Medullary Respiratory Neurons Research Articles

In vivo evidence for the cellular basis of central hypoventilation of Rett syndrome and pharmacological correction in the rat model.

Rett syndrome (RTT) is a neurodevelopmental disorder caused mostly by mutations in the MECP2 gene. RTT patients show periodical hypoventilation attacks. The breathing disorder contributing to the high incidence of sudden death is thought to be due to depressed central inspiratory (I) activity via unknown cellular processes. Demonstration of such processes may lead to targets for pharmacological control of the RTT-type hypoventilation. We performed in vivo recordings from medullary respiratory neurons on the RTT rat model. To our surprise, both I and expiratory (E) neurons in the ventral respiratory column (VRC) increased their firing activity in Mecp2-null rats with severe hypoventilation. These I neurons including E-I phase-spanning and other I neurons remained active during apneas. Consistent with enhanced central I drive, ectopic phrenic discharges during expiration as well as apnea were observed in the Mecp2-null rats. Considering the increased I neuronal firing and ectopic phrenic activity, the RTT-type hypoventilation does not seem to be caused by depression in central I activity, neither reduced medullary I premotor output. This as well as excessive E neuronal firing as shown in our previous studies suggests inadequate synaptic inhibition for phase transition. We found that the abnormal respiratory neuronal firing, ectopic phrenic discharge as well as RTT-type hypoventilation all can be corrected by enhancing GABAergic inhibition. More strikingly, Mecp2-null rats reaching humane endpoints with severe hypoventilation can be rescued by GABAergic augmentation. Thus, defective GABAergic inhibition among respiratory neurons is likely to play a role in the RTT-type hypoventilation, which can be effectively controlled with pharmacological agents.

Oxidative stress in the medullary respiratory neurons contributes to respiratory dysfunction in the 6-OHDA model of Parkinson's disease.

Parkinson's disease (PD) is associated with respiratory dysfunction. In the 6-OHDA rat model of PD this is seen as a reduction in respiratory frequency and minute ventilation during normoxia and hypercapnia stimulus. Respiratory dysfunction is caused by neuronal death of medullary respiratory nuclei in the 6-OHDA model of PD. Oxidative stress can be considered a strong candidate for neurodegeneration via miR-34c downregulation and pro-apoptotic signalling in respiratory neurons, preceding the functional impairment observed in the 6-OHDA model of PD. Parkinson's disease (PD) is a neurodegenerative disease caused by dopaminergic neuron death in the substantia nigra (SN). New evidence has revealed that this neurodegeneration is the result of complex interactions between genetic abnormalities, environmental toxins, mitochondrial dysfunction and disruption of the blood-brain barrier (BBB) in the SN. In addition to classic symptoms, PD patients also exhibit respiratory failure. Here, we investigated whether oxidative stress was associated with neurodegeneration in a respiratory group (RG) of 6-OHDA-treated rats, which act as a model of PD. We analysed how oxidative stress affected apoptotic signalling in the RG 30days after 6-OHDA treatment, shortly before commencement of breathing impairment (40days). After 30days, a dihydroethidium assay showed increased oxidative stress in the RG, anti-apoptotic signalling, as shown by an increase in p-Akt and BcL-2 and a decrease in Bax in the caudal aspect of the nucleus of the solitary tract (cNTS), and a decrease in p-p38 and Bax levels in the retrotrapezoid nucleus (RTN); pro-apoptotic signalling was indicated by a decrease in p-Akt and BcL-2 and an increase in Bax in the rostral ventral respiratory group (rVRG) and pre-Botzinger complex (preBotC). miR-34c, a known oxidative stress protector, was downregulated in 6-OHDA animals in the RC. After 40days of 6-OHDA, the NTS, rVRG, preBotC and RTN exhibited reduced NeuN immunoreactivity, no BBB disruption and an increase in thiobarbituric acid reactivity. We conclude that in the 6-OHDA model of PD, oxidative stress contributes to neurodegeneration in medullary respiratory neurons.

Distinct dampening effects of progesterone on the activity of nucleus tractus solitarii neurons in rat pups.

