Purinergic signaling mediates neuroglial interactions to modulate sighs.

Sighs prevent the collapse of alveoli in the lungs, initiate arousal under hypoxic conditions, and are an expression of sadness and relief. Sighs are periodically superimposed on normal breaths, known as eupnea. Implicated in the generation of these rhythmic behaviors is the preBötzinger complex (preBötC). Our experimental evidence suggests that purinergic signaling is necessary to generate spontaneous and hypoxia-induced sighs in a mouse model. Our results demonstrate that driving calcium increases in astrocytes through pharmacological methods robustly increases sigh, but not eupnea, frequency. Calcium imaging of preBötC slices corroborates this finding with an increase in astrocytic calcium upon application of sigh modulators, increasing intracellular calcium through g-protein signaling. Moreover, photo-activation of preBötC astrocytes is sufficient to elicit sigh activity, and this response is blocked with purinergic antagonists. We conclude that sighs are modulated through neuron-glia coupling in the preBötC network, where the distinct modulatory responses of neurons and glia allow for both rhythms to be independently regulated.

Molecular heterogeneity of C. elegans glia across sexes.

A comprehensive description of nervous system function, and sex dimorphism within, is incomplete without clear assessment of the diversity of its component cell types, neurons and glia. C. elegans has an invariant nervous system with the first mapped connectome of a multicellular organism and single-cell atlas of component neurons. Here we present single nuclear RNA-seq evaluation of glia across the entire adult C. elegans nervous system, including both sexes. Machine learning models enabled us to identify both sex-shared and sex-specific glia and glial subclasses. We have identified and validated molecular markers in silico and in vivo for these molecular subcategories. Comparative analytics also reveals previously unappreciated molecular heterogeneity in anatomically identical glia between and within sexes, indicating consequent functional heterogeneity. Furthermore, our datasets reveal that while adult C. elegans glia express neuropeptide genes, they lack the canonical unc-31/CAPS-dependent dense core vesicle release machinery. Thus, glia employ alternate neuromodulator processing mechanisms. Overall, this molecular atlas, available at www.wormglia.org, reveals rich insights into heterogeneity and sex dimorphism in glia across the entire nervous system of an adult animal.

The sigh and related behaviors.

Breathing is a critical, complex, and highly integrated behavior. Normal rhythmic breathing, also referred to as eupnea, is interspersed with different breathing related behaviors. Sighing is one of such behaviors, essential for maintaining effective gas exchange by preventing the gradual collapse of alveoli in the lungs, known as atelectasis. Critical for the generation of both sighing and eupneic breathing is a region of the medulla known as the preBötzinger Complex (preBötC). Efforts are underway to identify the cellular pathways that link sighing as well as sneezing, yawning, and hiccupping with other brain regions to better understand how they are integrated and regulated in the context of other behaviors including chemosensation, olfaction, and cognition. Unraveling these interactions may provide important insights into the diverse roles of these behaviors in the initiation of arousal, stimulation of vigilance, and the relay of certain behavioral states. This chapter focuses primarily on the function of the sigh, how it is locally generated within the preBötC, and what the functional implications are for a potential link between sighing and cognitive regulation. Furthermore, we discuss recent insights gained into the pathways and mechanisms that control yawning, sneezing, and hiccupping.

Inspiratory rhythm generation is stabilized by Ih.

Cellular and network properties must be capable of generating rhythmic activity that is both flexible and stable. This is particularly important for breathing, a rhythmic behavior that dynamically adapts to environmental, behavioral, and metabolic changes from the first to the last breath. The pre-Bötzinger complex (preBötC), located within the ventral medulla, is responsible for producing rhythmic inspiration. Its cellular properties must be tunable, flexible as well as stabilizing. Here, we explore the role of the hyperpolarization-activated, nonselective cation current (Ih) for stabilizing PreBötC activity during opioid exposure and reduced excitatory synaptic transmission. Introducing Ih into an in silico preBötC network predicts that loss of this depolarizing current should significantly slow the inspiratory rhythm. By contrast, in vitro and in vivo experiments revealed that the loss of Ih minimally affected breathing frequency, but destabilized rhythmogenesis through the generation of incompletely synchronized bursts (burstlets). Associated with the loss of Ih was an increased susceptibility of breathing to opioid-induced respiratory depression or weakened excitatory synaptic interactions, a paradoxical depolarization at the cellular level, and the suppression of tonic spiking. Tonic spiking activity is generated by nonrhythmic excitatory and inhibitory preBötC neurons, of which a large percentage express Ih. Together, our results suggest that Ih is important for maintaining tonic spiking, stabilizing inspiratory rhythmogenesis, and protecting breathing against perturbations or changes in network state.NEW & NOTEWORTHY The Ih current plays multiple roles within the preBötC. This current is important for promoting intrinsic tonic spiking activity in excitatory and inhibitory neurons and for preserving rhythmic function during conditions that dampen network excitability, such as in the context of opioid-induced respiratory depression. We therefore propose that the Ih current expands the dynamic range of rhythmogenesis, buffers the preBötC against network perturbations, and stabilizes rhythmogenesis by preventing the generation of unsynchronized bursts.

