PCO2 (P(a),CO(2)) increased. 5. The onset of tongue muscle activity relative to the onset of IIC muscle activity advanced with increases in P(a),CO(2) but was unaffected by lung inflation. This suggests that hypoglossal and external intercostal motoneuron pools are controlled by different circuits or have different sensitivities to CO(2), lung inflation and/or anaesthetic agents. 6. We conclude that hypoglossal motoneuronal activity is more strongly influenced by chemoreceptor-mediated facilitation than by lung volume-mediated inhibition. Hypoglossal motoneurons driving tongue protrudor and retractor muscles respond identically to these stimuli.
• Brennick, M. J., Trouard, T. P., Gmitro, A. F., & Fregosi, R. F. (2001). MRI study of pharyngeal airway changes during stimulation of the hypoglossal nerve branches in rats. Journal of applied physiology (Bethesda, Md. : 1985), 90(4), 1373-84.
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  The medial branch (Med) of the hypoglossal nerve innervates the tongue protrudor muscles, whereas the lateral branch (Lat) innervates tongue retractor muscles. Our previous finding that pharyngeal airflow increased during either selective Med stimulation or whole hypoglossal nerve (WHL) stimulation (coactivation of protrudor and retractor muscles) led us to examine how WHL, Med, or Lat stimulation affected tongue movements and nasopharyngeal (NP) and oropharyngeal (OP) airway volume. Electrical stimulation of either WHL, Med, or Lat nerves was performed in anesthetized, tracheotomized rats while magnetic resonance images of the NP and OP were acquired (slice thickness 0.5 mm, in-plane resolution 0.25 mm). NP and OP volume was greater during WHL and Med stimulation vs. no stimulation (P < 0.05). Ventral tongue depression (measured in the midsagittal images) and OP volume were greater during Med stimulation than during WHL stimulation (P < 0.05). Lat stimulation did not alter NP volume (P = 0.39). Our finding that either WHL or Med stimulation dilates the NP and OP airways sheds new light on the control of pharyngeal airway caliber by extrinsic tongue muscles and may lead to new treatments for patients with obstructive sleep apnea.
• Goodwin, J. L., Enright, P. L., Kaemingk, K. L., Rosen, G. M., Morgan, W. J., Fregosi, R. F., & Quan, S. F. (2001). Feasibility of using unattended polysomnography in children for research--report of the Tucson Children's Assessment of Sleep Apnea study (TuCASA). Sleep, 24(8), 937-44.
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  The Tucson Children's Assessment of Sleep Apnea study (TuCASA) is designed to investigate the prevalence and correlates of objectively measured sleep-disordered breathing in pre-adolescent children. This paper documents the methods and feasibility of attaining quality unattended polysomnograms in the first 162 TuCASA children recruited.
• Fuller, D. D., & Fregosi, R. F. (2000). Fatiguing contractions of tongue protrudor and retractor muscles: influence of systemic hypoxia. Journal of applied physiology (Bethesda, Md. : 1985), 88(6), 2123-30.
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  The influence of systemic hypoxia on the endurance performance of tongue protrudor and retractor muscles was examined in anesthetized, ventilated rats. Tongue protrudor (genioglossus) or retractor (hyoglossus and styloglossus) muscles were activated via medial or lateral XII nerve branch stimulation (0.1-ms pulse; 40 Hz; 330-ms trains; 1 train/s). Maximal evoked potentials (M waves) of genioglossus and hyoglossus were monitored with electromyography. Fatigue tests were performed under normoxic and hypoxic (arterial PO(2) = 50 +/- 1 Torr) conditions in separate animals. The fatigue index (FI; %initial force) after 5 min of normoxic stimulation was 85 +/- 6 and 79 +/- 7% for tongue protrudor and retractor muscles, respectively; these values were significantly lower during hypoxia (protrudor FI = 52 +/- 10, retractor FI = 18 +/- 6%; P < 0.05). Protrudor and retractor muscle M-wave amplitude declined over the course of the hypoxic fatigue test but did not change during normoxia (P < 0.05). We conclude that hypoxia attenuates tongue protrudor and retractor muscle endurance performance; potential mechanisms include neuromuscular transmission failure and/or diminished sarcolemmal excitability.
• Janssen, P. L., & Fregosi, R. F. (2000). No evidence for long-term facilitation after episodic hypoxia in spontaneously breathing, anesthetized rats. Journal of applied physiology (Bethesda, Md. : 1985), 89(4), 1345-51.
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  Repeated electrical or hypoxic stimulation of peripheral chemoreceptors has been shown to cause a persistent poststimulus increase in respiratory motoneuron activity, termed long-term facilitation (LTF). LTF after episodic hypoxia has been demonstrated most consistently in anesthetized, vagotomized, paralyzed, artificially ventilated rats. Evidence for LTF in spontaneously breathing animals and humans after episodic hypoxia is equivocal and may have been influenced by the awake state of the subjects in these studies. The present study was designed to test the hypothesis that LTF is evoked in respiratory-related tongue muscle and inspiratory pump muscle activities after episodic hypoxia in 10 spontaneously breathing, anesthetized, vagotomized rats. The animals were exposed to three (5-min) episodes of isocapnic hypoxia, separated by 5 min of hyperoxia (50% inspired oxygen). Genioglossus, hyoglossus, and inspiratory intercostal EMG activities, along with respiratory-related tongue movements and esophageal pressure, were recorded before, during, and for 60 min after the end of episodic isocapnic hypoxia. We found no evidence for LTF in tongue muscle (genioglossus, hyoglossus) or inspiratory pump muscle (inspiratory intercostal) activities after episodic hypoxia. Rather, the primary poststimulus effect of episodic hypoxia was diminished respiratory frequency, which contributed to a reduction in ventilatory drive.