What is the central question of the study? Progesterone is considered a respiratory stimulant drug, but its effect on medullary respiratory neurons are poorly documented. We investigated whether progesterone alters spontaneous activity of neurons in the nucleus of the solitary tract (NTS). What is the main finding and its importance? In NTS neurons, progesterone decreases the action potential firing frequency in response to current injections and the amplitude of excitatory postsynaptic currents. Based on the established neuroprotective effect of progesterone against excitotoxicity resulting from insults, this inhibitory effect is likely to reflect inhibition of ion fluxes. These results are important because they further our understanding of the mechanisms underlying the diversity of respiratory effects of progesterone. Progesterone is known to stimulate breathing, but its actions on the respiratory control system have received limited attention. We addressed this issue at the cellular level by testing the hypothesis that progesterone augments excitatory currents at the level of the nucleus tractus solitarii (NTS). Medullary slices from juvenile male rats (14-17days of age) containing the commissural region of the NTS (NTScom) were incubated with progesterone (1μm) or vehicle (0.004% DMSO) for 60min. We performed whole-cell voltage-clamp recordings of spontaneous excitatory postsynaptic currents (EPSCs) in the NTScom and determined membrane properties by applying depolarizing current steps. In comparison to vehicle-treated cells, progesterone exposure attenuates the firing frequency response to current injection and reduces the EPSC amplitude without modifying the EPSC frequency or the basal membrane properties. These data do not support our hypothesis, because they indicate that incubation with progesterone attenuates intrinsic action potential generation and inhibits excitatory synaptic inputs in the NTS. Given that these results are more in line with the protective effect of progesterone against excitotoxicity resulting from various insults, we propose that progesterone acts via inhibition of ionic flux.

Firing properties of ventral medullary respiratory neurons in sino-aortic denervated rats.

What is the central question of this study? After sino-aortic denervation (SAD), rats present normal levels of mean arterial pressure (MAP), high MAP variability and changes in breathing. However, mechanisms involved in SAD-induced respiratory changes and their impact on the modulation of sympathetic activity remain unclear. Herein, we characterized the firing frequency of medullary respiratory neurons after SAD. What is the main finding and its importance? Sino-aortic denervation-induced prolonged inspiration was associated with a reduced interburst frequency of pre-inspiratory/inspiratory neurons and an increased long-term variability of late inspiratory neurons, but no changes were observed in the ramp-inspiratory and post-inspiratory neurons. This imbalance in the respiratory network might contribute to the modulation of sympathetic activity after SAD. In previous studies, we documented that after sino-aortic denervation (SAD) in rats there are significant changes in the breathing pattern, but no significant changes in sympathetic activity and mean arterial pressure compared with sham-operated rats. However, the neural mechanisms involved in the respiratory changes after SAD and the extent to which they might contribute to the observed normal sympathetic activity and mean arterial pressure remain unclear. Here, we hypothesized that after SAD, rats present with changes in the firing frequency of the ventral medullary inspiratory and post-inspiratory neurons. To test this hypothesis, male Wistar rats underwent SAD or sham surgery and 3days later were surgically prepared for an in situ experiment. The duration of inspiration significantly increased in SAD rats. During inspiration, the total firing frequency of ramp-inspiratory, pre-inspiratory/inspiratory and late-inspiratory neurons was not different between groups. During post-inspiration, the total firing frequency of post-inspiratory neurons was also not different between groups. Furthermore, the data demonstrate a reduced interburst frequency of pre-inspiratory/inspiratory neurons and an increased long-term variability of late-inspiratory neurons in SAD compared with sham-operated rats. These findings indicate that the SAD-induced prolongation of inspiration was not accompanied by alterations in the total firing frequency of the ventral medullary respiratory neurons, but it was associated with changes in the long-term variability of late-inspiratory neurons. We suggest that the timing imbalance in the respiratory network in SAD rats might contribute to the modulation of presympathetic neurons after removal of baroreceptor afferents.

