Contributions of carotid bodies, retrotrapezoid nucleus neurons and preBötzinger complex astrocytes to the CO2 -sensitive drive for breathing.

Current models of respiratory CO2 chemosensitivity are centred around the function of a specific population of neurons residing in the medullary retrotrapezoid nucleus (RTN). However, there is significant evidence suggesting that chemosensitive neurons exist in other brainstem areas, including the rhythm-generating region of the medulla oblongata - the preBötzinger complex (preBötC). There is also evidence that astrocytes, non-neuronal brain cells, contribute to central CO2 chemosensitivity. In this study, we reevaluated the relative contributions of the RTN neurons, the preBötC astrocytes, and the carotid body chemoreceptors in mediating the respiratory responses to CO2 in experimental animals (adult laboratory rats). To block astroglial signalling via exocytotic release of transmitters, preBötC astrocytes were targeted to express the tetanus toxin light chain (TeLC). Bilateral expression of TeLC in preBötC astrocytes was associated with ∼20% and ∼30% reduction of the respiratory response to CO2 in conscious and anaesthetized animals, respectively. Carotid body denervation reduced the CO2 respiratory response by ∼25%. Bilateral inhibition of RTN neurons transduced to express Gi-coupled designer receptors exclusively activated by designer drug (DREADDGi ) by application of clozapine-N-oxide reduced the CO2 response by ∼20% and ∼40% in conscious and anaesthetized rats, respectively. Combined blockade of astroglial signalling in the preBötC, inhibition of RTN neurons and carotid body denervation reduced the CO2 -induced respiratory response by ∼70%. These data further support the hypothesis that the CO2 -sensitive drive to breathe requires inputs from the peripheral chemoreceptors and several central chemoreceptor sites. At the preBötC level, astrocytes modulate the activity of the respiratory network in response to CO2 , either by relaying chemosensory information (i.e. they act as CO2  sensors) or by enhancing the preBötC network excitability to chemosensory inputs. KEY POINTS: This study reevaluated the roles played by the carotid bodies, neurons of the retrotrapezoid nucleus (RTN) and astrocytes of the preBötC in mediating the CO2 -sensitive drive to breathe. The data obtained show that disruption of preBötC astroglial signalling, blockade of inputs from the peripheral chemoreceptors or inhibition of RTN neurons similarly reduce the respiratory response to hypercapnia. These data provide further support for the hypothesis that the CO2 -sensitive drive to breathe is mediated by the inputs from the peripheral chemoreceptors and several central chemoreceptor sites.

Astrocytic modulation of central pattern generating motor circuits.

Central pattern generators (CPGs) generate the rhythmic and coordinated neural features necessary for the proper conduction of complex behaviors. In particular, CPGs are crucial for complex motor behaviors such as locomotion, mastication, respiration, and vocal production. While the importance of these networks in modulating behavior is evident, the mechanisms driving these CPGs are still not fully understood. On the other hand, accumulating evidence suggests that astrocytes have a significant role in regulating the function of some of these CPGs. Here, we review the location, function, and role of astrocytes in locomotion, respiration, and mastication CPGs and propose that, similarly, astrocytes may also play a significant role in the vocalization CPG.

Regulation of myelin structure and conduction velocity by perinodal astrocytes.

The speed of impulse transmission is critical for optimal neural circuit function, but it is unclear how the appropriate conduction velocity is established in individual axons. The velocity of impulse transmission is influenced by the thickness of the myelin sheath and the morphology of electrogenic nodes of Ranvier along axons. Here we show that myelin thickness and nodal gap length are reversibly altered by astrocytes, glial cells that contact nodes of Ranvier. Thrombin-dependent proteolysis of a cell adhesion molecule that attaches myelin to the axon (neurofascin 155) is inhibited by vesicular release of thrombin protease inhibitors from perinodal astrocytes. Transgenic mice expressing a dominant-negative fragment of VAMP2 in astrocytes, to reduce exocytosis by 50%, exhibited detachment of adjacent paranodal loops of myelin from the axon, increased nodal gap length, and thinning of the myelin sheath in the optic nerve. These morphological changes alter the passive cable properties of axons to reduce conduction velocity and spike-time arrival in the CNS in parallel with a decrease in visual acuity. All effects were reversed by the thrombin inhibitor Fondaparinux. Similar results were obtained by viral transfection of tetanus toxin into astrocytes of rat corpus callosum. Previously, it was unknown how the myelin sheath could be thinned and the functions of perinodal astrocytes were not well understood. These findings describe a form of nervous system plasticity in which myelin structure and conduction velocity are adjusted by astrocytes. The thrombin-dependent cleavage of neurofascin 155 may also have relevance to myelin disruption and repair.