• Janssen, P. L., Williams, J. S., & Fregosi, R. F. (2000). Consequences of periodic augmented breaths on tongue muscle activities in hypoxic rats. Journal of applied physiology (Bethesda, Md. : 1985), 88(5), 1915-23.
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  This study was designed to investigate the influence of hypoxia-evoked augmented breaths (ABs) on respiratory-related tongue protrudor and retractor muscle activities and inspiratory pump muscle output. Genioglossus (GG) and hyoglossus (HG) electromyogram (EMG) activities and respiratory-related tongue movements were compared with peak esophageal pressure (Pes; negative change in pressure during inspiration) and minute Pes (Pes x respiratory frequency = Pes/min) before and after ABs evoked by sustained poikilocapnic, isocapnic, and hypercapnic hypoxia in spontaneously breathing, anesthetized rats. ABs evoked by poikilocapnic and isocapnic hypoxia triggered long-lasting (duration at least 10 respiratory cycles) reductions in GG and HG EMG activities and tongue movements relative to pre-AB levels, but Pes was reduced transiently (duration of
• Williams, J. S., Janssen, P. L., Fuller, D. D., & Fregosi, R. F. (2000). Influence of posture and breathing route on neural drive to upper airway dilator muscles during exercise. Journal of applied physiology (Bethesda, Md. : 1985), 89(2), 590-8.
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  Our purpose was to determine the influence of posture and breathing route on electromyographic (EMG) activities of nasal dilator (NDM) and genioglossus (GG) muscles during exercise. Nasal and oral airflow rates and EMG activities of the NDM and GG were recorded in 10 subjects at rest and during upright and supine incremental cycling exercise to exhaustion. EMG activities immediately before and after the switch from nasal to oronasal breathing were also determined for those subjects who demonstrated a clear switch point (n = 7). NDM and GG EMG activities were significantly correlated with increases in nasal, oral, and total ventilatory rates during exercise, and these relationships were not altered by posture. In both upright and supine exercise, NDM activity rose more sharply as a function of nasal inspired ventilation compared with total or oral inspired ventilation (P < 0.01), but GG activity showed no significant breathing-route dependence. Peak NDM integrated EMG activity decreased (P = 0.008), and peak GG integrated EMG activity increased (P = 0.032) coincident with the switch from nasal to oronasal breathing. In conclusion, 1) neural drive to NDM and GG increases as a function of exercise intensity, but the increase is unaltered by posture; 2) NDM activity is breathing-route dependent in steady-state exercise, but GG activity is not; and 3) drive to both muscles changes significantly at the switch point, but the change in GG activity is more variable and is often transient. This suggests that factors other than the breathing route dominate drive to the GG soon after the initial changes in the configuration of the oronasal airway are made.
• Fuller, D. D., Williams, J. S., Janssen, P. L., & Fregosi, R. F. (1999). Effect of co-activation of tongue protrudor and retractor muscles on tongue movements and pharyngeal airflow mechanics in the rat. The Journal of physiology, 519 Pt 2, 601-13.
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  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.
• Fuller, D., Mateika, J. H., & Fregosi, R. F. (1998). Co-activation of tongue protrudor and retractor muscles during chemoreceptor stimulation in the rat. The Journal of physiology, 507 ( Pt 1), 265-76.
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  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.
• Mateika, J. H., Essif, E. G., Dellorusso, C., & Fregosi, R. F. (1998). Contractile properties of human nasal dilator motor units. Journal of neurophysiology, 79(1), 371-8.
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  The technique of intramuscular microstimulation was used to activate facial nerve fibers while acquiring simultaneous twitch force measurements to measure the contractile properties and force-frequency responses of human nasal dilator (ND) motor units. Twitch force amplitude (TF), contraction time (CT), half-relaxation time (HRT), and the maximal rate of rise of force normalized to the peak force (maximum contraction rate, MCR) were recorded from 98 ND motor units in 37 subjects. The average CT, HRT, MCR, and TF were 47.9 +/- 1.8 ms, 42.6 +/- 2.1 ms, 28.6 +/- 1.8 s-1, and 1.06 +/- 0.1 mN, respectively. Neither CT nor HRT were significantly correlated with TF. The average CT and HRT were similar to values recorded for small muscles of the hand but were faster than the values recorded from human toe extensor motor units. However the lack of an association between twitch force and CT or HRT was similar to the findings obtained for both human hand and foot muscles. Force-frequency curves were recorded from eight ND motor units. The force produced by the eight motor units was recorded in response to stimuli delivered at 1, 5, 10, 15, 20, 25, 30, 35, and 40 Hz to assess force-frequency relationships. The mean twitch force of the eight motor units was 0.91 +/- 0.3 mN and the average tetanic force was 8.1 +/- 1.8 mN. Therefore the average twitch force was equal to 12.7% of the tetanic force. Fifty percent of the unit tetanic force was achieved at an average frequency of 16. 4 +/- 1.7 Hz, which is greater than the value recorded for human toe extensor motor units (9.6 Hz). Thus the force produced by the ND motor units was more sensitive to changes in discharge frequency over the range of approximately 10-30 Hz and less sensitive to changes in the range of 0-10 Hz because of their fast contractile properties. The mean slope of the regression lines that were fit to the steep portion of each force-frequency curve was 5.15 +/- 0.5% change in force/Hz. This value was greater than the slope measured for human toe extensor muscles (4.2% change in force/Hz). These observations suggest that force gradation by ND motor units is more sensitive to changes in stimulation frequency than human toe extensor motor units. We conclude that most ND motor units have fast contractile properties and that rate coding may play a significant role in the gradation of force produced by the ND muscle. Furthermore, the findings of this investigation have demonstrated that contractile speed and TF in a human facial muscle are not correlated. This supports previous findings obtained from human hand and foot muscles and suggests that there may be a fundamental difference in the contractile speed-twitch force relationship between many human muscles and most muscles of other mammals.