Mecp2 Disruption in Rats Causes Reshaping in Firing Activity and Patterns of Brainstem Respiratory Neurons

People with Rett Syndrome (RTT), a neurodevelopmental disorder caused by mutations in the MECP2 gene, have breathing abnormalities manifested as periodical hypoventilation with compensatory hyperventilation, which are attributable to a high incidence of sudden death. Similar breathing abnormalities have been found in animal models with Mecp2 disruptions. Although RTT-type hypoventilation is believed to be due to depressed central inspiratory activity, whether this is true remains unknown. Here we show evidence for reshaping in firing activity and patterns of medullary respiratory neurons in RTT-type hypoventilation without evident depression in inspiratory neuronal activity. Experiments were performed in decerebrate rats in vivo. In Mecp2-null rats, abnormalities in breathing patterns were apparent in both decerebrate rats and awake animals, suggesting that RTT-type breathing abnormalities take place in the brainstem without forebrain input. In comparison to their wild-type counterparts, both inspiratory and expiratory neurons in Mecp2-null rats extended their firing duration, and fired more action potentials during each burst. No changes in inspiratory or expiratory neuronal distributions were found. Most inspiratory neurons started firing in the middle of expiration and changed their firing pattern to a phase-spanning type. The proportion of post-inspiratory neurons was reduced in the Mecp2-null rats. With the increased firing activity of both inspiratory and expiratory neurons in null rats, phrenic discharges shifted to a slow and deep breathing pattern. Thus, the RTT-type hypoventilation appears to result from reshaping of firing activity of both inspiratory and expiratory neurons without evident depression in central inspiratory activity.

Inputs to medullary respiratory neurons from a pontine subregion that controls breathing frequency

Neurons in a subregion of the medial parabrachial (PB) complex control expiratory duration (TE) and the inspiratory on-switch. To better understanding the underlying mechanisms, this study aimed to determine the types of medullary neurons in the rhythmogenic preBötzinger/Bötzinger Complex (preBötC/BötC) and adjacent areas that receive synaptic inputs from the PB subregion and whether these inputs are excitatory or inhibitory in nature. Highly localized electrical stimuli in the PB subregion combined with multi-electrode recordings from respiratory neurons and phrenic nerve activities were used to generate stimulus-to-spike event histograms to detect correlations in decerebrate, vagotomized dogs during isocapnic hyperoxia. Short-time scale correlations were found in 237/442 or ∼54% of the ventral respiratory column (VRC) neurons. Inhibition of E-neurons was ∼2.5X greater than for I-neurons, while Pre-I and I-neurons were excited. These findings indicate that the control of TE and the inspiratory on-switch by the PB subregion are mediated by a marked inhibition of BötC E-neurons combined with an excitation of I-neurons, especially pre-I neurons.

Sustained cell body reactivity and loss of NeuN in a subset of axotomized bulbospinal neurons after a chronic high cervical spinal cord injury

Following central nervous system lesion, the ability of injured axons to regrowth may depend on the level and duration of the injured cell body response (CBR). Therefore, to investigate whether axotomized brainstem neurons maintain a durable growth-competent state after spinal cord injury, we studied the effect of a chronic C2 hemisection in rats on the expression of various CBR markers involved in axon regeneration, such as c-Jun, ATF-3, HSP27, NO synthase (NOS), and also of the neural mature phenotype marker NeuN, in the bulbospinal respiratory neurons as compared to the gigantocellularis nucleus. Both at 7 and 30days post-lesion (DPL), c-Jun and HSP27 were present in, respectively, ~60 and ~20% of the axotomized respiratory neurons, whereas the apoptotic factor caspase 3 was not detected in these cells. NOS appeared belatedly, and it was detected in ~20% of the axotomized respiratory neurons at 30DPL. At 30DPL, these different CBR markers were strongly colocalized in a sub-population of axotomized respiratory neurons and also in a sub-population of injured neurons within the gigantocellularis nucleus. Such CBR was also accompanied by a sustained alteration of the neural mature phenotype, as indicated by a loss of NeuN immunoreactivity selectively in HSP27+ bulbospinal neurons at 7DPL and 30DPL. Altogether, this study shows that a subset of axotomized medullary respiratory neurons remains in a growth-competent state after a chronic injury, suggesting that they may play a preferential role in long-lasting respiratory neuroplasticity processes.

