Respiratory circuits: development, function and models

Breathing is a rhythmic motor behavior generated and controlled by hindbrain neuronal networks. Respiratory motor output arises from two distinct, but functionally interacting, rhythmogenic networks: the pre-B?tzinger complex (preB?tC) and the retrotrapezo?d nucleus/parafacial respiratory group (RTN/pFRG). This review outlines recent advances in delineating the genetic specification of the neuronal constituents of these two rhythmogenic networks, their respective roles in respiratory function and how they interact to constitute a functional respiratory circuit ensemble. The often lethal consequences of disruption to these networks found in naturally occurring developmental disorders, transgenic animals, and highly specific lesion studies are described. In addition, we discuss how recent computational models enhance our understanding of how respiratory networks generate and regulate respiratory behavior.     

A Closed-Loop Model of the Respiratory System: Focus on Hypercapnia and Active Expiration

Breathing is a vital process providing the exchange of gases between the lungs and atmosphere. During quiet breathing, pumping air from the lungs is mostly performed by contraction of the diaphragm during inspiration, and muscle contraction during expiration does not play a significant role in ventilation. In contrast, during intense exercise or severe hypercapnia forced or active expiration occurs in which the abdominal “expiratory” muscles become actively involved in breathing. The mechanisms of this transition remain unknown. To study these mechanisms, we developed a computational model of the closed-loop respiratory system that describes the brainstem respiratory network controlling the pulmonary subsystem representing lung biomechanics and gas (O₂ and CO₂) exchange and transport. The lung subsystem provides two types of feedback to the neural subsystem: a mechanical one from pulmonary stretch receptors and a chemical one from central chemoreceptors. The neural component of the model simulates the respiratory network that includes several interacting respiratory neuron types within the Bötzinger and pre-Bötzinger complexes, as well as the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) representing the central chemoreception module targeted by chemical feedback. The RTN/pFRG compartment contains an independent neural generator that is activated at an increased CO₂ level and controls the abdominal motor output. The lung volume is controlled by two pumps, a major one driven by the diaphragm and an additional one activated by abdominal muscles and involved in active expiration. The model represents the first attempt to model the transition from quiet breathing to breathing with active expiration. The model suggests that the closed-loop respiratory control system switches to active expiration via a quantal acceleration of expiratory activity, when increases in breathing rate and phrenic amplitude no longer provide sufficient ventilation. The model can be used for simulation of closed-loop control of breathing under different conditions including respiratory disorders.     

Nogo/RTN4 isoforms and RTN3 expression protect SH-SY5Y cells against multiple death insults

Among the members of the reticulon (RTN) family, Nogo-A/RTN4A, a prominent myelin-associated neurite growth inhibitory protein, and RTN3 are highly expressed in neurons. However, neuronal cell-autonomous functions of Nogo-A, as well as other members of the RTN family, are unclear. We show here that SH-SY5Y neuroblastoma cells stably over-expressing either two of the three major isoforms of Nogo/RTN4 (Nogo-A and Nogo-B) or a major isoform of RTN3 were protected against cell death induced by a battery of apoptosis-inducing agents (including serum deprivation, staurosporine, etoposide, and H₂O₂) compared to vector-transfected control cells. Nogo-A, -B, and RTN3 are particularly effective in terms of protection against H₂O₂-induced increase in intracellular reactive oxygen species levels and ensuing apoptotic and autophagic cell death. Expression of these RTNs upregulated basal levels of Bax, activated Bax, and activated caspase 3, but did not exhibit an enhanced ER stress response. The protective effect of RTNs is also not dependent on classical survival-promoting signaling pathways such as Akt and Erk kinase pathways. Neuron-enriched Nogo-A/Rtn4A and RTN3 may, therefore, exert a protective effect on neuronal cells against death stimuli, and elevation of their levels during injury may have a cell-autonomous survival-promoting function.     

Hoxa2 and Hoxb2 control dorsoventral patterns of neuronal development in the rostral hindbrain

Little is known about how the generation of specific neuronal types at stereotypic positions within the hindbrain is linked to Hox gene-mediated patterning. Here, we show that during neurogenesis, Hox paralog group 2 genes control both anteroposterior (A-P) and dorsoventral (D-V) patterning. Hoxa2 and Hoxb2 differentially regulate, in a rhombomere-specific manner, the expression of several genes in broad D-V-restricted domains or narrower longitudinal columns of neuronal progenitors, immature neurons, and differentiating neuronal subtypes. Moreover, Hoxa2 and Hoxb2 can functionally synergize in controlling the development of ventral neuronal subtypes in rhombomere 3 (r3). Thus, in addition to their roles in A-P patterning, Hoxa2 and Hoxb2 have distinct and restricted functions along the D-V axis during neurogenesis, providing insights into how neuronal fates are assigned at stereotypic positions within the hindbrain.     