• Fregosi, R. F., & Fuller, D. D. (1997). Respiratory-related control of extrinsic tongue muscle activity. Respiration physiology, 110(2-3), 295-306.
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  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.
• Mateika, J. H., & Fregosi, R. F. (1997). Long-term facilitation of upper airway muscle activities in vagotomized and vagally intact cats. Journal of applied physiology (Bethesda, Md. : 1985), 82(2), 419-25.
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  The primary purpose of the present investigation was to determine whether long-term facilitation (LTF) of upper airway muscle activities occurs in vagotomized and vagally intact cats. Tidal volume and diaphragm, genioglossus, and nasal dilator muscle activities were recorded before, during, and after one carotid sinus nerve was stimulated five times with 2-min trains of constant current. Sixty minutes after stimulation, nasal dilator and genioglossus muscle activities were significantly greater than control in the vagotomized cats but not in the vagally intact cats. Tidal volume recorded from the vagotomized and vagally intact cats was significantly greater than control during the poststimulation period. In contrast, diaphragm activities were not significantly elevated in the poststimulation period in either group of animals. We conclude that 1) LTF of genioglossus and nasal dilator muscle activities can be evoked in vagotomized cats; 2) vagal mechanisms inhibit LTF in upper airway muscles; and 3) LTF can be evoked in accessory inspiratory muscles because LTF of inspired tidal volume was greater than LTF of diaphragm activity.
• Fuller, D., Sullivan, J., & Fregosi, R. F. (1996). Expiratory muscle endurance performance after exhaustive submaximal exercise. Journal of applied physiology (Bethesda, Md. : 1985), 80(5), 1495-502.
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  The aim of our study was to describe the endurance capacity of the expiratory muscles and to determine whether it is altered after exhaustive cycling exercise. Subjects performed repeated maximal expiratory efforts against a closed breathing valve, with and without prior exercise performed at a work rate that elicited 75% of the maximum ventilation rate. Each expiratory effort lasted 6 s, was separated by 10 s of rest, and was initiated from the end-expiratory lung volume. Endurance performance was assessed by measuring the decline in area under the pressure*time curve over 39 contractions. Prior exhaustive exercise attenuated the ability to generate and sustain maximal expiratory pressure (P = 0.013) and resulted in significant declines in the integrated electromyogram of the rectus abdominis (P = 0.005) and external oblique (P = 0.036) abdominal muscles. Each subject also performed a handgrip endurance task before and after exhaustive exercise on a separate day. Prior exercise had no effect on handgrip endurance performance, suggesting that the decline in expiratory muscle performance after exercise was not the result of reduced motivation. We conclude that the ability to maximally activate the abdominal expiratory muscles and to generate maximum expiratory pressure is impaired after exhaustive exercise. Declines in the surface integrated electromyogram despite maximal effort is consistent with findings in limb muscles and is thought to be due to a slowing of motoneuron firing rates or to neuromuscular transmission failure.
• Mateika, J. H., Essif, E., & Fregosi, R. F. (1996). Effect of hypoxia on abdominal motor unit activities in spontaneously breathing cats. Journal of applied physiology (Bethesda, Md. : 1985), 81(6), 2428-35.
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  These experiments were designed to examine the behavior of external oblique motor units in spontaneously breathing cats during hypoxia and to estimate the contribution of recruitment and rate coding to changes in the integrated external oblique electromyogram (iEMG). Motor unit activities in the external oblique muscle were identified while the cats expired against a positive end-expiratory pressure (PEEP) of 1-2.5 cmH2O. After localization of unit activity, PEEP was removed, and recordings were made continuously for 3-4 min during hyperoxia, normoxia, and hypoxia. A total of 35 single motor unit activities were recorded from 10 cats. At each level of fractional concentration of end-tidal O2, the motor unit activity was characterized by an abrupt increase in mean discharge frequency, at approximately 30% of expiratory time, which then continued to increase gradually or remained constant before declining abruptly at the end of expiration. The transition from hyperoxia to normoxia and hypoxia was accompanied by an increase in the number of active motor units (16 of 35, 20 of 35, and 29 of 35, respectively) and by an increase in the mean discharge frequency of those units active during hyperoxia. The changes in motor unit activity recorded during hypoxia were accompanied by a significant increase in the average peak amplitude of the abdominal iEMG. Linear regression analysis revealed that motor unit rate coding was responsible for close to 60% of the increase in peak iEMG amplitude. The changes in abdominal motor unit activity and the external oblique iEMG that occurred during hypoxia were abolished if the arterial PCO2 was allowed to fall. We conclude that external oblique motor units are activated during the latter two-thirds of expiration and that rate coding and recruitment contribute almost equally to the increase in expiratory muscle activity that occurs with hypoxia. In addition, the excitation of abdominal motor units during hypoxia is critically dependent on changes in CO2 and/or tidal volume.