In vivo Studies of Brainstem Neurons with Respiratory Rhythm Activity in a Rat Model of Rett Syndrome

People with Rett Syndrome (RTT), a neurodevelopmental disorder caused by mutations of the MECP2 gene, show breathing abnormalities that are attributable to the high incidence of sudden death. To understand the underlying mechanisms for the breathing disorders, direct in vivo recordings from brainstem neurons with respiratory rhythm are needed, which is a challenge in mouse RTT models. The newly available rat model may allow such a study. Therefore, we performed electrophysiological recording from individual neurons of the medullary ventral respiratory group (VRG) in decerebrate Mecp2‐null and wild‐type (WT) rats. In both groups of rats, neurons with expiratory rhythm were recorded from the rostral and caudal VRG, and cells with inspiratory rhythm were found in between. Firing frequency of the inspiratory neurons was significantly lower in null rats than in WT rats, while expiratory neurons in both rostral and caudal VRG did not show such a difference. Firing durations of both expiratory and inspiratory neurons were elongated in null rats compared with the WT. The ratio of inspiratory to expiratory firing duration (Ti/Te) was lower in null rats. A large number of cells showed E‐I phase‐spanning in null rats. The null rats had a frequent breathing pattern of apneusis followed by apnea, during which inspiratory neurons showed increased firing frequency and caudal expiratory neurons showed decreased firing frequency, whereas no significant change was observed in rostral expiratory neurons. Although this breathing pattern of apneusis followed by apnea was also seen in WT rats, the firing frequency of either respiratory neurons did not seem to change. These findings suggest that Mecp2 disruption appears to cause a large scale of changes in firing activity and pattern of medullary respiratory neurons consistent with the broad range of breathing abnormalities seen in people with RTT and animal models.Support or Funding InformationThis work was supported by NIH grant R01‐NS‐073875 and International Rett Syndrome Foundation.

Oscillation patterns are enhanced and firing threshold is lowered in medullary respiratory neuron discharges by threshold doses of a μ-opioid receptor agonist.

μ-Opioid receptors are distributed widely in the brain stem respiratory network, and opioids with selectivity for μ-type receptors slow in vivo respiratory rhythm in lowest effective doses. Several studies have reported μ-opioid receptor effects on the three-phase rhythm of respiratory neurons, but there are until now no reports of opioid effects on oscillatory activity within respiratory discharges. In this study, effects of the μ-opioid receptor agonist fentanyl on spike train discharge properties of several different types of rhythm-modulating medullary respiratory neuron discharges were analyzed. Doses of fentanyl that were just sufficient for prolongation of discharges and slowing of the three-phase respiratory rhythm also produced pronounced enhancement of spike train properties. Oscillation and burst patterns detected by autocorrelation measurements were greatly enhanced, and interspike intervals were prolonged. Spike train properties under control conditions and after fentanyl were uniform within each experiment, but varied considerably between experiments, which might be related to variability in acid-base balance in the brain stem extracellular fluid. Discharge threshold was shifted to more negative levels of membrane potential. The effects on threshold are postulated to result from opioid-mediated disinhibition and postsynaptic enhancement of N-methyl-d- aspartate receptor current. Lowering of firing threshold, enhancement of spike train oscillations and bursts and prolongation of discharges by lowest effective doses of fentanyl could represent compensatory adjustments in the brain stem respiratory network to override opioid blunting of CO2/pH chemosensitivity.

Changes in the Firing Frequency of Medullary Respiratory Neurons of Juvenile Female Rats Exposed to Chronic Intermittent Hypoxia

Juvenile female rats exposed to chronic intermittent hypoxia (CIH) presented a significant reduction in the time of inspiration and increase in sympathetic activity during this phase of respiratory cycle, characterizing changes in the respiratory‐sympathetic coupling. There is evidence that the firing frequency of the pre‐sympathetic neurons is modulated by the respiratory neurons activity. In this context, we hypothesized that: in CIH‐female rats, the enhanced sympathetic activity during inspiration is due to the increased inspiratory neurons activity that modulates the pre‐sympathetic neurons firing frequency. We aimed to evaluate the respiratory neurons activity in the ventral surface of the medulla in juvenile female rats exposed to CIH. For this purpose, juvenile female rats (P20–21) were exposed to CIH (FiO2=6%) for 10 days and after this protocol we performed extracellular single unit recordings of medullary respiratory neurons using the working heart‐brainstem preparation (ventral approach). The respiratory neurons were identified and classified by their activity (firing frequency) in relation to the phrenic nerve activity. All experimental protocols were approved by the institutional ethical committee (#091/2013). Pre‐inspiratory/Inspiratory (Pre‐I/I) neurons of CIH‐female rats (n=12) presented a reduced firing frequency (39.3 ±5 vs 61.5 ±8 Hz, p=0.027) when compared to control (n=10). Ramp‐I, Post‐I and Aug‐E neurons presented no significant changes in their firing frequency in CIH‐female rats compared to control. We concluded that Pre‐I/I neurons presented a reduced firing frequency in CIH‐female rats, which may explain the reduced time of inspiration.Support or Funding InformationFAPESP, CAPES and CNPq.