Brainstem respiratory oscillators develop independently of neuronal migration defects in the Wnt/PCP mouse mutant looptail

The proper development and maturation of neuronal circuits require precise migration of component neurons from their birthplace (germinal zone) to their final positions. Little is known about the effects of aberrant neuronal position on the functioning of organized neuronal groups, especially in mammals. Here, we investigated the formation and properties of brainstem respiratory neurons in looptail (Lp) mutant mice in which facial motor neurons closely apposed to some respiratory neurons fail to migrate due to loss of function of the Wnt/Planar Cell Polarity (PCP) protein Vangl2. Using calcium imaging and immunostaining on embryonic hindbrain preparations, we found that respiratory neurons constituting the embryonic parafacial oscillator (e-pF) settled at the ventral surface of the medulla in Vangl2^Lp/+ and Vangl2^Lp/Lp embryos despite the failure of tangential migration of its normally adjacent facial motor nucleus. Anatomically, the e-pF neurons were displaced medially in Lp/+ embryos and rostro-medially Lp/Lp embryos. Pharmacological treatments showed that the e-pF oscillator exhibited characteristic network properties in both Lp/+ and Lp/Lp embryos. Furthermore, using hindbrain slices, we found that the other respiratory oscillator, the preBötzinger complex, was also anatomically and functionally established in Lp mutants. Importantly, the displaced e-pF oscillator established functional connections with the preBötC oscillator in Lp/+ mutants. Our data highlight the robustness of the developmental processes that assemble the neuronal networks mediating an essential physiological function.     

Multiple rostral medullary nuclei can influence breathing in awake goats.

The purpose of this study was to determine the effect on breathing of neuronal dysfunction in the retrotrapezoid (RTN), facial (FN), gigantocellularis reticularis (RGN), or vestibular (VN) nuclei of adult awake goats. Microtubules were chronically implanted to induce neuronal dysfunction by microinjection of an excitatory amino acid (EAA) receptor antagonist or a neurotoxin. The EAA receptor antagonist had minimal effect on eupneic breathing, but 8--10 days after injection of the neurotoxin, 7 of 10 goats hypoventilated (arterial PCO(2) increased 3.2 +/- 0.7 Torr). Overall there were no significant (P > 0.10) effects of the EAA receptor antagonist on CO(2) sensitivity. However, for all nuclei, > or =66% of the antagonist injections altered CO(2) sensitivity by more than the normal 12.7 +/- 1.6% day-to-day variation. These changes were not uniform, inasmuch as the antagonist increased (RTN, n = 2; FN, n = 7; RGN, n = 6; VN, n = 1) or decreased (RTN, n = 2; RGN, n = 3; VN, n = 2) CO(2) sensitivity. Ten days after injection of the neurotoxin into the FN (n = 3) or RGN (n = 5), CO(2) sensitivity was also reduced. Neuronal dysfunction also did not have a uniform effect on the exercise arterial PCO(2) response, and there was no correlation between effects on CO(2) sensitivity and the exercise hyperpnea. We conclude that there is a heterogeneous population of neurons in these rostral medullary nuclei (or adjacent tissue) that can affect breathing in the awake state, possibly through chemoreception or chemoreceptor-related mechanisms.     

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering the molecular signals that cooperatively guide neuronal migration in the embryonic brain is therefore important to understand how the complex neural networks form which later support postnatal life. Facial branchiomotor (FBM) neurons in the mouse embryo hindbrain migrate from rhombomere (r) 4 caudally to form the paired facial nuclei in the r6-derived region of the hindbrain. Here we provide a detailed protocol for wholemount ex vivo culture of mouse embryo hindbrains suitable to investigate the signaling pathways that regulate FBM migration. In this method, hindbrains of E11.5 mouse embryos are dissected and cultured in an open book preparation on cell culture inserts for 24 hr. During this time, FBM neurons migrate caudally towards r6 and can be exposed to function-blocking antibodies and small molecules in the culture media or heparin beads loaded with recombinant proteins to examine roles for signaling pathways implicated in guiding neuronal migration.     

The role of spiking and bursting pacemakers in the neuronal control of breathing

Breathing is controlled by a distributed network involving areas in the neocortex, cerebellum, pons, medulla, spinal cord, and various other subcortical regions. However, only one area seems to be essential and sufficient for generating the respiratory rhythm: the preBötzinger complex (preBötC). Lesioning this area abolishes breathing and following isolation in a brain slice the preBötC continues to generate different forms of respiratory activities. The use of slice preparations led to a thorough understanding of the cellular mechanisms that underlie the generation of inspiratory activity within this network. Two types of inward currents, the persistent sodium current (I_NaP) and the calcium-activated non-specific cation current (I_CAN), play important roles in respiratory rhythm generation. These currents give rise to autonomous pacemaker activity within respiratory neurons, leading to the generation of intrinsic spiking and bursting activity. These membrane properties amplify as well as activate synaptic mechanisms that are critical for the initiation and maintenance of inspiratory activity. In this review, we describe the dynamic interplay between synaptic and intrinsic membrane properties in the generation of the respiratory rhythm and we relate these mechanisms to rhythm generating networks involved in other behaviors.     