• Sullivan, J., Fuller, D., & Fregosi, R. F. (1996). Control of nasal dilator muscle activities during exercise: role of nasopharyngeal afferents. Journal of applied physiology (Bethesda, Md. : 1985), 80(5), 1520-7.
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  Our primary aim was to determine whether reducing the activity of nasal airway receptors would influence drive to the nasal dilator muscles (NDMs) during exercise. We used lidocaine (2%) or nasal splints to diminish afferent airway receptor activity and measured the electromyogram (EMG) activity of the NDMs during incremental bicycle exercise in subjects who breathed nasally. NDM EMG activities increased as a function of exercise intensity but were not changed by lidocaine and were only slightly reduced by splinting. Similarly, neither intervention altered the normal decrease in NDM EMG activity associated with reductions in airway resistance evoked by He-O2 breathing. We also compared the NDM EMG response to exercise with that evoked by CO2 rebreathing at rest to determine whether the nature of the ventilatory stimulus influences drive to the NDMs; comparisons were made at constant levels of nasal inspired ventilation and, therefore, constant total ventilatory output. The increase in EMG activity was much higher during exercise compared with hyperoxic hypercapnia. In conclusion, 1) desensitizing the nasal airway does not alter NDM activity significantly during exercise and 2) exercise results in much greater increases in NDM activity compared with hypercapnia, indicating that different ventilatory stimuli can evoke more or less activation of upper airway motoneurons, even when comparisons are made at constant levels of total ventilatory output.
• Fregosi, R. F., & Lansing, R. W. (1995). Neural drive to nasal dilator muscles: influence of exercise intensity and oronasal flow partitioning. Journal of applied physiology (Bethesda, Md. : 1985), 79(4), 1330-7.
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  Our aim was to test the following hypotheses: 1) neural drive to the muscles of the alae nasi (AN) is proportional to nasal airflow and is independent of the overall level of central respiratory drive, and 2) the switch from nasal to oronasal breathing corresponds to the onset of marked flow turbulence in the nasal airway. Total and nasal inspired ventilation rates (VI) and the electromyogram (EMG) of the AN muscles were measured in seven subjects during progressive-intensity bicycling exercise. In separate experiments in six subjects the nasal VI corresponding to the transition from laminar to turbulent airflow was determined by measuring the pressure-flow relationship of the nasal airway with anterior rhinomanometry. Nasal VI accounted for 70 +/- 11% of total VI at rest and 27 +/- 8% (SE) at 90% of the maximal attainable power (max). Nasal VI and integrated AN EMG activities increased linearly with exercise intensity up to 60% of the max power, but both variables plateaued at this level even though total VI (and central respiratory drive) began to increase exponentially as exercise intensity increased to 90% max. The onset of the exponential rise in total VI was associated with a sharp increase in oral VI and with the onset of marked flow turbulence in the nasal airway. The results suggest that during incremental exercise 1) changes in AN EMG activities are highly correlated with changes in nasal VI, 2) turbulent flow in the nose may be the stimulus for the switch to oronasal breathing so that total pulmonary resistance is minimized, and 3) the correlation between nasal airflow and neural drive to the AN muscles is probably mediated by mechanisms that monitor airway resistance. Although these mechanisms were not identified, the most likely possibilities are receptors in the upper and/or lower airways that are sensitive to negative transmural pressure, or to effort sensations leading to greater corollary motor discharge to nasal dilator muscle motoneurons.
• Fuller, D., Sullivan, J., Essif, E., Personius, K., & Fregosi, R. F. (1995). Measurement of the EMG-force relationship in a human upper airway muscle. Journal of applied physiology (Bethesda, Md. : 1985), 79(1), 270-8.
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  The upper airway muscles play an important role in breathing, swallowing, and speaking, but little is known about the electromyogram (EMG)-force relationship of these muscles. We have measured the peak integrated EMG activity (iEMG) and force of human nasal dilator muscles (NDM) with a custom-designed headpiece that was attached via the forehead and upper lip. The headpiece contains a micromanipulator that holds a rod with a load cell mounted on its tip. The reproducibility of the force measurements was examined by measuring the lateral or "flaring" force of the NDM in multiple trials on two separate occasions in 13 subjects. For these studies the subjects were instructed to perform maximal voluntary contractions (MVCs). Test-retest reproducibility averaged 8.3% (coefficient of variation) for within-day comparisons and 13.7% between days. We also measured iEMG and NDM force during an incremental exercise test in nine of the subjects; they were instructed to breathe nasally throughout one 30-s epoch at rest and at each workload. The iEMG and force during peak exercise (175-275 W) averaged 81 +/- 26% (SD) MVC and 235 +/- 127 mN (approximately 75% MVC), respectively. The iEMG during incremental exercise was linearly related to the peak force (r = 0.90, P < 0.001). Contractile properties were measured in seven of the subjects by application of single supramaximal shocks (0.1-ms pulse) to the facial nerve. Twitch force averaged 9 +/- 6% MVC, and the time to peak force was 62 +/- 13 ms, which is considerably faster than that in human diaphragm or elbow flexors.(ABSTRACT TRUNCATED AT 250 WORDS)
• Halseth, A. E., Fogt, D. L., Fregosi, R. F., & Henriksen, E. J. (1995). Metabolic responses of rat respiratory muscles to voluntary exercise training. Journal of applied physiology (Bethesda, Md. : 1985), 79(3), 902-7.