Insights gleaned from pharmaco‐genetic dissection and modelling of cardio‐respiratory neural networks

This special issue consists of a series of papers based on presentations from a sym-posium we organised at the Experim-ental Biology meeting in 2014, San Diego, CA, USA, entitled ‘Insights gleaned from pharmaco-genetic dissection and modelling of cardio-respiratory neural networks’. This symposium followed two symposia we previously chaired in 2003 (‘Identifying genes and targets in cardiovascular autonomic pathophysiological states’) and 2008 (‘Smarter targeting of genes for cardiovascular genomics’). Our vision for these previous symposia and indeed the current one presented here was to showcase contemporary science with the very latest experimental technology. To this end we asked four speakers to demonstrate how rapidly changing technology is pushing their research into unexplored areas. Below we provide the original titles of these talks and a brief synopsis of what the papers report. First, Dr Abdala presented ‘Defining a role for glycinergic inhibition within the respiratory network using optogenetics’. Her article reviews the role of fast inhib-itory synaptic transmission for generation of a physiological respiratory pattern, while discussing some of the opportunities provided by emerging optogenetic approaches as high-resolution functional analysis tools of genotypically identified inhibitory respiratory neurones. It offers the reader helpful insight on merits and limitations of this new technology, especially in regard of the unique challenges provided by the respiratory circuitry (Abdala et al. 2015). Second, Dr Bub's presentation was entitled ‘Macro-micro imaging of cardiac-neural circuits in co-cultures from normal and diseased hearts’. A variety of life threatening cardiac arrhythmias have been attributed to formation of large scale patterns of excitation such as the formation and break up of spiral waves. These may be initiated by stimulation of myocytes by sympathetic neurons. An understa-nding of arrhythmogenesis can depend on measurement of complex activation patterns, which require methods for recording activity over long time periods. Optical mapping techniques, based on imaging fluorescence from voltage or ion sensitive dyes using fast cameras have emerged as vital tools for elucidating arrhythmogenic mechanisms at the organ and tissue levels. Gil Bub and Rebecca Burton (Bub & Burton, 2015) present their recent work on developing a simplified in vitro cell-culture model of neurally mediated arrhythmogenesis using dye-free high-speed optical mapping methods. This imaging technique allows us to measure experimentally important cardiac parameters at high speeds and simultaneously measure fine structural detail to relate structure and function. Additionally, this imaging modality also allows the investigator to utilise optogenetic tools to allow specific perturbations of different cells. Third, Dr David Moraes (Moraes et al. 2015) spoke on ‘Brainstem channelopathy of neurogenic hypertension’. A novel idea was presented showing that sympathetic overactivity in rodent models of neurogenic hypertension is dependent on alterations in the electrical excitability of medullary respiratory neurones that modulate sympatho-excitatory networks. The current dogma of increased excitability of pre-motor sympathetic neurones in neurogenic hypertension was therefore contested. Also presented were new data showing that the changes in the medullary respiratory neurone excitability were due to changes in ion channel expression – so-called ‘respiratory neurone channelopathy of hypertension’. The latter was dependent on carotid body input supporting the notion of targeting the carotid body as a potential novel therapeutic strategy for controlling hypertension. Finally, Dr Osborn presented a talk on ‘A new computational model for mechanisms of salt sensitive hypertension: a role for the sympathetic nervous system’. The causes of salt-sensitive hypertension are complex and mathematical models can elucidate potential mechanisms. The previous model of Guyton and Coleman was used to explain long-term control of arterial pressure. This classic model makes the assumption that sodium excretion is driven by renal perfusion pressure and that all forms of hypertension are due to a shift of the renal function curve to a higher operating pressure. Based on experimental data from numerous rodent models and development of a new mathematical model, which they term the ‘neurogenic model’, Osborn et al. (2015) re-examined the Guyton and Coleman model. This report discusses that salt-sensitive hypertension does not have to be due solely to renal dysfunction, as the Guyton–Coleman model predicts but could also result from sympathetic activation to the kidney.