Scientists, society, and stuttering.

More than 70 million people worldwide are affected by developmental stuttering. It is important to reach out to the public, scientific and medical communities, and those who stutter with a goal to raise awareness about stuttering. In this short perspective, we argue that to educate, advocate, and spread awareness about stuttering, we need role models, support, and opportunities.

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

KEY POINTS: 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. ABSTRACT: 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.

Brain metabolic sensing and metabolic signaling at the level of an astrocyte.

Astrocytes support neuronal function by providing essential structural and nutritional support, neurotransmitter trafficking and recycling and may also contribute to brain information processing. In this article we review published results and report new data suggesting that astrocytes function as versatile metabolic sensors of central nervous system (CNS) milieu and play an important role in the maintenance of brain metabolic homeostasis. We discuss anatomical and functional features of astrocytes that allow them to detect and respond to changes in the brain parenchymal levels of metabolic substrates (oxygen and glucose), and metabolic waste products (carbon dioxide). We report data suggesting that astrocytes are also sensitive to circulating endocrine signals-hormones like ghrelin, glucagon-like peptide-1 and leptin, that have a major impact on the CNS mechanisms controlling food intake and energy balance. We discuss signaling mechanisms that mediate communication between astrocytes and neurons and consider how these mechanisms are recruited by astrocytes activated in response to various metabolic challenges. We review experimental data suggesting that astrocytes modulate the activities of the respiratory and autonomic neuronal networks that ensure adaptive changes in breathing and sympathetic drive in order to support the physiological and behavioral demands of the organism in ever-changing environmental conditions. Finally, we discuss evidence suggesting that altered astroglial function may contribute to the pathogenesis of disparate neurological, respiratory and cardiovascular disorders such as Rett syndrome and systemic arterial hypertension.

Functional Oxygen Sensitivity of Astrocytes.

In terrestrial mammals, the oxygen storage capacity of the CNS is limited, and neuronal function is rapidly impaired if oxygen supply is interrupted even for a short period of time. However, oxygen tension monitored by the peripheral (arterial) chemoreceptors is not sensitive to regional CNS differences in partial pressure of oxygen (PO2 ) that reflect variable levels of neuronal activity or local tissue hypoxia, pointing to the necessity of a functional brain oxygen sensor. This experimental animal (rats and mice) study shows that astrocytes, the most numerous brain glial cells, are sensitive to physiological changes in PO2 . Astrocytes respond to decreases in PO2 a few millimeters of mercury below normal brain oxygenation with elevations in intracellular calcium ([Ca(2+)]i). The hypoxia sensor of astrocytes resides in the mitochondria in which oxygen is consumed. Physiological decrease in PO2 inhibits astroglial mitochondrial respiration, leading to mitochondrial depolarization, production of free radicals, lipid peroxidation, activation of phospholipase C, IP3 receptors, and release of Ca(2+) from the intracellular stores. Hypoxia-induced [Ca(2+)]i increases in astrocytes trigger fusion of vesicular compartments containing ATP. Blockade of astrocytic signaling by overexpression of ATP-degrading enzymes or targeted astrocyte-specific expression of tetanus toxin light chain (to interfere with vesicular release mechanisms) within the brainstem respiratory rhythm-generating circuits reveals the fundamental physiological role of astroglial oxygen sensitivity; in low-oxygen conditions (environmental hypoxia), this mechanism increases breathing activity even in the absence of peripheral chemoreceptor oxygen sensing. These results demonstrate that astrocytes are functionally specialized CNS oxygen sensors tuned for rapid detection of physiological changes in brain oxygenation. Significance statement: Most, if not all, animal cells possess mechanisms that allow them to detect decreases in oxygen availability leading to slow-timescale, adaptive changes in gene expression and cell physiology. To date, only two types of mammalian cells have been demonstrated to be specialized for rapid functional oxygen sensing: glomus cells of the carotid body (peripheral respiratory chemoreceptors) that stimulate breathing when oxygenation of the arterial blood decreases; and pulmonary arterial smooth muscle cells responsible for hypoxic pulmonary vasoconstriction to limit perfusion of poorly ventilated regions of the lungs. Results of the present study suggest that there is another specialized oxygen-sensitive cell type in the body, the astrocyte, that is tuned for rapid detection of physiological changes in brain oxygenation.