The psychophysiology of the sigh: I: The sigh from the physiological perspective.

Breathing is composed of multiple, distinct behaviors that are bidirectionally regulated through autonomic and voluntary mechanisms. One behavioral component is the sigh, which serves distinct physiological and psychological roles. In two accompanying reviews we will discuss these roles. The present review focuses on the physiological function, where sighs play a critical role in controlling lung compliance by preventing the collapse of alveoli. Implicated in the generation of sighs and normal breathing is the preBötzinger Complex, a rhythmogenic network in the medulla. Although sighs and normal inspiration are generated within the same network, they show distinct temporal characteristics. While sighs occur every few minutes, normal breathing is generated in the range of seconds. Both are differentiated by distinct modulatory and synaptic mechanisms, and recent evidence indicates that these mechanisms are regulated by inputs from different regions of the brain. An important modulator of sighs is hypoxia, implicating sighs in the arousal response.

Impact of obesity and ovariectomy on respiratory function in female mice.

Obesity and the corresponding variations in female sex hormones are associated with severe lung disease. We determined the potential effects of obesity and sex hormones in female mice by investigating changes in lung structure and respiratory function in an obesity model induced by postnatal overnutrition. Obese female mice exhibited pronounced weight gain, abdominal fat accumulation and collagen type I deposition in the airways. However, neither elastic tissue nor estrogen receptors-α/-ß were affected in obese female mice after ovariectomy or sham-operated mice. Bronchoconstriction in response to methacholine challenge in obese sham-operated mice was higher than in the obese group after ovariectomy. Our results suggest that the coexistence of obesity and ovariectomy impacted on respiratory system and airway resistance (attenuates bronchoconstriction after methacholine), on collagen I deposition and on airway estrogen ß-receptors of mice.

Neuron-astrocyte networking: astrocytes orchestrate and respond to changes in neuronal network activity across brain states and behaviors.

Astrocytes are known to play many important roles in brain function. However, research underscoring the extent to which astrocytes modulate neuronal activity is still underway. Here we review the latest evidence regarding the contribution of astrocytes to neuronal oscillations across the brain, with a specific focus on how astrocytes respond to changes in brain state (e.g., sleep, arousal, stress). We then discuss the general mechanisms by which astrocytes signal to neurons to modulate neuronal activity, ultimately driving changes in behavior, followed by a discussion of how astrocytes contribute to respiratory rhythms in the medulla. Finally, we contemplate the possibility that brain stem astrocytes could modulate brainwide oscillations by communicating the status of oxygenation to higher cortical areas.

A spatially dynamic network underlies the generation of inspiratory behaviors.

The ability of neuronal networks to reconfigure is a key property underlying behavioral flexibility. Networks with recurrent topology are particularly prone to reconfiguration through changes in synaptic and intrinsic properties. Here, we explore spatial reconfiguration in the reticular networks of the medulla that generate breathing. Combined results from in vitro and in vivo approaches demonstrate that the network architecture underlying generation of the inspiratory phase of breathing is not static but can be spatially redistributed by shifts in the balance of excitatory and inhibitory network influences. These shifts in excitation/inhibition allow the size of the active network to expand and contract along a rostrocaudal medullary column during behavioral or metabolic challenges to breathing, such as changes in sensory feedback, sighing, and gasping. We postulate that the ability of this rhythm-generating network to spatially reconfigure contributes to the remarkable robustness and flexibility of breathing.