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  Voluntary wheel running for 4 or 8 wk was used to assess whether a volitional training stimulus would induce adaptations in the oxidative capacity [citrate synthase activity (CS)], glucose phosphorylation capacity [hexokinase activity (HK)], and glucose transporter protein level (GLUT-4) of rat respiratory muscles. Running distances averaged approximately 10-13 km/day over the final 5 wk of training. Peak oxygen consumption by the trained animals was 17% greater (P < 0.05) than by age-matched sedentary control animals after 8 wk. CS, HK, and GLUT-4 in soleus and plantaris muscles all increased because of exercise training. CS increased in the rectus abdominis (+17%), external oblique (+28%), and internal oblique (+17%) but not in the costal or crural diaphragm after 4 wk of training. However, after 8 wk, CS in the costal diaphragm was 39% greater than control but was unchanged in the crural diaphragm. Whereas HK was significantly greater than control in the costal diaphragm (+18%) and rectus abdominis (+54%) after 4 wk, 8 wk of running were required for increases in HK in the external oblique (+17%) and internal oblique (+14%). HK in the crural diaphragm was not significantly altered by the exercise training. GLUT-4 did not change significantly in any of the respiratory muscles studied. These results indicate that significant adaptations in the glucose phosphorylation capacity and oxidative capacity of both inspiratory and expiratory muscles can take place in response to voluntary exercise. However, this same stimulus is not sufficient to cause an adaptive response in GLUT-4 protein level in these respiratory muscles.
• Fregosi, R. F. (1994). Changes in the neural drive to abdominal expiratory muscles in hemorrhagic hypotension. The American journal of physiology, 266(6 Pt 2), H2423-9.
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  The purpose of this study was to test the hypothesis that hemorrhage-induced hypotension increases the neural drive to the abdominal expiratory muscles in chloralose-urethan-anesthetized cats that are studied under conditions of constant arterial PCO2 (PaCO2) and hyperoxia. A secondary aim was to describe in detail the concomitant changes in inspired pulmonary ventilation (VI) and the pattern of breathing under these conditions. The rectified and integrated electromyogram (EMG) of the external oblique and rectus abdominis muscles and VI were recorded in moderate and severe hemorrhagic hypotension, leading to reductions in mean blood pressure of approximately 30 and 60%, respectively. The PaCO2 was prevented from falling, and the arterial PO2 was maintained at a hyperoxic level (> 200 mmHg) by adding CO2 and O2 to the inspired gas mixture. VI increased by 2.5- and 5-fold in moderate and severe hypotension (P < 0.05). The changes in VI were mediated exclusively by changes in tidal volume, indicating that the reflex did not alter the activity of respiratory rhythm-generating structures. The EMG of external oblique muscles averaged 2, 44, and 100% in control conditions and in moderate and severe hypotension, respectively; corresponding values in rectus abdominis muscles were 10, 28, and 100% (P < 0.05 for both muscles). Bilateral cervical vagotomy caused a one- to three-fold decrease in the ventilatory response to hemorrhage and abolished the increase in abdominal muscle EMG activities. In conclusion, hemorrhagic hypotension reflexly increases pulmonary ventilation and the neural drive to the abdominal muscles. The reflex is vagally mediated, but the location of the receptors was not identified.
• Fregosi, R. F. (1994). Influence of hypoxia and carotid sinus nerve stimulation on abdominal muscle activities in the cat. Journal of applied physiology (Bethesda, Md. : 1985), 76(2), 602-9.
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  Experiments were designed to test two hypotheses regarding the influence of isocapnic hypoxia on the expiratory activity of the abdominal muscles: 1) brain hypoxia attenuates the increased drive to the abdominal muscles that is elicited by hypoxic stimulation of peripheral chemoreceptor afferents, and 2) activation of the abdominal muscles in hypoxia requires vagal afferent feedback. The measurements included inspired ventilation (VI) and the electromyogram (EMG) of the external and internal oblique and transversus abdominis muscles in 12 supine cats that were anesthetized with chloralose (50 mg/kg) and breathed spontaneously. Changes in respiratory drive were evoked with isocapnic hypoxia or electrical stimulation of a carotid sinus nerve. Although both stimuli increased abdominal motor output, carotid sinus nerve stimulation evoked a significantly greater increase in the external and internal oblique EMG than hypoxia when comparisons were made at an equivalent level of VI. Neither stimulus changed the abdominal EMG significantly after bilateral cervical vagotomy. Separate experiments revealed that, at a given level of VI, hypercapnia evoked a significantly greater increase in abdominal activity than isocapnic hypoxia. The results suggest that the increased drive to the abdominal muscles elicited by stimulation of the peripheral and central chemoreceptors can be antagonized by an inhibitory input that is triggered by brain hypoxia. Moreover the decrease in expiratory motor activity often observed during hypoxia in vagotomized animals is due to the removal of an excitatory mechanism that is mediated by vagal afferent feedback.