Morphological deficits of glial cells in a transgenic mouse model for developmental stuttering.

Vocal production involves intricate neural coordination across various brain regions. Stuttering, a common speech disorder, has genetic underpinnings, including mutations in lysosomal-targeting pathway genes. Using a Gnptab-mutant mouse model linked to stuttering, we examined neuron and glial cell morphology in vocal production circuits. Our findings revealed altered astrocyte and microglia processes in these circuits in Gnptab-mutant mice, while control regions remained unaffected. Our results shed light on the potential role of glial cells in stuttering pathophysiology and highlight their relevance in modulating vocal production behaviors.

Stuttering as a spectrum disorder: A hypothesis.

Childhood-onset fluency disorder, commonly referred to as stuttering, affects over 70 million adults worldwide. While stuttering predominantly initiates during childhood and is more prevalent in males, it presents consistent symptoms during conversational speech. Despite these common clinical manifestations, evidence suggests that stuttering, may arise from different etiologies, emphasizing the need for personalized therapy approaches. Current research models often regard the stuttering population as a singular, homogenous group, potentially overlooking the inherent heterogeneity. This perspective consolidates both historical and recent observations to emphasize that stuttering is a heterogeneous condition with diverse causes. As such, it is crucial that both therapeutic research and clinical practices consider the potential for varied etiologies leading to stuttering. Recognizing stuttering as a spectrum disorder embraces its inherent variability, allowing for a more nuanced categorization of individuals based on the underlying causes. This perspective aligns with the principles of precision medicine, advocating for tailored treatments for distinct subgroups of people who stutter, ultimately leading to personalized therapeutic approaches.

Whole-brain analysis of CO2 chemosensitive regions and identification of the retrotrapezoid and medullary raphé nuclei in the common marmoset (Callithrix jacchus).

Respiratory chemosensitivity is an important mechanism by which the brain senses changes in blood partial pressure of CO2 (PCO2). It is proposed that special neurons (and astrocytes) in various brainstem regions play key roles as CO2 central respiratory chemosensors in rodents. Although common marmosets (Callithrix jacchus), New-World non-human primates, show similar respiratory responses to elevated inspired CO2 as rodents, the chemosensitive regions in marmoset brain have not been defined yet. Here, we used c-fos immunostainings to identify brain-wide CO2-activated brain regions in common marmosets. In addition, we mapped the location of the retrotrapezoid nucleus (RTN) and raphé nuclei in the marmoset brainstem based on colocalization of CO2-induced c-fos immunoreactivity with Phox2b, and TPH immunostaining, respectively. Our data also indicated that, similar to rodents, marmoset RTN astrocytes express Phox2b and have complex processes that create a meshwork structure at the ventral surface of medulla. Our data highlight some cellular and structural regional similarities in brainstem of the common marmosets and rodents.

Morphometric analysis of astrocytes in vocal production circuits of common marmoset (Callithrix jacchus).