Carotid body overactivity induces respiratory neurone channelopathy contributing to neurogenic hypertension.

Why sympathetic activity rises in neurogenic hypertension remains unknown. It has been postulated that changes in the electrical excitability of medullary pre-sympathetic neurones are the main causal mechanism for the development of sympathetic overactivity in experimental hypertension. Here we review recent data suggesting that enhanced sympathetic activity in neurogenic hypertension is, at least in part, dependent on alterations in the electrical excitability of medullary respiratory neurones and their central modulation of sympatho-excitatory networks. We also present results showing a critical role for carotid body tonicity in the aetiology of enhanced central respiratory modulation of sympathetic activity in neurogenic hypertension. We propose a novel hypothesis of respiratory neurone channelopathy induced by carotid body overactivity in neurogenic hypertension that may contribute to sympathetic excess. Moreover, our data support the notion of targeting the carotid body as a potential novel therapeutic approach for reducing sympathetic vasomotor tone in neurogenic hypertension.

Electrophysiological Properties of Ventral Medullary Respiratory Neurons in Rats Submitted to Chronic Intermittent Hypoxia

The central network controlling breathing exhibits plasticity in response to environmental challenges, such as chronic intermittent hypoxia (CIH). For a better understanding of the effects of CIH on the central generation of breathing, we combined single cell qRT-PCR, nerve and whole cell patch clamp recordings of Bötzinger Complex (BötC) and pre-Bötzinger Complex (pre-BötC) respiratory neurons in in situ preparations from control and CIH rats. In the BötC, we recorded 32 augmenting-expiratory (aug-E; glicinergic) and 24 post-inspiratory (post-I; 19 glicinergic and 5 glutamatergic) neurons, which present distinct intrinsic electrophysiological properties. CIH decreased the superior laryngeal nerve (SLN) post-inspiratory activity, by increasing potassium-mediated leak current in the post-I neurons, and increased the duration of expiration by changes in the aug-E neurons. In the pre-BötC, we recorded four types of neurons: pre-inspiratory (pre-I; 15 glutamatergic, 9 GABAergic and 11 glicinergic), early-inspiratory (19 glicinergic neurons), ramp-inspiratory (13 glicinergic and 9 GABAergic) and late-inspiratory (7 GABAergic and 9 glicinergic). CIH enhanced SLN pre-I activity, by increasing the inward riluzole-sensitive persistent sodium current in the intrinsically bursting glutamatergic pre-I neurons. These data show that CIH triggers a reconfiguration of synaptic interaction within the respiratory network in rats, which seems to be an adaptive process required for appropriated pulmonary ventilation and regulation of upper airway resistance. Support: FAPESP, CAPES and CNPQ.

Daily isoflurane exposure increases barbiturate insensitivity in medullary respiratory and cortical neurons via expression of ε-subunit containing GABA ARs.

The parameters governing GABAA receptor subtype expression patterns are not well understood, although significant shifts in subunit expression may support key physiological events. For example, the respiratory control network in pregnant rats becomes relatively insensitive to barbiturates due to increased expression of ε-subunit-containing GABAARs in the ventral respiratory column. We hypothesized that this plasticity may be a compensatory response to a chronic increase in inhibitory tone caused by increased central neurosteroid levels. Thus, we tested whether increased inhibitory tone was sufficient to induce ε-subunit upregulation on respiratory and cortical neurons in adult rats. Chronic intermittent increases in inhibitory tone in male and female rats was induced via daily 5-min exposures to 3% isoflurane. After 7d of treatment, phrenic burst frequency was less sensitive to barbiturate in isoflurane-treated male and female rats in vivo. Neurons in the ventral respiratory group and cortex were less sensitive to pentobarbital in vitro following 7d and 30d of intermittent isoflurane-exposure in both male and female rats. The pentobarbital insensitivity in 7d isoflurane-treated rats was reversible after another 7d. We hypothesize that increased inhibitory tone in the respiratory control network and cortex causes a compensatory increase in ε-subunit-containing GABAARs.

Stimulation of Respiratory Motor Output and Ventilation in a Murine Model of Pompe Disease by Ampakines.

