Inspiratory Neurons Research Articles

Bifurcation analysis of mixed bursting in the pre-Bötzinger complex

• We show through dynamical analysis that the neuron in pre-Bötzinger complexcan exhibit multiple mixed bursting patterns. • Mechanisms of the mixed bursting patterns are revealed. • Influences of certain parameters on the mixed bursting are explored. Bursting activities of certain neurons in the pre-Bötzinger complex (pre-BötC) of the mammalian brainstem have been extensively studied both experimentally and computationally due to their relationship with the inspiratory phase of respiration. Investigations shown that both the persistent sodium current ( I NaP ) and the intracellular Ca 2 + actived nonspecific cation ( I CAN ) can drive the bursting in the pre-BötC. The two mechanisms can act separately or jointly. Bursting evoked by the joint action of the two mechanisms is often called the somato-dendritic bursting. One special somato-dendritic bursting observed experimentally is the so called mixed bursting (MB) characterized by combining two or more different types of short bursts within each cycle of a periodic bursting solution. In this paper, we explore MB solutions in a single-compartment model of a pre-BötC inspiratory neuron. We show that the system can exhibit multiple patterns of MB that containing two or three different types of bursts in one periodic cycle. Using the methods of fast-slow decomposition and bifurcation analysis , we study the dynamical mechanisms underlying those MB, and investigate how the sodium conductance ( g Na ) and the potassium conductance ( g K ) affect MB patterns of the pre-BötC. Furthermore, we also elucidate the effect of parameters g K and g L on the number of somatic bursts in an MB solution.

Kinetic properties of persistent Na+ current orchestrate oscillatory bursting in respiratory neurons.

The rhythmic pattern of breathing depends on the pre-Bötzinger complex (preBötC) in the brainstem, a vital circuit that contains a population of neurons with intrinsic oscillatory bursting behavior. Here, we investigate the specific kinetic properties that enable voltage-gated sodium channels to establish oscillatory bursting in preBötC inspiratory neurons, which exhibit an unusually large persistent Na+ current (INaP). We first characterize the kinetics of INaP in neonatal rat brainstem slices in vitro, using whole-cell patch-clamp and computational modeling, and then test the contribution of INaP to rhythmic bursting in live neurons, using the dynamic clamp technique. We provide evidence that subthreshold activation, persistence at suprathreshold potentials, slow inactivation, and slow recovery from inactivation are kinetic features of INaP that regulate all aspects of intrinsic rhythmic bursting in preBötC neurons. The slow and cumulative inactivation of INaP during the burst active phase controls burst duration and termination, while the slow recovery from inactivation controls the duration of the interburst interval. To demonstrate this mechanism, we develop a Markov state model of INaP that explains a comprehensive set of voltage clamp data. By adding or subtracting a computer-generated INaP from a live neuron via dynamic clamp, we are able to convert nonbursters into intrinsic bursters, and vice versa. As a control, we test a model with inactivation features removed. Adding noninactivating INaP into nonbursters results in a pattern of random transitions between sustained firing and quiescence. The relative amplitude of INaP is the key factor that separates intrinsic bursters from nonbursters and can change the fraction of intrinsic bursters in the preBötC. INaP could thus be an important target for regulating network rhythmogenic properties.

Cell Type-Dependent Activation Sequence During Rhythmic Bursting in the PreBötzinger Complex in Respiratory Rhythmic Slices From Mice.

Spontaneous respiratory rhythmic burst activity can be preserved in the preBötzinger Complex (preBötC) of rodent medullary transverse slices. It is known, that the activation sequence of inspiratory neurons in the preBötC stochastically varies from cycle to cycle. To test whether the activation timing of an inspiratory neuron depends on its neurotransmitter, we performed calcium imaging of preBötC neurons using double-transgenic mice expressing EGFP in GlyT2+ neurons and tdTomato in GAD65+ neurons. Five types of inspiratory neurons were identified using the fluorescence protein expression and the maximum cross-correlation coefficient between neuronal calcium fluctuation and field potential. Regarding the activation sequence, irregular type putative excitatory (GlyT2-/GAD65-) neurons and irregular type glycinergic (GlyT2+/GAD65-) neurons tended to be activated early, while regular type putative excitatory neurons, regular type glycinergic neurons tended to be activated later. In conclusion, the different cell types define a general framework for the stochastically changing activation sequence of inspiratory neurons in the preBötC.

