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 which, via regulation of breathing, maintains constant levels of blood and brain PCO 2 /pH. We found that astrocytes of the brainstem chemoreceptor areas are highly chemosensitive. They responded to physiological decreases in pH with vigorous elevations in intracellular Ca 2+ and release of ATP. ATP propagated astrocytic Ca 2+ excitation, activated chemoreceptor neurons, and induced adaptive increases in breathing. Mimicking pHevoked Ca 2+ responses by optogenetic stimulation of astrocytes expressing channelrhodopsin-2 activated chemoreceptor neurons via 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.The role of astrocytes in the brain is by no means limited to just providing structural and metabolic support to neurons. Astrocytes are closely associated with cerebral blood vessels and are thought to regulate cerebrovascular tone adjusting blood supply to match local metabolic demands(1-6). A single astrocyte may enwrap several neuronal somata(7) and make contact with thousands of synapses(8), potentially regulating synaptic strength and information processing (4,(9)(10)(11)(12)(13)(14). However, direct evidence demonstrating the functional role of astrocytes in complex behaviors is only starting to emerge(15).Astrocytes provide a vascular-neuronal interface and are in a position to quickly relay bloodborne stimuli to the activities of neuronal networks. Does this have a functional significance for the detection of the relevant stimuli by brain chemosensors which monitor key homeostatic parameters including glucose concentration, pH and PCO 2 ? Here we tested the * This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS. Fig 1a). Subsequent histological examination of the chemoresponsive areas confirmed contacts of transduced astrocytes with pia mater and penetrating arterioles ( fig. S3). Supporting Online MaterialPropagating Ca 2+ excitation of ventral medullary astrocytes in response to acidification was also observed in different in vitro preparations such as acute horizontal brainstem slices of adult rats (n = 56 slices; Fig In brainstem slices of adult rats, in which blood vessels were visualized with lectin, many pH-sensitive astrocytes were found to be located adjacent to the VS vasculature (Fig. 1f).Acidification-induced Ca 2+ excitation of VS astrocytes is unlikely to be secondary to increased activity of local neurons. To minimize ne...

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 (P O 2 ) 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 P

Astrocytes produce and release L-lactate as a potential source of energy for neurons. Here we present evidence that L-lactate, independently of its caloric value, serves as an astrocytic signalling molecule in the locus coeruleus (LC). The LC is the principal source of norepinephrine to the frontal brain and thus one of the most influential modulatory centers of the brain. Optogenetically activated astrocytes release L-lactate, which excites LC neurons and triggers release of norepinephrine. Exogenous L-lactate within the physiologically relevant concentration range mimics these effects. L-lactate effects are concentration-dependent, stereo-selective, independent of L-lactate uptake into neurons and involve a cAMP-mediated step. In vivo injections of L-lactate in the LC evokes arousal similar to the excitatory transmitter, L-glutamate. Our results imply the existence of an unknown receptor for this ‘glio-transmitter’.

BackgroundUsing cell-type-specific promoters to restrict gene expression to particular cells is an attractive approach for gene therapy, but often hampered by insufficient transcriptional activity of these promoters. Previous studies have shown that transcriptional amplification strategy (TAS) can be used to enhance the activity of such promoters without loss of cell type specificity. Originally TAS involved the use of two copies of a cell-specific promoter leading to generation of large expression cassettes, which can be hard to use given the space limitations of the conventional viral gene expression vectors.ResultsWe have now developed a new bidirectional lentiviral vector system, based on TAS that can enhance the transcriptional activity of human synapsin-1 (SYN) promoter and the compact glial fibrillary acidic protein (GfaABC1D) promoter. In the opposite orientation, a minimal core promoter (65 bp) derived from the human cytomegalovirus (CMV) was joined upstream of the SYN promoter or GfaABC1D promoter. This led to the formation of synthetic bidirectional promoters which were flanked with two gene expression cassettes. The 5' cassette transcribed the artificial transcriptional activator. The downstream cassette drove the synthesis of the gene of interest. Studies in both cell cultures and in vivo showed that the new bidirectional promoters greatly increased the expression level of the reporter gene. In vivo studies also showed that transgene expression was enhanced without loss of cell specificity of both SYN and GfaABC1D promoters.ConclusionThis work establishes a novel approach for creating compact TAS-amplified cell-specific promoters, a feature important for their use in viral backbones. This improved approach should prove useful for the development of powerful gene expression systems based on weak cell-specific promoters.

Astrocytes are implicated in modulation of neuronal excitability and synaptic function, but it remains unknown if these glial cells can directly control activities of motor circuits to influence complex behaviors in vivo. This study focused on the vital respiratory rhythmgenerating circuits of the preBötzinger complex (preBötC) and determined how compromised function of local astrocytes affects breathing in conscious experimental animals (rats). Vesicular release mechanisms in astrocytes were disrupted by virally driven expression of either the dominant-negative SNARE protein or light chain of tetanus toxin. We show that blockade of vesicular release in preBötC astrocytes reduces the resting breathing rate and frequency of periodic sighs, decreases rhythm variability, impairs respiratory responses to hypoxia and hypercapnia, and dramatically reduces the exercise capacity. These findings indicate that astrocytes modulate the activity of CNS circuits generating the respiratory rhythm, critically contribute to adaptive respiratory responses in conditions of increased metabolic demand and determine the exercise capacity.