The Relative Contribution of Glycine-GABA Cotransmission in the Core of the Respiratory Network.

The preBötzinger complex (preBötC) and the Bötzinger complex (BötC) are interconnected neural circuits that are involved in the regulation of breathing in mammals. Fast inhibitory neurotransmission is known to play an important role in the interaction of these two regions. Moreover, the corelease of glycine and GABA has been described in the respiratory network, but the contribution of the individual neurotransmitter in different pathways remains elusive. In sagittal brainstem slices of neonatal mice, we employed a laser point illumination system to activate glycinergic neurons expressing channelrhodopsin-2 (ChR2). This approach allowed us to discern the contribution of glycine and GABA to postsynaptic currents of individual whole-cell clamped neurons in the preBötC and BötC through the application of glycine and GABA receptor-specific antagonists. In more than 90% of the recordings, both transmitters contributed to the evoked IPSCs, with the glycinergic component being larger than the GABAergic component. The GABAergic component appeared to be most prominent when stimulation and recording were both performed within the preBötC. Taken together, our data suggest that GABA-glycine cotransmission is the default mode in the respiratory network of neonatal mice with regional differences that may be important in tuning the network activity.

In-vivo optogenetic identification and electrophysiology of glycinergic neurons in pre-Bötzinger complex of mice.

Breathing requires distinct patterns of neuronal activity in the brainstem. The most critical part of the neuronal network responsible for respiratory rhythm generation is the preBötzinger Complex (preBötC), located in the ventrolateral medulla. This area contains both rhythmogenic glutamatergic neurons and also a high number of inhibitory neurons. Here, we aimed to analyze the activity of glycinergic neurons in the preBötC in anesthetized mice. To identify inhibitory neurons, we used a transgenic mouse line that allows expression of Channelrhodopsin 2 in glycinergic neurons. Using juxtacellular recordings and optogenetic activation via a single recording electrode, we were able to identify neurons as inhibitory and define their activity pattern in relation to the breathing rhythm. We could show that the activity pattern of glycinergic respiratory neurons in the preBötC was heterogeneous. Interestingly, only a minority of the identified glycinergic neurons showed a clear phase-locked activity pattern in every respiratory cycle. Taken together, we could show that neuron identification is possible by a combination of juxtacellular recordings and optogenetic activation via a single recording electrode.

Longitudinal x-ray based lung function measurement for monitoring Nintedanib treatment response in a mouse model of lung fibrosis.

Lung fibrosis (LF) is a chronic progressive, incurable, and debilitating condition of the lung, which is associated with different lung disease. Treatment options are still sparse. Nintedanib, an oral tyrosine kinase inhibitor, significantly slows the LF progression. However, there is a strong need of further research and the development of novel therapies. In this study, we used a correlative set-up that combines X-ray based lung function (XLF) with microCT and whole body plethysmography (WBP) for a comprehensive functional and structural evaluation of lung fibrosis (LF) as well as for monitoring response to orally administered Nintedanib in the mouse model of bleomycin induced LF. The decline in lung function as early as one week after intratracheal bleomycin instillation was reliably detected by XLF, revealing the lowest decay rate in the LF mice compared to healthy ones. Simultaneously performed microCT and WBP measurements corroborated XLF findings by exhibiting reduced lung volume [Formula: see text] and tidal volume [Formula: see text]. In LF mice XLF also revealed profound improvement in lung function one week after Nintedanib treatment. This positive response to Nintedanib therapy was further substantiated by microCT and WBP measurements which also showed significantly improved [Formula: see text] and [Formula: see text] in the Nintedanib treated mice. By comparing the XLF data to structural features assessing the extent of fibrosis obtained by ex-vivo high-resolution synchrotron radiation-based imaging and classical histology we demonstrate that: (1) a simple low dose x-ray measurement like XLF is sensitive enough to pick up treatment response, (2) Nintedanib treatment successfully improved lung function in a bleomycin induced LF mouse model and (3) differences between the fully restored lung function and the partially reduced fibrotic burden compared to healthy and untreated mice. The presented analysis pipeline underlines the importance of a combined functional and anatomical readout to reliably measure treatment response and could easily be adapted to other preclinical lung disease models.