Neurons in the dorsomedial medulla contribute to swallow pattern generation: Evidence of inspiratory activity during swallow.

Active contraction of the diaphragm and other inspiratory pump muscles during swallow create a negative thoracic pressure to improve the movement of the bolus (food/liquid) into the esophagus. We tested the hypothesis that dorsomedial medullary inspiratory neurons, including the nucleus tractus solitarius (NTS, pre-motor to the phrenic) would be active during swallow induced by oral water infusion. We recorded neurons in the NTS and medial reticular formation in anesthetized spontaneously breathing cats, and induced swallow by injection of water into the oropharynx. Our results indicate that: 1) a majority of inspiratory cells in the dorsomedial medulla are active during swallow, 2) expiratory neurons are present in the medial reticular formation (deeper to the NTS) in unparalyzed cats and a majority of these cells decreased firing frequency during swallow. Our findings suggest that the dorsomedial medulla is a source of inspiratory motor drive during swallow and that a novel population of breathing-modulated neurons that also are modulated during swallowing exist in the medial reticular formation in unparalyzed animals.

Integration of the Proprioceptive and Central Inspiratory Inhibitory Afferent Inputs by Pontine Noradrenergic A5 Neurons in Rats.

Inhibitory afferent inputs to pontine A5 noradrenergic neurons (A5 NN) are not known, except partial baroreceptor input. In spontaneously breathing pentobarbital-anesthetized rats, we registered 35 A5 NN that were activated by hypoxia (100% N2, 10 sec) by more than 5 times in comparison with the background. Cooling of retrotrapezoid nucleus (15°C, 6 sec) completely blocked the motor inspiratory output and A5 NN discharge frequency increased (23/23) by more than 7 times in comparison with the background values. The beginning of A5 NN activation coincided with cessation of inspiratory activity. Short-term passive stretching of the shin muscles (1 sec, 100 g) caused BP drop and complete inhibition of A5 NN (12/12) activated by hypoxia. Inhibitory afferent inputs from proprioceptors and central inspiratory neurons that can limit A5 NN activity were demonstrated.

Acute exposure to zinc oxide nanoparticles critically disrupts operation of the respiratory neural network in neonatal rat

Due to their extremely small size that gives them unique physicochemical properties, nanoparticles (NPs) are used in the production of everyday materials. However, NPs can accumulate in body organs and could cause various diseases. Moreover, NPs that cross biological membranes such as the blood-brain barrier can aggregate in the brain and potentially produce neuronal damage. Although studies have reported the effects of diverse NPs on the bioelectrical properties of individual neurons, their potential influences on the operation of whole neuronal networks have not been documented. Here, we aimed to evaluate the effects of an acute exposure to zinc oxide (ZnO) NPs on the central neural networks responsible for mammalian respiratory rhythm generation. Using an isolated ex vivo brainstem-spinal cord preparation from neonatal rat in which the circuitry for the central respiratory command remained intact, we show that ZnO NPs accelerate, then profoundly disrupt respiratory-related activity produced by the pre-Bötzinger complex (preBötC) responsible for inspiratory rhythm generation. Consequently, a sudden and definitive cessation of respiratory-related activity occurs in ZnO NPs-exposed preparations. Part of these effects is related to zinc ions released from NPs. Using brainstem slice preparations containing the preBötC network, whole-cell patch-clamp recordings revealed that ZnO NPs depolarize preBötC inspiratory neurons and affect their bioelectrical properties by reducing the amplitude of action potentials, thereby leading to a depression of intra-network activity and the ultimate termination of respiratory rhythmogenesis. These findings support the conclusion that ZnO NPs may have deleterious effects on the central respiratory centers of newborn mammals.