Pompe disease results from a mutation in the acid α-glucosidase gene leading to lysosomal glycogen accumulation. Respiratory insufficiency is common, and the current U.S. Food and Drug Administration-approved treatment, enzyme replacement, has limited effectiveness. Ampakines are drugs that enhance α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor responses and can increase respiratory motor drive. Recent work indicates that respiratory motor drive can be blunted in Pompe disease, and thus pharmacologic stimulation of breathing may be beneficial. Using a murine Pompe model with the most severe clinical genotype (the Gaa(-/-) mouse), our primary objective was to test the hypothesis that ampakines can stimulate respiratory motor output and increase ventilation. Our second objective was to confirm that neuropathology was present in Pompe mouse medullary respiratory control neurons. The impact of ampakine CX717 on breathing was determined via phrenic and hypoglossal nerve recordings in anesthetized mice and whole-body plethysmography in unanesthetized mice. The medulla was examined using standard histological methods coupled with immunochemical markers of respiratory control neurons. Ampakine CX717 robustly increased phrenic and hypoglossal inspiratory bursting and reduced respiratory cycle variability in anesthetized Pompe mice, and it increased inspiratory tidal volume in unanesthetized Pompe mice. CX717 did not significantly alter these variables in wild-type mice. Medullary respiratory neurons showed extensive histopathology in Pompe mice. Ampakines stimulate respiratory neuromotor output and ventilation in Pompe mice, and therefore they have potential as an adjunctive therapy in Pompe disease.

電気生理学的アプローチによる喉頭基礎研究

Laryngeal movements including breathing, phonation, and airway protective reflexes, such as swallowing and coughing, are mainly controlled by the neuronal networks in the brain stem. To understand the mechanisms underlying these movements, the morphological and functional properties of the brain stem neurons that participate in the production of these movements should be investigated. We previously examined the functional roles of the brain stem neurons that contribute to the generation of laryngeal movements using electrophysiological and morphological procedures. The findings of two studies are introduced in the presentation. In the first study, we reported the projections of the swallow-related neurons in the swallowing central pattern generator in the medulla oblongata using a tracer injection in anesthetized paralyzed guinea pigs. This study indicated that the swallowrelated neurons were broadly distributed in the nucleus tractus solitarius and reticular formation, and that there appeared to be complex interconnections amongst the nucleus tractus solitarius, the reticular formation, and the cranial motor nuclei. The other study showed that the activity of the respiratory neurons in the ventrolateral medulla, in particular between the Bötzinger complex and the rostral ventral respiratory group, was altered during fictive vocalization, swallowing, and coughing in a type-specific manner, and thus supported the hypothesis that the medullary respiratory neurons are multifunctional and that the respiratory center in the network circuitry underlying these behaviors can be shared.

Specific Respiratory Neuron Types Have Increased Excitability That Drive Presympathetic Neurones in Neurogenic Hypertension

A major aspect of hypertension is excessive sympathetic activity but the reasons for this remain elusive. Sympathetic tone is increased in the spontaneously hypertensive (SH) rat reflecting, in part, enhanced respiratory-sympathetic coupling. We aimed to identify which respiratory cells might have altered properties. Using the working heart-brain stem preparation, we monitored simultaneously sympathetic and respiratory nerve activity in combination with intracellular recordings of physiologically characterized medullary presympathetic or respiratory neurons. In SH rats, respiratory modulation of both inspiratory and postinspiratory phases of sympathetic activity was larger relative to Wistar rats. An additional burst of sympathetic activity in the preinspiratory phase was also present in SH rats. After synaptic isolation of rostral medullary presympathetic neurons, there was no difference in their excitability compared with neurons in Wistar rats. Rather, both pre-Bötzinger preinspiratory and Bötzinger postinspiratory neurons had increased neuronal excitability in SH rats relative to Wistar rats; this was attributed to higher input resistance/reduced leak current in preinspiratory neurons and reduced calcium activated potassium conductance in postinspiratory neurons. Thus, the respiratory network of the SH rat is reconfigured to a pattern dominated by heightened excitability of preinspiratory and postinspiratory neurons. These neurons both provide augmented excitatory synaptic drive to rostral medullary presympathetic neurons contributing to excessive sympathetic nerve activity associated with hypertension in the in situ SH rat. Our data indicate selective modulation of potassium conductances in 2 subsets of respiratory neurons contributing to neurogenic hypertension.