Fiber optical imaging of astroglial calcium signaling in the respiratory network in the working heart brainstem preparation.

The neuronal activity in the respiratory network strongly depends on a variety of different neuromodulators. Given the essential role of astrocytes in stabilizing respiratory network activity generated by neurons in the preBötzinger complex (preBötC), our aim was to investigate astrocytic calcium signaling in the working heart brainstem preparation using fiber-optical imaging. By using transgenic mice that express GCaMP6s specifically in astrocytes, we successfully recorded astrocytic calcium signals in response to norepinephrine from individual astrocytes.

Increase of breathing rate mediated by unilateral optogenetic inactivation of inhibitory neurons in the preBötzinger Complex in vivo.

Brainstem neural circuits located in the preBötzinger complex (preBötC) and Bötzinger complex (BötC) play a critical role in the control of breathing. In this study, glycinergic preBötC and BötC neurons were inactivated with optogenetics in vivo using mice with Cre inducible expression of eNpHR3.0-EYFP. Unilateral inhibition of glycinergic neurons in the preBötC, and to a lower extend also in the BötC, led to a higher respiratory rate. It can be concluded that functional inactivation of inhibitory neurons leads to a disinhibition of preBötC excitatory neurons and thus an increase in the respiratory drive of the network.

Angiotensin II increases respiratory rhythmic activity in the preBötzinger complex without inducing astroglial calcium signaling.

Angiotensin II (Ang II) is the primary modulator of the renin-angiotensin system and has been widely studied for its effect on the cardiovascular system. While a few studies have also indicated an involvement of Ang II in the regulation of breathing, very little is known in this regard and its effect on brainstem respiratory regions such as the preBötzinger complex (preBötC), the kernel for inspiratory rhythm generation, has not been investigated yet. This study reports that Ang II temporarily increases phrenic nerve activity in the working heart-brainstem preparation, indicating higher central respiratory drive. Previous studies have shown that the carotid body is involved in mediating this effect and we revealed that the preBötC also plays a part, using acute slices of the brainstem. It appears that Ang II is increasing the respiratory drive in an AT1R-dependent manner by optimizing the interaction of inhibitory and excitatory neurons of the preBötC. Thus, Ang II-mediated effects on the preBötC are potentially involved in dysregulating breathing in patients with acute lung injury.

Cell types and synchronous-activity patterns of inspiratory neurons in the preBötzinger complex of mouse medullary slices during early postnatal development.

To examine whether and how the inspiratory neuronal network in the preBötzinger complex (preBötC) develops during the early postnatal period, we quantified the composition of the population of inspiratory neurons between postnatal day 1 (p1) and p10 by applying calcium imaging to medullary transverse slices in double-transgenic mice expressing fluorescent marker proteins. We found that putative excitatory and glycinergic neurons formed a majority of the population of inspiratory neurons, and the composition rates of these two inspiratory neurons inverted at p5-6. We also found that the activity patterns of these two types of inspiratory neurons became significantly well-synchronized with the inspiratory rhythmic bursting pattern in the preBötC within the first postnatal week. GABAergic and GABA-glycine cotransmitting inspiratory neurons formed only a small population just after birth, which almost disappeared until p10. In conclusion, the inspiratory neuronal network in the preBötC matures at the level of both neuronal population and neuronal activities during early postnatal development.

Non-Invasive Optical Motion Tracking Allows Monitoring of Respiratory Dynamics in Dystrophin-Deficient Mice.