GABAB receptors suppress burst-firing in reticular thalamic neurons

Burst-firing in thalamic neurons is known to play a key role in mediating thalamocortical (TC) oscillations that are associated with non-REM sleep and some types of epileptic seizure. Within the TC system the primary output of GABAergic neurons in the reticular thalamic nucleus (RTN) is thought to induce the de-inactivation of T-type calcium channels in thalamic relay (TR) neurons, promoting burst-firing drive to the cortex and the propagation of TC network activity. However, RTN neurons also project back onto other neurons within the RTN. The role of this putative negative feedback upon the RTN itself is less well understood, although is hypothesized to induce de-synchronization of RTN neuron firing leading to the suppression of TC oscillations. Here we tested two hypotheses concerning possible mechanisms underlying TC oscillation modulation. Firstly, we assessed the burst-firing behavior of RTN neurons in response to GABA_B receptor activation using acute brain slices. The selective GABA_B receptor agonist baclofen was found to induce suppression of burst-firing concurrent with effects on membrane input resistance. Secondly, RTN neurons express Ca_V3.2 and Ca_V3.3 T-type calcium channel isoforms known to contribute toward TC burst-firing and we examined the modulation of these channels by GABA_B receptor activation. Utilizing exogenously expressed T-type channels we assessed whether GABA_B receptor activation could directly alter T-type calcium channel properties. Overall, GABA_B receptor activation had only modest effects on Ca_V3.2 and Ca_V3.3 isoforms. The only effect that could be predicted to suppress burst-firing was a hyperpolarized shift in the voltage-dependence of inactivation, potentially causing lower channel availability at membrane potentials critical for burst-firing. Conversely, other effects observed such as a hyperpolarized shift in the voltage-dependence of activation of both Ca_V3.2 and Ca_V3.3 as well as increased time constant of activation of the Ca_V3.3 isoform would be expected to enhance burst-firing. Together, we hypothesize that GABA_B receptor activation mediates multiple downstream effectors that combined act to suppress burst-firing within the RTN. It appears unlikely that direct GABA_B receptor-mediated modulation of T-type calcium channels is the major mechanistic contributor to this suppression.     

Neuronal subtype specification in the cerebellum and dorsal hindbrain

In the nervous system, there are hundreds to thousands of neuronal cell types that have morphologically, physiologically, and histochemically different characteristics and this diversity may enable us to elicit higher brain function. A better understanding of the molecular machinery by which neuron subtype specification occurs is thus one of the most important issues in brain science. The dorsal hindbrain, including the cerebellum, is a good model system to study this issue because a variety of types of neurons are produced from this region. Recently developed genetic lineage-tracing methods in addition to gene-transfer technologies have clarified a fate map of neurons produced from the dorsal hindbrain and accelerated our understanding of the molecular machinery of neuronal subtype specification in the nervous system.     

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP¹, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons^2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding⁶ (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER⁷ with a calcium imaging technique⁸ using GCaMP3.0^{1, 9}, I have established an experimental system, where we can monitor activity of neurons in the feeding center – the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca²⁺ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.     

Ventilatory effects of impaired glial function in a brain stem chemoreceptor region in the conscious rat.

Glia are thought to regulate ion homeostasis, including extracellular pH; however, their role in modulating central CO2 chemosensitivity is unclear. Using a push-pull cannula in chronically instrumented and conscious rats, we administered a glial toxin, fluorocitrate (FC; 1 mM) into the retrotrapezoid nucleus (RTN), a putative chemosensitive site, during normocapnia and hypercapnia. FC exposure significantly increased expired minute ventilation (VE) to a value 38% above the control level during normocapnia. During hypercapnia, FC also significantly increased both breathing frequency and expired VE. During FC administration, maximal ventilation was achieved at approximately 4% CO2, compared with 8-10% CO2 during control hypercapnic trials. RTN perfusion of control solutions had little effect on any ventilatory measures (VE, tidal volume, or breathing frequency) during normocapnic or hypercapnic conditions. We conclude that unilateral impairment of glial function in the RTN of the conscious rat results in stimulation of respiratory output.     