Decreased spinal synaptic inputs to phrenic motor neurons elicit localized inactivity-induced phrenic motor facilitation

Phrenic motor neurons receive rhythmic synaptic inputs throughout life. Since even brief disruption in phrenic neural activity is detrimental to life, on-going neural activity may play a key role in shaping phrenic motor output. To test the hypothesis that spinal mechanisms sense and respond to reduced phrenic activity, anesthetized, ventilated rats received micro-injections of procaine in the C2 ventrolateral funiculus (VLF) to transiently (~30min) block axon conduction in bulbospinal axons from medullary respiratory neurons that innervate one phrenic motor pool; during procaine injections, contralateral phrenic neural activity was maintained. Once axon conduction resumed, a prolonged increase in phrenic burst amplitude was observed in the ipsilateral phrenic nerve, demonstrating inactivity-induced phrenic motor facilitation (iPMF). Inhibition of tumor necrosis factor alpha (TNFα) and atypical PKC (aPKC) activity in spinal segments containing the phrenic motor nucleus impaired ipsilateral iPMF, suggesting a key role for spinal TNFα and aPKC in iPMF following unilateral axon conduction block. A small phrenic burst amplitude facilitation was also observed contralateral to axon conduction block, indicating crossed spinal phrenic motor facilitation (csPMF). csPMF was independent of spinal TNFα and aPKC. Ipsilateral iPMF and csPMF following unilateral withdrawal of phrenic synaptic inputs were associated with proportional increases in phrenic responses to chemoreceptor stimulation (hypercapnia), suggesting iPMF and csPMF increase phrenic dynamic range. These data suggest that local, spinal mechanisms sense and respond to reduced synaptic inputs to phrenic motor neurons. We hypothesize that iPMF and csPMF may represent compensatory mechanisms that assure adequate motor output is maintained in a physiological system in which prolonged inactivity ends life.

The effects of doxapram on medullary respiratory neurones in brainstem–spinal cord preparations from newborn rats

Doxapram is the only dedicated respiratory stimulant used to aid recovery of breathing after major surgery. Doxapram acts on peripheral chemoreceptors and although the central action of doxapram has been suggested, its detailed neuronal mechanism is unknown. We assessed doxapram-induced changes in spontaneous cervical nerve (C4) inspiratory activity and the firing of action potentials in pre-inspiratory and inspiratory neurones in the medulla. Experiments were performed in neonatal rat brainstem-spinal cord preparations, which can produce respiratory rhythm for several hours under in vitro conditions. Doxapram application (for 15 min) increased the frequency and amplitude of C4 activity dose-dependently. Doxapram induced changes in the electrophysiological properties of pre-inspiratory and inspiratory neurones. Our results suggest that respiratory activity enhancement was likely to be induced via effects on the potassium channels of pre-inspiratory and inspiratory neurones and indicate the central actions of doxapram.

Activity of respiratory neurons in the rostral medulla during vocalization, swallowing, and coughing in guinea pigs

To examine the relationship between the neuronal networks underlying respiration and non-respiratory behaviors such as vocalization and airway defensive reflexes, we compared the activity of respiratory neurons in the ventrolateral medulla during breathing with that during non-respiratory behaviors including vocalization, swallowing, and coughing in guinea pigs. During fictive vocalization the activity of augmenting expiratory neurons ceased, whereas the other types of expiratory neurons did not show a consistent tendency of increasing or decreasing activity. All inspiratory neurons discharged in synchrony with the phrenic nerve activity. Most of the phase-spanning neurons were activated throughout the vocal phase. During fictive swallowing, many expiratory and inspiratory neurons were silent, whereas many phase-spanning neurons were activated. During fictive coughing, many expiratory neurons were activated during the expiratory phase of coughing. Most inspiratory neurons discharged in parallel with the phrenic nerve activity during coughing. Many phase-spanning neurons were activated during the expiratory phase of coughing. These findings indicate that the medullary respiratory neurons help shape respiratory muscle nerve activity not only during breathing but also during these non-respiratory behaviors, and thus suggest that at least some of the respiratory neurons are shared among the neuronal circuits underlying the generation of breathing and non-respiratory behaviors.