Astrocytes, the star-shaped glial cells, are the most abundant non-neuronal cell population in the central nervous system. They play a key role in modulating activities of neural networks, including those involved in complex motor behaviors. Common marmosets (Callithrix jacchus), the most vocal non-human primate (NHP), have been used to study the physiology of vocalization and social vocal production. However, the neural circuitry involved in vocal production is not fully understood. In addition, even less is known about the involvement of astrocytes in this circuit. To understand the role, that astrocytes may play in the complex behavior of vocalization, the initial step may be to study their structural properties in the cortical and subcortical regions that are known to be involved in vocalization. Here, in the common marmoset, we identify all astrocytic subtypes seen in other primate's brains, including intralaminar astrocytes. In addition, we reveal detailed structural characteristics of astrocytes and perform morphometric analysis of astrocytes residing in the cortex and midbrain regions that are associated with vocal production. We found that cortical astrocytes in these regions illustrate a higher level of complexity when compared to those in the midbrain. We hypothesize that this complexity that is expressed in cortical astrocytes may reflect their functions to meet the metabolic/structural needs of these regions.

An open-source tool for automated analysis of breathing behaviors in common marmosets and rodents.

The respiratory system maintains homeostatic levels of oxygen (O2) and carbon dioxide (CO2) in the body through rapid and efficient regulation of breathing frequency and depth (tidal volume). The commonly used methods of analyzing breathing data in behaving experimental animals are usually subjective, laborious, and time-consuming. To overcome these hurdles, we optimized an analysis toolkit for the unsupervised study of respiratory activities in animal subjects. Using this tool, we analyzed breathing behaviors of the common marmoset (Callithrix jacchus), a New World non-human primate model. Using whole-body plethysmography in room air as well as acute hypoxic (10% O2) and hypercapnic (6% CO2) conditions, we describe breathing behaviors in awake, freely behaving marmosets. Our data indicate that marmosets' exposure to acute hypoxia decreased metabolic rate and increased sigh rate. However, the hypoxic condition did not augment ventilation. Hypercapnia, on the other hand, increased both the frequency and depth (i.e., tidal volume) of breathing.

Investigation of Risperidone Treatment Associated With Enhanced Brain Activity in Patients Who Stutter.

Stuttering is a childhood onset fluency disorder that leads to impairment in speech. A randomized, double-blinded placebo-controlled study was conducted with 10 adult subjects to observe the effects of risperidone (a dopamine receptor 2/serotonin receptor 2 antagonist) on brain metabolism, using [18F] deoxyglucose as the marker. At baseline and after 6 weeks of taking risperidone (0.5-2.0 mg/day) or a placebo pill, participants were assigned to a solo reading aloud task for 30 min and subsequently underwent a 90-min positron emission tomography scan. Paired t-tests were performed to compare the pre-treatment vs. post-treatment in groups. After imaging and analysis, the blind was broken, which revealed an equal number of subjects of those on risperidone and those on placebo. There were no significant differences in the baseline scans taken before medication randomization. However, scans taken after active treatment demonstrated higher glucose uptake in the specific regions of the brain for those in the risperidone treatment group (p < 0.05). Risperidone treatment was associated with increased metabolism in the left striatum, which consists of the caudate and putamen, and the Broca's area. The current study strengthens previous research that suggests the role of elevated dopamine activity and striatal hypometabolism in stuttering. We propose that the mechanism of risperidone's action in stuttering, in part, involves increased metabolism of striatal astrocytes. We conclude that using neuroimaging techniques to visualize changes in the brain of those who stutter can provide valuable insights into the pathophysiology of the disorder and guide the development of future interventions.

Dopamine, vocalization, and astrocytes.

Dopamine, the main catecholamine neurotransmitter in the brain, is predominately produced in the basal ganglia and released to various brain regions including the frontal cortex, midbrain and brainstem. Dopamine's effects are widespread and include modulation of a number of voluntary and innate behaviors. Vigilant regulation and modulation of dopamine levels throughout the brain is imperative for proper execution of motor behaviors, in particular speech and other types of vocalizations. While dopamine's role in motor circuitry is widely accepted, its unique function in normal and abnormal speech production is not fully understood. In this perspective, we first review the role of dopaminergic circuits in vocal production. We then discuss and propose the conceivable involvement of astrocytes, the numerous star-shaped glia cells of the brain, in the dopaminergic network modulating normal and abnormal vocal productions.