Duchenne muscular dystrophy (DMD) is the most common x-chromosomal inherited dystrophinopathy which leads to progressive muscle weakness and a premature death due to cardiorespiratory dysfunction. The mdx mouse lacks functional dystrophin protein and has a comparatively human-like diaphragm phenotype. To date, diaphragm function can only be inadequately mapped in preclinical studies and a simple reliable translatable method of tracking the severity of the disease still lacks. We aimed to establish a sensitive, reliable, harmless and easy way to assess the effects of respiratory muscle weakness and subsequent irregularity in breathing pattern. Optical respiratory dynamics tracking (ORDT) was developed utilising a camera to track the movement of paper markers placed on the thoracic-abdominal region of the mouse. ORDT successfully distinguished diseased mdx phenotype from healthy controls by measuring significantly higher expiration constants (k) in mdx mice compared to wildtype (wt), which were also observed in the established X-ray based lung function (XLF). In contrast to XLF, with ORDT we were able to distinguish distinct fast and slow expiratory phases. In mdx mice, a larger part of the expiratory marker displacement was achieved in this initial fast phase as compared to wt mice. This phenomenon could not be observed in the XLF measurements. We further validated the simplicity and reliability of our approach by demonstrating that it can be performed using free-hand smartphone acquisition. We conclude that ORDT has a great preclinical potential to monitor DMD and other neuromuscular diseases based on changes in the breathing patterns with the future possibility to track therapy response.

Synergistic Control of Transmitter Turnover at Glycinergic Synapses by GlyT1, GlyT2, and ASC-1.

In addition to being involved in protein biosynthesis and metabolism, the amino acid glycine is the most important inhibitory neurotransmitter in caudal regions of the brain. These functions require a tight regulation of glycine concentration not only in the synaptic cleft, but also in various intracellular and extracellular compartments. This is achieved not only by confining the synthesis and degradation of glycine predominantly to the mitochondria, but also by the action of high-affinity large-capacity glycine transporters that mediate the transport of glycine across the membranes of presynaptic terminals or glial cells surrounding the synapses. Although most cells at glycine-dependent synapses express more than one transporter with high affinity for glycine, their synergistic functional interaction is only poorly understood. In this review, we summarize our current knowledge of the two high-affinity transporters for glycine, the sodium-dependent glycine transporters 1 (GlyT1; SLC6A9) and 2 (GlyT2; SLC6A5) and the alanine-serine-cysteine-1 transporter (Asc-1; SLC7A10).

Evaluation of a mechanical lung model to test small animal whole body plethysmography.

Whole-body plethysmography (WBP) is an established method to determine physiological parameters and pathophysiological alteration of breathing in animals and animal models of a variety of diseases. Although frequently used, there is ongoing debate about what exactly is measured by whole-body-plethysmography and how reliable the data derived from this method are. Here, we designed an artificial lung model that enables a thorough evaluation of different predictions about and around whole-body plethysmography. Using our lung model, we confirmed that during WBP two components contribute to the pressure changes detected in the chamber: (1) the increase in the pressure due to heating and moistening of the air during inspiration, termed conditioning; (2) changes in the chamber pressure that depend on airway resistance. Both components overlap and contribute to the temporal pressure-profile measured in the chamber or across the wall of the chamber, respectively. Our data showed that a precise measurement of the breathing volume appears to be hindered by at least two factors: (1) the unknown relative contribution of each of these two components; (2) not only the air in the inspired volume is conditioned during inspiration, but also air within the residual volume and dead space that is recruited during inspiration. Moreover, our data suggest that the expiratory negative pressure peak that is used to determine the enhanced pause (Penh) parameter is not a measure for airway resistance as such but rather a consequence of the animal's response to the airway resistance, using forced or active expiration to overcome the resistance by a higher thoracic pressure.

Inspiratory Off-Switch Mediated by Optogenetic Activation of Inhibitory Neurons in the preBötzinger Complex In Vivo.