• Fregosi, R. F., & Mitchell, G. S. (1994). Long-term facilitation of inspiratory intercostal nerve activity following carotid sinus nerve stimulation in cats. The Journal of physiology, 477 ( Pt 3), 469-79.
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  1. Repeated carotid sinus nerve (CSN) stimulation evokes a serotonin-dependent long-term facilitation (LTF) of phrenic nerve activity in cats. To determine whether CSN stimulation-evoked LTF is a general property of spinal inspiratory motoneurones, phrenic and inspiratory internal intercostal (IIC) nerve activities were recorded in nine cats (eight anaesthetized; one decerebrate), which were vagotomized, paralysed, thoracotomized and ventilated with O2; airway CO2 was controlled by means of of a servo-respirator. Baseline conditions were established by setting the arterial CO2 pressure (Pa,CO2) at approximately 2 mmHg above the threshold for IIC activity. One CSN was stimulated (3 times threshold, 25 Hz, 0.5 ms duration) with five (2 min) trains, each separated by 5 min. 2. The peak integrated phrenic activity was elevated by 33% whereas IIC activity was elevated by 226% above baseline, 90 min post-stimulation (P < 0.05). The results were similar when expressed as a percentage of the maximal neural activities (elicited by combined hypercapnia and CSN stimulation), although differences between the nerves were less pronounced. The burst frequency was not change following stimulation. 3. In five additional cats that were pretreated with the serotonin receptor antagonist, methysergide maleate (0.5-1 mg kg-1, I.V.), the CO2 thresholds of the phrenic (12 mmHg) and IIC nerves (22 mmHg) were increased (P < 0.05), and LTF could not be elicited in either neurogram. 4. Successive CSN stimulation episodes evoked a previously undescribed phenomenon. Although the peak integrated phrenic activity was unchanged (90-95% of maximal), IIC activity increased progressively during successive stimulus episodes (66-90% of maximal; P < 0.05). However, after methysergide treatment, the initial stimulus-evoked phrenic response decreased to 58% of maximal and both neurograms exhibited progressive augmentation of the stimulus-evoked response. As stimulus-evoked augmentation does not require serotonin, it is independent of LTF. 5. We conclude that CSN stimulation-evoked LTF of IIC activity exceeds that of phrenic activity. Since LTF requires the neuromodulator serotonin and is expressed predominantly by changes in burst pattern formation versus rhythm generation, serotonin may exert a greater influence on IIC relative to phrenic respiratory motor output. A unique mechanism is described whereby successive CSN stimulus episodes cause progressively increasing responses in both neurograms.
• Connel, D. C., & Fregosi, R. F. (1993). Influence of nasal airflow and resistance on nasal dilator muscle activities during exercise. Journal of applied physiology (Bethesda, Md. : 1985), 74(5), 2529-36.
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  Our purpose was to assess the separate effects of nasal airflow and resistance on the activity of the nasal dilator [alae nasi (AN)] muscles. Nasal airflow and the AN electromyogram were recorded at rest and during progressive-intensity exercise at 60, 120, and 150-180 W in 10 healthy subjects who breathed nasally under all conditions. The activity of the AN muscles increased linearly as a function of the increase in nasal minute ventilation evoked by progressive-intensity exercise (r = 0.99, P < 0.002). Reciprocal changes in nasal airflow and resistance were produced by surreptitious substitution of 12-15 breaths of 79% He-21% O2 for air at rest and during exercise. The switch to He-O2 decreased airway resistance (anterior rhinomanometry) by approximately 30% at rest and 40-60% during exercise. He-O2 did not change nasal flow or AN activities significantly under resting conditions. In contrast, He-O2 increased nasal flow and decreased the AN electromyogram by 25-50% during exercise (P < 0.05). The results suggest that AN muscle activities during nasal breathing are regulated by mechanisms that track airway resistance or the level of flow turbulence. The increase in AN activities during exercise probably helps ensure nasal airway patency in the face of the considerable collapsing pressures that prevail under these conditions.
• Fregosi, R. F., & Seals, D. R. (1993). Hypoxic potentiation of the ventilatory response to dynamic forearm exercise. Journal of applied physiology (Bethesda, Md. : 1985), 74(5), 2365-72.
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  The slope of the relationship between ventilation (VI) and O2 consumption, as derived in progressive-intensity exercise tests, is increased markedly by systemic hypoxia. The mechanisms underlying the hypoxic potentiation of the ventilatory response to exercise have not been established, partly because several factors that can increase respiratory drive (e.g., metabolic rate, cardiac output, circulating catecholamine levels) change significantly and simultaneously under these conditions. In an effort to avoid these confounding changes, we sought to determine whether hypoxia potentiates the ventilatory response to dynamic forearm exercise in humans. Forearm exercise increased the O2 consumption by only 80-90 ml/min; nevertheless, hypoxia resulted in a significant potentiation of VI that was mediated by a marked increase in breathing frequency. These observations led us to hypothesize that the hypoxic potentiation of VI is due to an exaggerated stimulation of chemosensitive afferent nerve endings within the exercising muscles ("muscle chemoreceptors"). We tested this hypothesis in separate experiments under conditions of forearm ischemia so that the stimulus to the muscle chemoreceptors in normoxic and hypoxic exercise would be the same. The magnitude of the change in VI evoked by hypoxic ischemic exercise was significantly greater than the sum of the separate changes evoked by normoxic ischemic exercise and hypoxic ischemic rest. We conclude that the combination of dynamic forearm exercise and hypoxia potentiates VI and that this effect is mediated by neural structures that govern respiratory frequency. Moreover the potentiated ventilatory response cannot be attributed to an exaggerated stimulation of intramuscular chemoreceptors.