Physiology: New Insights into Central Oxygen Sensing.

Astrocytes, the star-shaped brain cells, are known chemosensitive cells in the respiratory system. A new study shows that trafficking of TRPA1 channels in and out of the cell membrane in brainstem astrocytes contributes to their role as central respiratory oxygen sensors.

Synuclein Deficiency Results in Age-Related Respiratory and Cardiovascular Dysfunctions in Mice.

Synuclein (α, ß, and γ) proteins are highly expressed in presynaptic terminals, and significant data exist supporting their role in regulating neurotransmitter release. Targeting the gene encoding α-synuclein is the basis of many animal models of Parkinson's disease (PD). However, the physiological role of this family of proteins in not well understood and could be especially relevant as interfering with accumulation of α-synuclein level has therapeutic potential in limiting PD progression. The long-term effects of their removal are unknown and given the complex pathophysiology of PD, could exacerbate other clinical features of the disease, for example dysautonomia. In the present study, we sought to characterize the autonomic phenotypes of mice lacking all synucleins (α, ß, and γ; αßγ-/-) in order to better understand the role of synuclein-family proteins in autonomic function. We probed respiratory and cardiovascular reflexes in conscious and anesthetized, young (4 months) and aged (18-20 months) αßγ-/- male mice. Aged mice displayed impaired respiratory responses to both hypoxia and hypercapnia when breathing activities were recorded in conscious animals using whole-body plethysmography. These animals were also found to be hypertensive from conscious blood pressure recordings, to have reduced pressor baroreflex gain under anesthesia, and showed reduced termination of both pressor and depressor reflexes. The present data demonstrate the importance of synuclein in the normal function of respiratory and cardiovascular reflexes during aging.

Astrocytes monitor cerebral perfusion and control systemic circulation to maintain brain blood flow.

Astrocytes provide neurons with essential metabolic and structural support, modulate neuronal circuit activity and may also function as versatile surveyors of brain milieu, tuned to sense conditions of potential metabolic insufficiency. Here we show that astrocytes detect falling cerebral perfusion pressure and activate CNS autonomic sympathetic control circuits to increase systemic arterial blood pressure and heart rate with the purpose of maintaining brain blood flow and oxygen delivery. Studies conducted in experimental animals (laboratory rats) show that astrocytes respond to acute decreases in brain perfusion with elevations in intracellular [Ca2+]. Blockade of Ca2+-dependent signaling mechanisms in populations of astrocytes that reside alongside CNS sympathetic control circuits prevents compensatory increases in sympathetic nerve activity, heart rate and arterial blood pressure induced by reductions in cerebral perfusion. These data suggest that astrocytes function as intracranial baroreceptors and play an important role in homeostatic control of arterial blood pressure and brain blood flow.

The Role Of Parafacial Neurons In The Control Of Breathing During Exercise.

Neuronal cell groups residing within the retrotrapezoid nucleus (RTN) and C1 area of the rostral ventrolateral medulla oblongata contribute to the maintenance of resting respiratory activity and arterial blood pressure, and play an important role in the development of cardiorespiratory responses to metabolic challenges (such as hypercapnia and hypoxia). In rats, acute silencing of neurons within the parafacial region which includes the RTN and the rostral aspect of the C1 circuit (pFRTN/C1), transduced to express HM4D (Gi-coupled) receptors, was found to dramatically reduce exercise capacity (by 60%), determined by an intensity controlled treadmill running test. In a model of simulated exercise (electrical stimulation of the sciatic or femoral nerve in urethane anaesthetised spontaneously breathing rats) silencing of the pFRTN/C1 neurons had no effect on cardiovascular changes, but significantly reduced the respiratory response during steady state exercise. These results identify a neuronal cell group in the lower brainstem which is critically important for the development of the respiratory response to exercise and, determines exercise capacity.