Naloxone application to the ventrolateral medulla enhances the respiratory response to inspired carbon dioxide

Previous studies have shown that systemic administration of the opiate antagonist naloxone potentiates the ventilatory response to inspired carbon dioxide. The present study was designed to localize the site of action of naloxone for increasing the respiratory chemosensitivity to inhaled carbon dioxide (CO2) in cats. Naloxone applied topically to the caudal chemosensitive area on the ventral medullary surface (VMS) during hypercapnic breathing produced a 75% greater increase in minute ventilation than hypercapnic breathing alone. Furthermore, hypercapnic breathing produced a 200% increase in neuronal activity of VMS chemosensitive cells; this was further increased 120% by naloxone. It is concluded that naloxone increases the sensitivity of neurons in the caudal respiratory chemosensitive area of cats to hypercapnia, and that endogenous opiates may act as modulators at VMS chemosensitive sites during hypercapnic breathing.     

Widespread contribution of Gdf7 lineage to cerebellar cell types and implications for hedgehog-driven medulloblastoma formation

The roof plate is a specialized embryonic midline tissue of the central nervous system that functions as a signaling center regulating dorsal neural patterning. In the developing hindbrain, roof plate cells express Gdf7 and previous genetic fate mapping studies showed that these cells contribute mostly to non-neural choroid plexus epithelium. We demonstrate here that constitutive activation of the Sonic hedgehog signaling pathway in the Gdf7 lineage invariably leads to medulloblastoma. Lineage tracing analysis reveals that Gdf7-lineage cells not only are a source of choroid plexus epithelial cells, but are also present in the cerebellar rhombic lip and contribute to a subset of cerebellar granule neuron precursors, the presumed cell-of-origin for Sonic hedgehog-driven medulloblastoma. We further show that Gdf7-lineage cells also contribute to multiple neuronal and glial cell types in the cerebellum, including glutamatergic granule neurons, unipolar brush cells, Purkinje neurons, GABAergic interneurons, Bergmann glial cells, and white matter astrocytes. These findings establish hindbrain roof plate as a novel source of diverse neural cell types in the cerebellum that is also susceptible to oncogenic transformation by deregulated Sonic hedgehog signaling.     

Dopamine Receptor 1 Modulates the Discharge Activities of Inspiratory and Biphasic Expiratory Neurons via cAMP-Dependent Pathways

Dopamine receptor 1 (D₁R) plays an essential role in regulating respiratory activity in mammals, however, little is known about how this receptor acts to modulate the basic respiratory rhythmogenesis. Here, by simultaneously recording the discharge activities of biphasic expiratory (biphasic E) neurons/inspiratory (I) neurons and the XII nerve rootlets from brainstem slices, we found that the application of D₁R agonist cis-(±)-1-(aminomethyl)-3,4-dihydro-3-phenyl-1H-2-benzopyran-5,6-diolhydrochloride (A68930, 5 μM), or forskolin, an intracellular cAMP-increasing agent, substantially decreased respiratory cycle and expiratory time of both types of neurons, and elevated the integral amplitude and frequency of XII nerve rootlets discharge. These changes were reversed by subsequent application of their antagonists SCH-23390 and Rp-Adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt hydrate (Rp-cAMPS), respectively. Importantly, after pretreatment with Rp-cAMPS, the effects of A68930 in both types of neurons were blocked, suggestive of a cAMP-dependent action of A68930. Thus, the current study indicates that D₁R may modulate basic breathing rhythmogenesis via cAMP-dependent mechanisms.     

Potency of Transgenic Effectors for Neurogenetic Manipulation in Drosophila Larvae

Genetic manipulations of neuronal activity are a cornerstone of studies aimed to identify the functional impact of defined neurons for animal behavior. With its small nervous system, rapid life cycle, and genetic amenability, the fruit fly Drosophila melanogaster provides an attractive model system to study neuronal circuit function. In the past two decades, a large repertoire of elegant genetic tools has been developed to manipulate and study neural circuits in the fruit fly. Current techniques allow genetic ablation, constitutive silencing, or hyperactivation of neuronal activity and also include conditional thermogenetic or optogenetic activation or inhibition. As for all genetic techniques, the choice of the proper transgenic tool is essential for behavioral studies. Potency and impact of effectors may vary in distinct neuron types or distinct types of behavior. We here systematically test genetic effectors for their potency to alter the behavior of Drosophila larvae, using two distinct behavioral paradigms: general locomotor activity and directed, visually guided navigation. Our results show largely similar but not equal effects with different effector lines in both assays. Interestingly, differences in the magnitude of induced behavioral alterations between different effector lines remain largely consistent between the two behavioral assays. The observed potencies of the effector lines in aminergic and cholinergic neurons assessed here may help researchers to choose the best-suited genetic tools to dissect neuronal networks underlying the behavior of larval fruit flies.