The Expression of Galanin in the Parafacial Respiratory Group and its Effects on Respiration in Neonatal Rats

The inhibitory peptide galanin is expressed within the retrotrapezoidal nucleus (RTN) – a key central chemoreceptor site that also contains the active expiratory oscillator. It was previously reported that microinjection of galanin into pre-Bötzinger complex – containing the inspiratory oscillator – exerts inhibitory effects on inspiratory motor output and respiratory rhythm. In neonatal rats, the present study aimed to investigate: (1) expression of galanin within the parafacial respiratory group (pFRG), which overlaps anatomically and functionally with the adult RTN, and; (2) effects of galanin on respiratory rhythm using the in vitro brainstem-spinal cord preparation. We showed that 14 ± 2% of Phox2b-immunoreactive (ir) neurons in the parafacial region were also galanin-ir. Galanin peptide expression was confirmed within 3/9 CO2-sensitive, Phox2b-ir Pre-Inspiratory neurons (Pre-I) recorded in parafacial region. Bath application of galanin (0.1–0.2 µM): (1) decreased the duration of membrane depolarization in both Pre-I and inspiratory pFRG neurons, and; (2) decreased the number of C4 bursts that were associated with each burst in Pre-I neurons within the pFRG. In preparations showing episodic breathing at baseline, the respiratory patterning reverted to the ‘normal’ pattern of single, uniformly rhythmic C4 bursts (n = 10). In preparations with normal respiratory patterning at baseline, slowing of C4 rhythm (n = 7) resulted although rhythmic bursting in recorded Pre-I neurons remained unperturbed (n = 6). This study therefore demonstrates that galanin is expressed within the pFRG of neonatal rats, including neurons that are intrinsically chemosensitive. Overall the peptide has an inhibitory effect on inspiratory motor output, as previously shown in adults.

Evidence that the preBotzinger complex does not participate in cough rhythmogenesis in the anesthetized cat.

The nucleus tractus solitarius (NTS) participates in cough phase timing in the anesthetized cat. The preBotzinger complex (preBotC) is key for breathing rhythmogenesis, but the role of this area in regulating cough rhythmogenesis is still unknown. We hypothesized that excitation of neurons in the preBotC would modulate shorten the duration of the inspiratory cough, consistent with the effects during breathing. To test this hypothesis, we unilaterally microinjected 5 mM DL‐Homocysteic acid (DLH) into the preBotC in anesthetized, spontaneously breathing adult cats (n=7). In 3 of these animals, 15 mM DLH also was injected into the preBotC. Criteria for data inclusion were multi‐unit inspiratory neuron recording at the injection site, apneusis or tachypnea as a result of DLH injection, and histological verification of the injection site. We recorded esophageal pressure (EsPr) and electromygrams (EMG) from inspiratory (parasternal and diaphragm) and expiratory (internal oblique) muscles. Tracheobronchial cough was induced via mechanical stimulation of the intrathoracic trachea. DLH microinjection significantly altered the breathing response: breathing frequency increased (18.0±1.4 to 46.9±7.4 bpm) due to changes in both inspiratory time (TI − 1.04±0.5 to 0.75±0.76 s) and expiratory time (TE −2.34±0.19 to 0.63±0.16 s). Diaphragm EMG magnitude did not change significantly. Microinjection of DLH (5 mM) into the preBotC did not alter cough phase timing (cough frequency: 23.3±4.0 to 22.9±4.4 coughs per minute; TI − 1.27±0.13 to 1.31±0.10 s; TE1 −0.26±0.7 to 0.26±0.07 s; TE2 − 1.35±0.24 to 1.29±0.49 s). While the magnitude of cough pressures and diaphragm EMGs were not altered (EsPr – control 49.4±5.8 to DLH 35.7±5.2 ; EMG amplitude ‐ control 70.8±3.5 to DLH 65.1±6.5), cough expiratory motor drive was significantly lower (abdominal – control 62.1±5.5 to DLH 40.0±5.9%, p <0.04). Microinjections of 15 mM DLH into the preBotC eliminated cough. In the 3 animals that had both 5 and 15 mM DLH injections, there was a trend for a longer recovery time for cough with the higher concentration of DLH (5 mM – 33.8±10.4 s; 15 mM – 66.8±27.5 s). Unlike for breathing, excitation of neurons via DLH in the region of the preBotC does not affect phase timing for cough, although DLH appears to exhibit a dose‐dependent cough suppression.Support or Funding InformationSupported by NIH HL 103415 and 1OT20D001983.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits.