• Stump, C. S., Woodman, C. R., Fregosi, R. F., & Tipton, C. M. (1993). Muscle glucose uptake in the rat after suspension with single hindlimb weight bearing. Journal of applied physiology (Bethesda, Md. : 1985), 74(5), 2072-8.
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  This study was designed to examine the effect of non-weight-bearing conditions and the systemic influences of simulated microgravity on rat hindlimb muscles. For this purpose, rats were suspended (SUS) in a head-down position (45 degrees) with the left hindlimb non-weight bearing (NWB) and the right hindlimb bearing 20% of presuspension body mass (WB). Weight bearing by the SUS-WB limb was accomplished by using a platform connected to a rod in sleeve, cable, and pulley apparatus to which weight could be added. Rats (250-325 g) were assigned to SUS or cage control (CC) conditions for 14 days. The angle between the foot and leg for SUS-WB and CC remained similar (20-30 degrees) throughout the experiment while the SUS-NWB hindlimbs extended to approximately 140 degrees by day 12. On day 14, the soleus, plantaris, and gastrocnemius muscles from the SUS-NWB limbs exhibited significantly lower (P < or = 0.05) masses than presuspension mass values (29, 11, and 21%, respectively). Weight bearing by the SUS-WB limbs prevented the loss of mass by these muscles. In separate groups of SUS and CC rats, 2-deoxyglucose uptake during hindlimb perfusion was significantly higher in both SUS-NWB and SUS-WB hindlimbs at 24,000 microU/ml of insulin compared with CC for all the muscles examined (21-80%). In addition, extracellular space (ml/g) was significantly greater in the soleus muscles from both the SUS-NWB and SUS-WB hindlimbs (64%) compared with CC muscles.(ABSTRACT TRUNCATED AT 250 WORDS)
• Fregosi, R. F., Hwang, J. C., Bartlett, D., & St John, W. M. (1992). Activity of abdominal muscle motoneurons during hypercapnia. Respiration physiology, 89(2), 179-94.
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  Our purpose was to examine the influence of hypercapnia on the activity of motoneurons innervating the transversus abdominis and internal oblique abdominal muscles, and of integrated phrenic and abdominal motor nerve activities. Studies were done in nine adult cats that were decerebrated, vagotomized, thoracotomized, paralyzed and ventilated mechanically. Of 42 motoneurons examined, 24 showed strong respiratory modulation (RM neurons), with the discharge confined primarily to the central expiratory period. The remaining 18 motoneurons discharged tonically, and failed to show respiratory modulation even at increased levels of central respiratory drive. Hyperoxic hypercapnia augmented the activities of the phrenic and abdominal nerves and increased the early expiratory discharge frequency of the RM neurons. The hypercapnia-induced increase in firing frequency during early expiration was accompanied by a corresponding decline in late expiration, and a virtual abolition of the inspiratory activity in the few neurons that discharged in this phase under normocapnic conditions. Finally, hypercapnia induced an increase in the number of spikes generated during each expiratory period in about half of the RM neurons, whereas the remaining cells showed a decrease. Thus, the increased peak activity of the integrated whole abdominal nerve burst with hypercapnia was brought about by a shift in the temporal pattern of motoneuron firing, or by an increase in the number of spikes generated during the expiratory period. The steep rate of rise and the pronounced early expiratory peak observed in the integrated abdominal nerve burst during hypercapnia in this preparation are consistent with the increase in motoneuron firing frequency during the early stages of the expiratory phase.
• Fregosi, R. F. (1991). Short-term potentiation of breathing in humans. Journal of applied physiology (Bethesda, Md. : 1985), 71(3), 892-9.
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  The purpose of this study was to determine if the increase in ventilation induced by hypoxic stimulation of the carotid bodies (CB) persists after cessation of the stimulus in humans. I reasoned that a short-term potentiation (STP) of breathing, sometimes called an "afterdischarge," could be unmasked by combining hypoxia with exercise, because ventilation increases synergistically under these conditions. Seven young healthy men performed mild bicycle exercise (30% peak power) while breathing O2 for 1.5 min ("control" state), and their CB were then stimulated by 1.5 min of hypoxic exercise (10% O2--balance N2). CB stimulation was then terminated by changing the inspirate back to O2 as exercise continued. Inspiratory and expiratory duration (TI and TE) and inspiratory flow and its time integral [tidal volume (VT)] were measured with a pneumotachometer. Inspired minute ventilation (VI) and mean inspiratory flow (VT/TI) declined exponentially after the cessation of CB stimulation, with first-order time constants of 28.6 +/- 6.7 and 24.6 +/- 1.6 (SD) s, respectively. The slow decay of VI was due primarily to potentiation of both TI and TE, although the effect on the latter predominated. Additional experiments in six subjects showed that brief intense CB stimulation with four to five breaths of N2 during mild exercise induced STP of similar magnitude to that observed in the hypoxic exercise experiments. Finally, the imposition of hyperoxia during air breathing exercise at a level of respiratory drive similar to that induced by the hypoxic exercise did not change VI significantly.(ABSTRACT TRUNCATED AT 250 WORDS)
• Seals, D. R., Johnson, D. G., & Fregosi, R. F. (1991). Hyperoxia lowers sympathetic activity at rest but not during exercise in humans. The American journal of physiology, 260(5 Pt 2), R873-8.