Advances in cellular and integrative control of oxygen homeostasis within the central nervous system.

Mammals must continuously regulate the levels of O2 and CO2 , which is particularly important for the brain. Failure to maintain adequate O2 /CO2 homeostasis has been associated with numerous disorders including sleep apnoea, Rett syndrome and sudden infant death syndrome. But, O2 /CO2 homeostasis poses major regulatory challenges, even in the healthy brain. Neuronal activities change in a differentiated, spatially and temporally complex manner, which is reflected in equally complex changes in O2 demand. This raises important questions: is oxygen sensing an emergent property, locally generated within all active neuronal networks, and/or the property of specialized O2 -sensitive CNS regions? Increasing evidence suggests that the regulation of the brain's redox state involves properties that are intrinsic to many networks, but that specialized regions in the brainstem orchestrate the integrated control of respiratory and cardiovascular functions. Although the levels of O2 in arterial blood and the CNS are very different, neuro-glial interactions and purinergic signalling are critical for both peripheral and CNS chemosensation. Indeed, the specificity of neuroglial interactions seems to determine the differential responses to O2 , CO2 and the changes in pH.

Targeting TWIST1 through loss of function inhibits tumorigenicity of human glioblastoma.

TWIST1 (TW) is a bHLH transcription factor (TF) and master regulator of the epithelial-to-mesenchymal transition (EMT). In vitro, TW promotes mesenchymal change, invasion, and self-renewal in glioblastoma (GBM) cells. However, the potential therapeutic relevance of TW has not been established through loss-of-function studies in human GBM cell xenograft models. The effects of TW loss of function (gene editing and knockdown) on inhibition of tumorigenicity of U87MG and GBM4 glioma stem cells were tested in orthotopic xenograft models and conditional knockdown in established flank xenograft tumors. RNAseq and the analysis of tumors investigated putative TW-associated mechanisms. Multiple bioinformatic tools revealed significant alteration of ECM, membrane receptors, signaling transduction kinases, and cytoskeleton dynamics leading to identification of PI3K/AKT signaling. We experimentally show alteration of AKT activity and periostin (POSTN) expression in vivo and/or in vitro. For the first time, we show that effect of TW knockout inhibits AKT activity in U87MG cells in vivo independent of PTEN mutation. The clinical relevance of TW and candidate mechanisms was established by analysis of the TCGA and ENCODE databases. TW expression was associated with decreased patient survival and LASSO regression analysis identified POSTN as one of top targets of TW in human GBM. While we previously demonstrated the role of TW in promoting EMT and invasion of glioma cells, these studies provide direct experimental evidence supporting protumorigenic role of TW independent of invasion in vivo and the therapeutic relevance of targeting TW in human GBM. Further, the role of TW driving POSTN expression and AKT signaling suggests actionable targets, which could be leveraged to mitigate the oncogenic effects of TW in GBM.

Twist1 mediated regulation of glioma tumorigenicity is dependent on mode of mouse neural progenitor transformation.

Twist1 is a master regulator of epithelial mesenchymal transition and carcinoma metastasis. Twist1 has also been associated with increased malignancy of human glioma. However, the impact of inhibiting Twist1 on tumorigenicity has not been characterized in glioma models in the context of different oncogenic transformation paradigms. Here we used an orthotopic mouse glioma model of transplanted transformed neural progenitor cells (NPCs) to demonstrate the effects of Twist1 loss of function on tumorigenicity. Decreased tumorigenicity was observed after shRNA mediated Twist knockdown in HPV E6/7 Ha-RasV12 transformed NPCs and Cre mediated Twist1 deletion in Twist1 fl/fl NPCs transformed by p53 knockdown and Ha-RasV12 expression. By contrast, Twist1 deletion had no effect on tumorigenicity of NPCs transformed by co-expression of Akt and Ha-RasV12. We demonstrated a dramatic off-target effect of Twist1 deletion with constitutive Cre expression, which was completely reversed when Twist1 deletion was achieved by transient administration of recombinant Cre protein. Together these findings demonstrate that the function of Twist1 in these models is highly dependent on specific oncogenic contexts of NPC transformation. Therefore, the driver mutational context in which Twist1 functions may need to be taken into account when evaluating mechanisms of action and developing therapeutic approaches to target Twist1 in human gliomas.