Transient receptor potential channel, TRPM4, the putative molecular substrate for Ca2+-activated nonselective cation current (ICAN), is hypothesized to generate bursting activity of pre-Bötzinger complex (pre-BötC) inspiratory neurons and critically contribute to respiratory rhythmogenesis. Another TRP channel, TRPC3, which mediates Na+/Ca2+ fluxes, may be involved in regulating Ca2+-related signaling, including affecting TRPM4/ICAN in respiratory pre-BötC neurons. However, TRPM4 and TRPC3 expression in pre-BötC inspiratory neurons and functional roles of these channels remain to be determined. By single-cell multiplex RT-PCR, we show mRNA expression for these channels in pre-BötC inspiratory neurons in rhythmically active medullary in vitro slices from neonatal rats and mice. Functional contributions were analyzed with pharmacological inhibitors of TRPM4 or TRPC3 in vitro as well as in mature rodent arterially perfused in situ brainstem–spinal cord preparations. Perturbations of respiratory circuit activity were also compared with those by a blocker of ICAN. Pharmacologically attenuating endogenous activation of TRPM4, TRPC3, or ICAN in vitro similarly reduced the amplitude of inspiratory motoneuronal activity without significant perturbations of inspiratory frequency or variability of the rhythm. Amplitude perturbations were correlated with reduced inspiratory glutamatergic pre-BötC neuronal activity, monitored by multicellular dynamic calcium imaging in vitro. In more intact circuits in situ, the reduction of pre-BötC and motoneuronal inspiratory activity amplitude was accompanied by reduced post-inspiratory motoneuronal activity, without disruption of rhythm generation. We conclude that endogenously activated TRPM4, which likely mediates ICAN, and TRPC3 channels in pre-BötC inspiratory neurons play fundamental roles in respiratory pattern formation but are not critically involved in respiratory rhythm generation.

Role of ventral medullary catecholaminergic neurons for respiratory modulation of sympathetic outflow in rats

Sympathetic activity displays rhythmic oscillations generated by brainstem inspiratory and expiratory neurons. Amplification of these rhythmic respiratory-related oscillations is observed in rats under enhanced central respiratory drive or during development of neurogenic hypertension. Herein, we evaluated the involvement of ventral medullary sympatho-excitatory catecholaminergic C1 neurons, using inhibitory Drosophila allatostatin receptors, for the enhanced expiratory-related oscillations in sympathetic activity in rats submitted to chronic intermittent hypoxia (CIH) and following activation of both peripheral (hypoxia) and central chemoreceptors (hypercapnia). Pharmacogenetic inhibition of C1 neurons bilaterally resulted in reductions of their firing frequency and amplitude of inspiratory-related sympathetic activity in rats in normocapnia, hypercapnia or after CIH. In contrast, hypercapnia or hypoxia-induced enhanced expiratory-related sympathetic oscillations were unaffected by C1 neuronal inhibition. Inhibition of C1 neurons also resulted in a significant fall in arterial pressure and heart rate that was similar in magnitude between normotensive and CIH hypertensive rats, but basal arterial pressure in CIH rats remained higher compared to controls. C1 neurons play a key role in regulating inspiratory modulation of sympathetic activity and arterial pressure in both normotensive and CIH hypertensive rats, but they are not involved in the enhanced late-expiratory-related sympathetic activity triggered by activation of peripheral or central chemoreceptors.

Carotid chemoreceptors tune breathing via multipath routing: reticular chain and loop operations supported by parallel spike train correlations.

