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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 (I(h)) for stabilizing PreBötC activity during opioid exposure and reduced excitatory synaptic transmission. Introducing I(h) 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 I(h) minimally affected breathing frequency, but destabilized rhythmogenesis through the generation of incompletely synchronized bursts (burstlets). Associated with the loss of I(h) 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 I(h). Together, our results suggest that I(h) is important for maintaining tonic spiking, stabilizing inspiratory rhythmogenesis, and protecting breathing against perturbations or changes in network state. NEW & NOTEWORTHY The I(h) 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 I(h) current expands the dynamic range of rhythmogenesis, buffers the preBötC against network perturbations, and stabilizes rhythmogenesis by preventing the generation of unsynchronized bursts.

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.

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.

Significance Breathing is a vital rhythmic behavior that originates from neural networks within the brainstem. It is hypothesized that the breathing rhythm is generated by spatially distinct networks localized to discrete kernels or compartments. Here, we provide evidence that the functional boundaries of these compartments expand and contract dynamically based on behavioral or physiological demands. The ability of these rhythmic networks to change in size may allow the breathing rhythm to be very reliable, yet flexible enough to accommodate the large repertoire of mammalian behaviors that must be integrated with breathing., 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.

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/-β 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 β-receptors of mice.

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. Open in a separate window

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 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.

Spinal cord trauma leads to destruction of the highly organized cytoarchitecture that carries information along the axis of the spinal column. Currently, there are no clinically accepted strategies that can help regenerate severed axons after spinal cord injury. Experimental neuroregenerative efforts include the creation of optimal biomaterials with aligned topography to support enhanced neuronal regeneration. Hydrogels are soft biomaterials with high water content that are widely used as scaffolds to interface with the central nervous system (CNS). Current available methods to create topography within the 3D amorphous hydrogels are typically complex. Here we examine a simple and reproducible method that results in consistently aligned fibrils within 3D matrices using thermally gelling biomimetic polymers that are compatible with neuronal cells. A collagen type I (Col)-based thermally gelling hydrogel system was used in combination with two other native extracellular matrix proteins: laminin I (LN), and hyaluronic acid (HA). Gelling kinetics for all gel types (Col, Col LN, Col HA) were examined, and we found that all three combinations of polymer formed consistent gels at 37°C. Col solution was faster to form gels (17 min), while Col LN and Col HA took longer (~22 minutes). A method of aspiration and ejection was used to produce Col-based hydrogels containing aligned fibrils. Gels were then examined using scanning electron microscopy (SEM). SEM images confirmed successful alignment in all gel types, and the size of fibers was consistent with reported values for collagen (~250 nm in diameter). We found that embryonic spinal cord neurons survive and produce processes aligned to collagen fibrils after 14 days in vitro. Next, we investigated the functionality of aligned and non-aligned Col hydrogels implanted acutely after a contusion type spinal cord injury to the thoracic spinal cord at T7/T8. Pan neuronal antibody-positive fibrils were found within all implants, aligned hydrogels supported neurite growth along the parallel direction of the implanted hydrogels. Our data indicate that thermally gelling biomimetic hydrogels can produce aligned matrices by a method of aspiration and ejection. The material composition and process of aligning hydrogels outlined here presents a novel platform for regenerative therapies for the CNS that is compatible with the survival and growth of neuronal cells in vitro and in vivo.

Mammals must continuously regulate the levels of O(2) and CO(2), which is particularly important for the brain. Failure to maintain adequate O(2)/CO(2) homeostasis has been associated with numerous disorders including sleep apnoea, Rett syndrome and sudden infant death syndrome. But, O(2)/CO(2) 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 O(2) demand. This raises important questions: is oxygen sensing an emergent property, locally generated within all active neuronal networks, and/or the property of specialized O(2)‐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 O(2) 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 O(2), CO(2) and the changes in pH. [Image: see text]