The role of inhibitory neurons in the respiratory network is a matter of ongoing debate. Conflicting and contradicting results are manifold and the question whether inhibitory neurons are essential for the generation of the respiratory rhythm as such is controversial. Inhibitory neurons are required in pulmonary reflexes for adapting the activity of the central respiratory network to the status of the lung and it is hypothesized that glycinergic neurons mediate the inspiratory off-switch. Over the years, optogenetic tools have been developed that allow for cell-specific activation of subsets of neurons in vitro and in vivo. In this study, we aimed to identify the effect of activation of inhibitory neurons in vivo. Here, we used a conditional transgenic mouse line that expresses Channelrhodopsin 2 in inhibitory neurons. A 200 µm multimode optical fiber ferrule was implanted in adult mice using stereotaxic surgery, allowing us to stimulate inhibitory, respiratory neurons within the core excitatory network in the preBötzinger complex of the ventrolateral medulla. We show that, in anesthetized mice, activation of inhibitory neurons by blue light (470 nm) continuously or with stimulation frequencies above 10 Hz results in a significant reduction of the respiratory rate, in some cases leading to complete cessation of breathing. However, a lower stimulation frequency (4-5 Hz) could induce a significant increase in the respiratory rate. This phenomenon can be explained by the resetting of the respiratory cycle, since stimulation during inspiration shortened the associated breath and thereby increased the respiratory rate, while stimulation during the expiratory interval reduced the respiratory rate. Taken together, these results support the concept that activation of inhibitory neurons mediates phase-switching by inhibiting excitatory rhythmogenic neurons in the preBötzinger complex.

Simple low dose radiography allows precise lung volume assessment in mice.

X-ray based lung function (XLF) as a planar method uses dramatically less X-ray dose than computed tomography (CT) but so far lacked the ability to relate its parameters to pulmonary air volume. The purpose of this study was to calibrate the functional constituents of XLF that are biomedically decipherable and directly comparable to that of micro-CT and whole-body plethysmography (WBP). Here, we developed a unique set-up for simultaneous assessment of lung function and volume using XLF, micro-CT and WBP on healthy mice. Our results reveal a strong correlation of lung volumes obtained from radiographic XLF and micro-CT and demonstrate that XLF is superior to WBP in sensitivity and precision to assess lung volumes. Importantly, XLF measurement uses only a fraction of the radiation dose and acquisition time required for CT. Therefore, the redefined XLF approach is a promising tool for preclinical longitudinal studies with a substantial potential of clinical translation.

CMTM6 expressed on the adaxonal Schwann cell surface restricts axonal diameters in peripheral nerves.

The velocity of nerve conduction is moderately enhanced by larger axonal diameters and potently sped up by myelination of axons. Myelination thus allows rapid impulse propagation with reduced axonal diameters; however, no myelin-dependent mechanism has been reported that restricts radial growth of axons. By label-free proteomics, STED-microscopy and cryo-immuno electron-microscopy we here identify CMTM6 (chemokine-like factor-like MARVEL-transmembrane domain-containing family member-6) as a myelin protein specifically localized to the Schwann cell membrane exposed to the axon. We find that disruption of Cmtm6-expression in Schwann cells causes a substantial increase of axonal diameters but does not impair myelin biogenesis, radial sorting or integrity of axons. Increased axonal diameters correlate with accelerated sensory nerve conduction and sensory responses and perturbed motor performance. These data show that Schwann cells utilize CMTM6 to restrict the radial growth of axons, which optimizes nerve function.

A Potential Role of the Renin-Angiotensin-System for Disturbances of Respiratory Chemosensitivity in Acute Respiratory Distress Syndrome and Severe Acute Respiratory Syndrome.

Acute respiratory distress syndrome (ARDS) represents an acute diffuse inflammation of the lungs triggered by different causes, uniformly leading to a noncardiogenic pulmonary edema with inhomogeneous densities in lung X-ray and lung CT scan and acute hypoxemia. Edema formation results in "heavy" lungs, inducing loss of compliance and the need to spend more energy to "move" the lungs. Consequently, an ARDS patient, as long as the patient is breathing spontaneously, has an increased respiratory drive to ensure adequate oxygenation and CO2 removal. One would expect that, once the blood gases get back to "physiological" values, the respiratory drive would normalize and the breathing effort return to its initial status. However, in many ARDS patients, this is not the case; their respiratory drive appears to be upregulated and fully or at least partially detached from the blood gas status. Strikingly, similar alteration of the respiratory drive can be seen in patients suffering from SARS, especially SARS-Covid-19. We hypothesize that alterations of the renin-angiotensin-system (RAS) related to the pathophysiology of ARDS and SARS are involved in this dysregulation of chemosensitive control of breathing.