We tested the hypothesis that carotid chemoreceptors tune breathing through parallel circuit paths that target distinct elements of an inspiratory neuron chain in the ventral respiratory column (VRC). Microelectrode arrays were used to monitor neuronal spike trains simultaneously in the VRC, peri-nucleus tractus solitarius (p-NTS)-medial medulla, the dorsal parafacial region of the lateral tegmental field (FTL-pF), and medullary raphe nuclei together with phrenic nerve activity during selective stimulation of carotid chemoreceptors or transient hypoxia in 19 decerebrate, neuromuscularly blocked, and artificially ventilated cats. Of 994 neurons tested, 56% had a significant change in firing rate. A total of 33,422 cell pairs were evaluated for signs of functional interaction; 63% of chemoresponsive neurons were elements of at least one pair with correlational signatures indicative of paucisynaptic relationships. We detected evidence for postinspiratory neuron inhibition of rostral VRC I-Driver (pre-Bötzinger) neurons, an interaction predicted to modulate breathing frequency, and for reciprocal excitation between chemoresponsive p-NTS neurons and more downstream VRC inspiratory neurons for control of breathing depth. Chemoresponsive pericolumnar tonic expiratory neurons, proposed to amplify inspiratory drive by disinhibition, were correlationally linked to afferent and efferent "chains" of chemoresponsive neurons extending to all monitored regions. The chains included coordinated clusters of chemoresponsive FTL-pF neurons with functional links to widespread medullary sites involved in the control of breathing. The results support long-standing concepts on brain stem network architecture and a circuit model for peripheral chemoreceptor modulation of breathing with multiple circuit loops and chains tuned by tegmental field neurons with quasi-periodic discharge patterns. NEW & NOTEWORTHY We tested the long-standing hypothesis that carotid chemoreceptors tune the frequency and depth of breathing through parallel circuit operations targeting the ventral respiratory column. Responses to stimulation of the chemoreceptors and identified functional connectivity support differential tuning of inspiratory neuron burst duration and firing rate and a model of brain stem network architecture incorporating tonic expiratory "hub" neurons regulated by convergent neuronal chains and loops through rostral lateral tegmental field neurons with quasi-periodic discharge patterns.

A V0 core neuronal circuit for inspiration

Breathing in mammals relies on permanent rhythmic and bilaterally synchronized contractions of inspiratory pump muscles. These motor drives emerge from interactions between critical sets of brainstem neurons whose origins and synaptic ordered organization remain obscure. Here, we show, using a virus-based transsynaptic tracing strategy from the diaphragm muscle in the mouse, that the principal inspiratory premotor neurons share V0 identity with, and are connected by, neurons of the preBötzinger complex that paces inspiration. Deleting the commissural projections of V0s results in left-right desynchronized inspiratory motor commands in reduced brain preparations and breathing at birth. This work reveals the existence of a core inspiratory circuit in which V0 to V0 synapses enabling function of the rhythm generator also direct its output to secure bilaterally coordinated contractions of inspiratory effector muscles required for efficient breathing.

Release of ATP by pre-Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca2+ -dependent P2Y1 receptor mechanism.

The ventilatory response to reduced oxygen (hypoxia) is biphasic, comprising an initial increase in ventilation followed by a secondary depression. Our findings indicate that, during hypoxia, astrocytes in the pre-Bötzinger complex (preBötC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y1 receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBötC involves P2Y1 receptor-mediated release of Ca2+ from intracellular stores. By identifying a role for gliotransmission and the sites, P2 receptor subtype, and signalling mechanisms via which ATP modulates breathing during hypoxia, these data advance our understanding of the mechanisms underlying the hypoxic ventilatory response and highlight the significance of purinergic signalling and gliotransmission in homeostatic control. Clinically, these findings are relevant to conditions in which hypoxia and respiratory depression are implicated, including apnoea of prematurity, sleep disordered breathing and congestive heart failure. The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady-state phase II) that can be life-threatening in premature infants who suffer from frequent apnoeas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the pre-Bötzinger Complex (preBötC) inspiratory rhythm-generating network acts via P2Y1 receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBötC contributes to the hypoxia-induced increases in ventilation. Unilateral blockade of P2Y1 receptors in the preBötC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y1 receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y1 receptor-mediated increase in fictive inspiratory frequency involves Ca2+ recruitment from intracellular stores leading to increases in intracellular Ca2+ ([Ca2+ ]i ) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y1 receptors on inspiratory neurons (and/or glia) to evoke Ca2+ release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.

Computational study on neuronal activities arising in the pre-Bötzinger complex.

