Mechanisms of CO2/H+ Sensitivity of Astrocytes.

Ventral regions of the medulla oblongata of the brainstem are populated by astrocytes sensitive to physiological changes in PCO2/[H+]. These astrocytes respond to decreases in pH with elevations in intracellular Ca2+ and facilitated exocytosis of ATP-containing vesicles. Released ATP propagates Ca2+ excitation among neighboring astrocytes and activates neurons of the brainstem respiratory network triggering adaptive increases in breathing. The mechanisms linking increases in extracellular and/or intracellular PCO2/[H+] with Ca2+ responses in chemosensitive astrocytes remain unknown. Fluorescent imaging of changes in [Na+]i and/or [Ca2+]i in individual astrocytes was performed in organotypic brainstem slice cultures and acute brainstem slices of adult rats. It was found that astroglial [Ca2+]i responses triggered by decreases in pH are preceded by Na+ entry, markedly reduced by inhibition of Na+/HCO3- cotransport (NBC) or Na+/Ca2+ exchange (NCX), and abolished in Na+-free medium or by combined NBC/NCX blockade. Acidification-induced [Ca2+]i responses were also dramatically reduced in brainstem astrocytes of mice deficient in the electrogenic Na+/HCO3- cotransporter NBCe1. Sensitivity of astrocytes to changes in pH was not affected by inhibition of Na+/H+ exchange or blockade of phospholipase C. These results suggest that in pH-sensitive astrocytes, acidification activates NBCe1, which brings Na+ inside the cell. Raising [Na+]i activates NCX to operate in a reverse mode, leading to Ca2+ entry followed by activation of downstream signaling pathways. Coupled NBC and NCX activities are, therefore, suggested to be responsible for functional CO2/H+ sensitivity of astrocytes that contribute to homeostatic regulation of brain parenchymal pH and control of breathing. SIGNIFICANCE STATEMENT: Brainstem astrocytes detect physiological changes in pH, activate neurons of the neighboring respiratory network, and contribute to the development of adaptive respiratory responses to the increases in the level of blood and brain PCO2/[H+]. The mechanisms underlying astroglial pH sensitivity remained unknown and here we show that in brainstem astrocytes acidification activates Na+/HCO3- cotransport, which brings Na+ inside the cell. Raising [Na+]i activates the Na+/Ca2+ exchanger to operate in a reverse mode leading to Ca2+ entry. This identifies a plausible mechanism of functional CO2/H+ sensitivity of brainstem astrocytes, which play an important role in homeostatic regulation of brain pH and control of breathing.

Astrocytes and Brain Hypoxia.

Astrocytes provide the structural and functional interface between the cerebral circulation and neuronal networks. They enwrap all intracerebral arterioles and capillaries, control the flux of nutrients as well as the ionic and metabolic environment of the neuropil. Astrocytes have the ability to adjust cerebral blood flow to maintain constant PO2 and PCO2 of the brain parenchyma. Release of ATP in the brainstem, presumably by local astrocytes, helps to maintain breathing and counteract hypoxia-induced depression of the respiratory network. Astrocytes also appear to be involved in mediating hypoxia-evoked changes in blood-brain barrier permeability, brain inflammation, and neuroprotection against ischaemic injury. Thus, astrocytes appear to play a fundamental role in supporting neuronal function not only in normal conditions but also in pathophysiological states when supply of oxygen to the brain is compromised.

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.

Brainstem hypoxia contributes to the development of hypertension in the spontaneously hypertensive rat.

Systemic arterial hypertension has been previously suggested to develop as a compensatory condition when central nervous perfusion/oxygenation is compromised. Principal sympathoexcitatory C1 neurons of the rostral ventrolateral medulla oblongata (whose activation increases sympathetic drive and the arterial blood pressure) are highly sensitive to hypoxia, but the mechanisms of this O2 sensitivity remain unknown. Here, we investigated potential mechanisms linking brainstem hypoxia and high systemic arterial blood pressure in the spontaneously hypertensive rat. Brainstem parenchymal PO2 in the spontaneously hypertensive rat was found to be ≈15 mm Hg lower than in the normotensive Wistar rat at the same level of arterial oxygenation and systemic arterial blood pressure. Hypoxia-induced activation of rostral ventrolateral medulla oblongata neurons was suppressed in the presence of either an ATP receptor antagonist MRS2179 or a glycogenolysis inhibitor 1,4-dideoxy-1,4-imino-d-arabinitol, suggesting that sensitivity of these neurons to low PO2 is mediated by actions of extracellular ATP and lactate. Brainstem hypoxia triggers release of lactate and ATP which produce excitation of C1 neurons in vitro and increases sympathetic nerve activity and arterial blood pressure in vivo. Facilitated breakdown of extracellular ATP in the rostral ventrolateral medulla oblongata by virally-driven overexpression of a potent ectonucleotidase transmembrane prostatic acid phosphatase results in a significant reduction in the arterial blood pressure in the spontaneously hypertensive rats (but not in normotensive animals). These results suggest that in the spontaneously hypertensive rat, lower PO2 of brainstem parenchyma may be associated with higher levels of ambient ATP and l-lactate within the presympathetic circuits, leading to increased central sympathetic drive and concomitant sustained increases in systemic arterial blood pressure.