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  The primary aim of this study was to determine the influence of systemic hyperoxia on sympathetic nervous system behavior at rest and during submaximal exercise in humans. In seven healthy subjects (aged 19-31 yr) we measured postganglionic sympathetic nerve activity to skeletal muscle (MSNA) in the leg, antecubital venous norepinephrine concentrations, heart rate, and arterial blood pressure during normoxic rest (control) followed by 3- to 4-min periods of either hyperoxic (100% O2 breathing) rest, normoxic exercise (rhythmic handgrips at 50% of maximum force), or hyperoxic exercise. During exercise, isocapnia was maintained by adding CO2 to the inspirate as necessary. At rest, hyperoxia lowered MSNA burst frequency (12-42%) and total activity (6-42%) in all subjects; the average reductions were 25 and 23%, respectively (P less than 0.05 vs. control). Heart rate also decreased during hyperoxia (6 +/- 1 beats/min, P less than 0.05), but arterial blood pressure was not affected. During hyperoxic compared with normoxic exercise, there were no differences in the magnitudes of the increases in MSNA burst frequency or total activity, plasma norepinephrine concentrations, or mean arterial blood pressure. In contrast, the increase in heart rate during hyperoxic exercise (13 +/- 2 beats/min) was less than the increase during normoxic exercise (20 +/- 2 beats/min; P less than 0.05). We conclude that, in healthy humans, systemic hyperoxia 1) lowers efferent sympathetic nerve activity to skeletal muscle under resting conditions without altering venous norepinephrine concentrations and 2) has no obvious modulatory effect on the nonactive muscle sympathetic nerve adjustments to rhythmic exercise.
• Seals, D. R., Johnson, D. G., & Fregosi, R. F. (1991). Hypoxia potentiates exercise-induced sympathetic neural activation in humans. Journal of applied physiology (Bethesda, Md. : 1985), 71(3), 1032-40.
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  Our purpose was to test the hypothesis that hypoxia potentiates exercise-induced sympathetic neural activation in humans. In 15 young (20-30 yr) healthy subjects, lower leg muscle sympathetic nerve activity (MSNA, peroneal nerve; microneurography), venous plasma norepinephrine (PNE) concentrations, heart rate, and arterial blood pressure were measured at rest and in response to rhythmic handgrip exercise performed during normoxia or isocapnic hypoxia (inspired O2 concn of 10%). Study I (n = 7): Brief (3-4 min) hypoxia at rest did not alter MSNA, PNE, or arterial pressure but did induce tachycardia [17 +/- 3 (SE) beats/min; P less than 0.05]. During exercise at 50% of maximum, the increases in MSNA (346 +/- 81 vs. 207 +/- 14% of control), PNE (175 +/- 25 vs. 120 +/- 11% of control), and heart rate (36 +/- 2 vs. 20 +/- 2 beats/min) were greater during hypoxia than during normoxia (P less than 0.05), whereas the arterial pressure response was not different (26 +/- 4 vs. 25 +/- 4 mmHg). The increase in MSNA during hypoxic exercise also was greater than the simple sum of the separate responses to hypoxia and normoxic exercise (P less than 0.05). Study II (n = 8): In contrast to study I, during 2 min of exercise (30% max) performed under conditions of circulatory arrest and 2 min of postexercise circulatory arrest (local ischemia), the MSNA and PNE responses were similar during systemic hypoxia and normoxia. Arm ischemia without exercise had no influence on any variable during hypoxia or normoxia.(ABSTRACT TRUNCATED AT 250 WORDS)
• Dempsey, J. A., & Fregosi, R. F. (1985). Adaptability of the pulmonary system to changing metabolic requirements. The American journal of cardiology, 55(10), 59D-67D.
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  The conventional view of the healthy pulmonary system during exercise is of a very precise and mechanically efficient homeostatic regulator of ventilation and gas exchange occurring within the reserves of a near ideal architecture of the lung and chest wall. These regulatory and architectural limits may be exceeded in the healthy pulmonary system when extremely high levels of metabolic demand are needed. For example, arterial hypoxemia will often occur at exercise intensities demanding greater than 25 liters/min cardiac output. This may be due to inadequate red cell transit time in the pulmonary capillary bed whose blood volume has been maximally recruited, thereby resulting in alveolar-end-capillary oxygen disequilibrium. At these extreme levels of exercise the hyperventilatory response may be minimal (and clearly inadequate in terms of alveolar oxygenation) despite substantial and progressive metabolic acidosis or hypoxemia or both. This evidence of compromised ventilatory response and inadequate gas exchange in the highly fit human suggests that the pulmonary system may not be reasonably designed or adaptable (with long-term physical training) to the extreme demands imposed on gas transport by a truly adapted cardiovascular system.