Experimental investigations have shown that the pre-Bötzinger complex (pre-BötC) within the mammalian brainstem generates the inspiratory phase of respiratory rhythm. Based on a single-compartment model of a pre-BötC inspiratory neuron, we, in this paper, use semi-analytical, numerical as well as fast-slow dynamical methods to investigate the effects of sodium conductance ([Formula: see text]) and potassium conductance ([Formula: see text]) on the firing activities of pre-BötC and try to reveal the dynamical mechanisms behind them. We show how [Formula: see text] and [Formula: see text] affect the bifurcations of the fast-subsystem and how the the firing patterns of pre-BötC transit according to the bifurcations.

Supraspinal respiratory plasticity following acute cervical spinal cord injury

Impaired breathing is a devastating result of high cervical spinal cord injuries (SCI) due to partial or full denervation of phrenic motoneurons, which innervate the diaphragm – a primary muscle of respiration. Consequently, people with cervical level injuries often become dependent on assisted ventilation and are susceptible to secondary complications. However, there is mounting evidence for limited spontaneous recovery of respiratory function following injury, demonstrating the neuroplastic potential of respiratory networks. Although many studies have shown such plasticity at the level of the spinal cord, much less is known about the changes occurring at supraspinal levels post-SCI. The goal of this study was to determine functional reorganization of respiratory neurons in the medulla acutely (>4h) following high cervical SCI. Experiments were conducted in decerebrate, unanesthetized, vagus intact and artificially ventilated rats. In this preparation, spontaneous recovery of ipsilateral phrenic nerve activity was observed within 4 to 6h following an incomplete, C2 hemisection (C2Hx). Electrophysiological mapping of the ventrolateral medulla showed a reorganization of inspiratory and expiratory sites ipsilateral to injury. These changes included i) decreased respiratory activity within the caudal ventral respiratory group (cVRG; location of bulbospinal expiratory neurons); ii) increased proportion of expiratory phase activity within the rostral ventral respiratory group (rVRG; location of inspiratory bulbo-spinal neurons); iii) increased respiratory activity within ventral reticular nuclei, including lateral reticular (LRN) and paragigantocellular (LPGi) nuclei. We conclude that disruption of descending and ascending connections between the medulla and spinal cord leads to immediate functional reorganization within the supraspinal respiratory network, including neurons within the ventral respiratory column and adjacent reticular nuclei.

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.

Firing properties of pre‐inspiratory neurons are altered in female rats exposed to chronic intermittent hypoxia

Female rats submitted to chronic intermittent hypoxia (CIH) presented a reduced time of inspiration, sympathetic overactivity and hypertension. The increase in sympathetic nerve discharge in CIH‐female rats is associated to an increase in the inspiratory modulation of sympathetic activity. In this context, we hypothesized that inspiratory neurons at the brainstem have change their activity in CIH‐female rats, which in turn, modulates the presympathetic neurons producing an increase in sympathetic activity during inspiration. To test this hypothesis, juvenile female Wistar rats (P20–21) underwent intermittent hypoxia for 10 days. One day after CIH, we performed the extracellular single unit recordings of respiratory neurons at the ventral surface of the medulla using the ventral approach of the working heart‐brainstem preparation. All the experimental protocols were approved by the institutional ethical committee (#091/2013). Pre‐inspiratory neurons (Pre‐I/I) of CIH‐female rats (n=16) have reduced their firing frequency (58.3 ± 9 vs 107.6 ± 12Hz, p=0.0027) compared to Pre‐I/I neurons of control (n=15). Synaptic blockade demonstrated that Pre‐I/I neurons (n=3) have auto‐depolarization property. Ramp‐I, Early‐I and Late‐I neurons presented no changes in their firing properties after CIH. Regarding the expiratory neurons, post‐inspiratory neurons (Post‐I) have increased the firing time relative to duration of expiration (92.2 ± 2 vs 81.3 ± 3 % of total duration of expiration, p=0.0127) in CIH‐female rats (n=15) compared to control (n=17). We conclude that CIH produces a reduction in the activity of pacemaker Pre‐I/I neurons that could explain the reduced time of inspiration and the increase in inspiratory modulation of sympathetic activity in CIH‐female rats. These findings contribute to a better understanding of the respiratory network activity associated with sympathetic overactivity and hypertension in female rats exposed to CIH.Support or Funding InformationFapesp, CAPES and CNPq

Effects of a TRPV1 agonist capsaicin on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats.