Silicon dioxide thin film mediated single cell nucleic acid isolation.

A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube.

Differential sensitivity of brainstem versus cortical astrocytes to changes in pH reveals functional regional specialization of astroglia.

Astrocytes might function as brain interoceptors capable of detecting different (chemo)sensory modalities and transmitting sensory information to the relevant neural networks controlling vital functions. For example, astrocytes that reside near the ventral surface of the brainstem (central respiratory chemosensitive area) respond to physiological decreases in pH with vigorous elevations in intracellular Ca(2+) and release of ATP. ATP transmits astroglial excitation to the brainstem respiratory network and contributes to adaptive changes in lung ventilation. Here we show that in terms of pH-sensitivity, ventral brainstem astrocytes are clearly distinct from astrocytes residing in the cerebral cortex. We monitored vesicular fusion in cultured rat brainstem astrocytes using total internal reflection fluorescence microscopy and found that ∼35% of them respond to acidification with an increased rate of exocytosis of ATP-containing vesicular compartments. These fusion events require intracellular Ca(2+) signaling and are independent of autocrine ATP actions. In contrast, the rate of vesicular fusion in cultured cortical astrocytes is not affected by changes in pH. Compared to cortical astrocytes, ventral brainstem astrocytes display higher levels of expression of genes encoding proteins associated with ATP vesicular transport and fusion, including vesicle-associated membrane protein-3 and vesicular nucleotide transporter. These results suggest that astrocytes residing in different parts of the rat brain are functionally specialized. In contrast to cortical astrocytes, astrocytes of the brainstem chemosensitive area(s) possess signaling properties that are functionally relevant-they are able to sense changes in pH and respond to acidification with enhanced vesicular release of ATP.

Astrocytes control breathing through pH-dependent release of ATP.

Astrocytes provide structural and metabolic support for neuronal networks, but direct evidence demonstrating their active role in complex behaviors is limited. Central respiratory chemosensitivity is an essential mechanism that, via regulation of breathing, maintains constant levels of blood and brain pH and partial pressure of CO2. We found that astrocytes of the brainstem chemoreceptor areas are highly chemosensitive. They responded to physiological decreases in pH with vigorous elevations in intracellular Ca2+ and release of adenosine triphosphate (ATP). ATP propagated astrocytic Ca2+ excitation, activated chemoreceptor neurons, and induced adaptive increases in breathing. Mimicking pH-evoked Ca2+ responses by means of optogenetic stimulation of astrocytes expressing channelrhodopsin-2 activated chemoreceptor neurons via an ATP-dependent mechanism and triggered robust respiratory responses in vivo. This demonstrates a potentially crucial role for brain glial cells in mediating a fundamental physiological reflex.

beta1-Adrenoceptor distribution in the rat brain: an immunohistochemical study.

Current knowledge of the central nervous system distribution of the beta(1)-adrenergic receptors (beta(1)-AR) is incomplete. Here we present a general map of the beta(1)-AR distribution in the rat brain. beta(1)-AR-immunoreactivity was detected throughout the entire rat brain, but particularly dense staining was observed in the cerebellar cortex and basal ganglia. Brainstem areas displaying significant beta(1)-AR-immunoreactivity include the ventrolateral medulla, nucleus ambiguus and the nucleus of the solitary tract. Within the hypothalamus, only the paraventricular nucleus and the median eminence (ME) showed beta(1)-AR immunostaining. Numerous beta(1)-AR-immunoreactive cells were also found in the hippocampus, basal ganglia and cerebral cortex. These results extend our knowledge of the expression profile of beta(1)-AR in the central nervous system. The identification of several distinct beta(1)-AR immunoreactive substrates linked with neuropathophysiological roles in cardiovascular disease supports the hypothesis that the therapeutic benefit of beta(1)-AR blockade may be conferred at least in part through central nervous system mechanisms.

Release of mitochondrial DNA fragments from brain mitochondria of irradiated mice.

Recently, we demonstrated that the release of mtDNA fragments from mitochondria occurs as a result of the opening of a non-specific pore in the inner mitochondrial membrane. Here, we show that irradiation of mice stimulates the appearance of mtDNA fragments in the cytosolic fractions of the brain. The fragments of mtDNA were found as early as 1h after irradiation, when no observable alteration of mitochondrial functioning occurred. The involvement of mitochondrial permeability transition in this process is discussed.