GABA-Glycine Cotransmitting Neurons in the Ventrolateral Medulla: Development and Functional Relevance for Breathing.

Inhibitory neurons crucially contribute to shaping the breathing rhythm in the brain stem. These neurons use GABA or glycine as neurotransmitter; or co-release GABA and glycine. However, the developmental relationship between GABAergic, glycinergic and cotransmitting neurons, and the functional relevance of cotransmitting neurons has remained enigmatic. Transgenic mice expressing fluorescent markers or the split-Cre system in inhibitory neurons were developed to track the three different interneuron phenotypes. During late embryonic development, the majority of inhibitory neurons in the ventrolateral medulla are cotransmitting cells, most of which differentiate into GABAergic and glycinergic neurons around birth and around postnatal day 4, respectively. Functional inactivation of cotransmitting neurons revealed an increase of the number of respiratory pauses, the cycle-by-cycle variability, and the overall variability of breathing. In summary, the majority of cotransmitting neurons differentiate into GABAergic or glycinergic neurons within the first 2 weeks after birth and these neurons contribute to fine-tuning of the breathing pattern.

The postnatal development of ultrasonic vocalization-associated breathing is altered in glycine transporter 2-deficient mice.

KEY POINTS: Newborn mice produce ultrasonic vocalization to communicate with their mother. The neuronal glycine transporter (GlyT2) is required for efficient loading of synaptic vesicles in glycinergic neurons. Mice lacking GlyT2 develop a phenotype that resembles human hyperekplexia and the mice die in the second postnatal week. In the present study, we show that GlyT2-knockout mice do not acquire adult ultrasonic vocalization-associated breathing patterns. Despite the strong impairment of glycinergic inhibition, they can produce sufficient expiratory airflow to produce ultrasonic vocalization. Because mouse ultrasonic vocalization is a valuable read-out in translational research, these data are highly relevant for a broad range of research fields. ABSTRACT: Mouse models are instrumental with respect to determining the genetic basis and neural foundations of breathing regulation. To test the hypothesis that glycinergic synaptic inhibition is required for normal breathing and proper post-inspiratory activity, we analysed breathing and ultrasonic vocalization (USV) patterns in neonatal mice lacking the neuronal glycine transporter (GlyT2). GlyT2-knockout (KO) mice have a profound reduction of glycinergic synaptic currents already at birth, develop a severe motor phenotype and survive only until the second postnatal week. At this stage, GlyT2-KO mice are smaller, have a reduced respiratory rate and still display a neonatal breathing pattern with active expiration for the production of USV. By contrast, wild-type mice acquire different USV-associated breathing patterns that depend on post-inspiratory control of air flow. Nonetheless, USVs per se remain largely indistinguishable between both genotypes. We conclude that GlyT2-KO mice, despite the strong impairment of glycinergic inhibition, can produce sufficient expiratory airflow to produce ultrasonic vocalization.

Probing the function of glycinergic neurons in the mouse respiratory network using optogenetics.

Glycine is a primary inhibitory transmitter in the ventral medullary respiratory network, but the functional role of glycinergic neurons for breathing remains a matter of debate. We applied optogenetics to selectively modulate glycinergic neuron activity within regions of the rostral ventral respiratory column (VRC). Responses of the phrenic nerve activity to the light-driven stimulation were studied in the working heart-brainstem preparation from adult glycine transporter 2 Cre mice (GlyT2-Cre), which received a unilateral injection of a Cre-dependent AAV virus into Bötzinger and preBötzinger Complex. Sustained light stimulation from the ventral medullary surface resulted in a substantial depression of the phrenic nerve (PN) frequency, which in most cases was compensated by an increase in PN amplitude. Periodic, burst stimulation with variable intervals could alter and reset respiratory rhythm. We conclude that unilateral activation of the rostral VRC glycinergic neurons can significantly affect respiratory pattern by lengthening the expiratory interval and modulating phase transition.

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

[This corrects the article DOI: 10.3389/fphys.2018.01219.].

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.