The heat-sensitive transient receptor potential vanilloid 1 (TRPV1) channels are expressed in the peripheral and central nervous systems. However, there is no report on how the activation of TRPV1 causes the modulation of neuronal activity in the medullary respiratory center. We examined effects of capsaicin, a specific agonist of TRPV1 channels, on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats. Capsaicin induced a biphasic response in the respiratory rhythm (a transient decrease followed by an increase in the C4 rate). The second-phase excitatory effect (but not the initial inhibitory effect) in the biphasic response was partly blocked by capsazepine or AMG9810 (TRPV1 antagonists). Capsaicin caused strong desensitization. After its washout, the strength of C4 burst inspiratory activity was augmented once per four to five respiratory cycles. The preinspiratory and inspiratory neurons showed tonic firings due to membrane depolarization during the initial inhibitory phase. In the presence of TTX, capsaicin increased the fluctuation of the membrane potential of the CO2-sensitive preinspiratory neurons in the parafacial respiratory group (pFRG), accompanied by slight depolarization. The C4 inspiratory activity did not stop, even 60-90min after the application of 50/100μM capsaicin. Voltage-sensitive dye imaging demonstrated that the spatiotemporal pattern of the respiratory rhythm generating networks after application of capsaicin (50μM, 70-90min) was highly similar to the control. A histochemical analysis using TRPV1 channel protein antibodies and mRNA demonstrated that the TRPV1 channel-positive cells were widely distributed in the reticular formation of the medulla, including the pFRG. Our results showed that the application of capsaicin in the medulla has various influences on the respiratory center: transient inhibitory and subsequent excitatory effects on the respiratory rhythm and periodical augmentation of the inspiratory burst pattern. The effects of capsaicin were partially blocked by TRPV1 antagonists but could be also induced at least partially via the non-specific action. Our results also suggested a minor contribution of the TRPV1 channels to central chemoreception.

Multiple timescale mixed bursting dynamics in a respiratory neuron model.

Experimental results in rodent medullary slices containing the pre-Bötzinger complex (pre-BötC) have identified multiple bursting mechanisms based on persistent sodium current (I NaP) and intracellular Ca2+. The classic two-timescale approach to the analysis of pre-BötC bursting treats the inactivation of I NaP, the calcium concentration, as well as the Ca2+-dependent inactivation of IP 3 as slow variables and considers other evolving quantities as fast variables. Based on its time course, however, it appears that a novel mixed bursting (MB) solution, observed both in recordings and in model pre-BötC neurons, involves at least three timescales. In this work, we consider a single-compartment model of a pre-BötC inspiratory neuron that can exhibit both I NaP and Ca2+ oscillations and has the ability to produce MB solutions. We use methods of dynamical systems theory, such as phase plane analysis, fast-slow decomposition, and bifurcation analysis, to better understand the mechanisms underlying the MB solution pattern. Rather surprisingly, we discover that a third timescale is not actually required to generate mixed bursting solutions. Through our analysis of timescales, we also elucidate how the pre-BötC neuron model can be tuned to improve the robustness of the MB solution.

Development of pacemaker properties and rhythmogenic mechanisms in the mouse embryonic respiratory network.

Breathing is a vital rhythmic behavior generated by hindbrain neuronal circuitry, including the preBötzinger complex network (preBötC) that controls inspiration. The emergence of preBötC network activity during prenatal development has been described, but little is known regarding inspiratory neurons expressing pacemaker properties at embryonic stages. Here, we combined calcium imaging and electrophysiological recordings in mouse embryo brainstem slices together with computational modeling to reveal the existence of heterogeneous pacemaker oscillatory properties relying on distinct combinations of burst-generating INaP and ICAN conductances. The respective proportion of the different inspiratory pacemaker subtypes changes during prenatal development. Concomitantly, network rhythmogenesis switches from a purely INaP/ICAN-dependent mechanism at E16.5 to a combined pacemaker/network-driven process at E18.5. Our results provide the first description of pacemaker bursting properties in embryonic preBötC neurons and indicate that network rhythmogenesis undergoes important changes during prenatal development through alterations in both circuit properties and the biophysical characteristics of pacemaker neurons.