No abstract

An extensive literature shows that astrocytes exhibit metabotropic glutamate receptor 5 (mGluR5)–dependent increases in cytosolic calcium ions (Ca2+) in response to glutamatergic transmission and, in turn, modulate neuronal activity by their Ca2+-dependent release of gliotransmitters. These findings, based on studies of young rodents, have led to the concept of the tripartite synapse, in which astrocytes actively participate in neurotransmission. Using genomic analysis, immunoelectron microscopy, and two-photon microscopy of astrocytic Ca2+ signaling in vivo, we found that astrocytic expression of mGluR5 is developmentally regulated and is undetectable after postnatal week 3. In contrast, mGluR3, whose activation inhibits adenylate cyclase but not calcium signaling, was expressed in astrocytes at all developmental stages. Neuroglial signaling in the adult brain may therefore occur in a manner fundamentally distinct from that exhibited during development.

Astrocytes have in recent years become the focus of intense experimental interest, yet markers for their definitive identification remain both scarce and imperfect. Astrocytes may be recognized as such by their expression of glial fibrillary acidic protein, glutamine synthetase, glutamate transporter 1 (GLT1), aquaporin-4, aldehyde dehydrogenase 1 family member L1, and other proteins. However, these proteins may all be regulated both developmentally and functionally, restricting their utility. To identify a nuclear marker pathognomonic of astrocytic phenotype, we assessed differential RNA expression by FACS-purified adult astrocytes and, on that basis, evaluated the expression of the transcription factor SOX9 in both mouse and human brain. We found that SOX9 is almost exclusively expressed by astrocytes in the adult brain except for ependymal cells and in the neurogenic regions, where SOX9 is also expressed by neural progenitor cells. Transcriptome comparisons of SOX9ϩ cells with GLT1ϩ cells showed that the two populations of cells exhibit largely overlapping gene expression. Expression of SOX9 did not decrease during aging and was instead upregulated by reactive astrocytes in a number of settings, including a murine model of amyotrophic lateral sclerosis (SOD1G93A), middle cerebral artery occlusion, and multiple ministrokes. We quantified the relative number of astrocytes using the isotropic fractionator technique in combination with SOX9 immunolabeling. The analysis showed that SOX9ϩ astrocytes constitute ϳ10 -20% of the total cell number in most CNS regions, a smaller fraction of total cell number than previously estimated in the normal adult brain. Significance StatementAstrocytes are traditionally identified immunohistochemically by antibodies that target cell-specific antigens in the cytosol or plasma membrane. We show here that SOX9 is an astrocyte-specific nuclear marker in all major areas of the CNS outside of the neurogenic regions. Based on SOX9 immunolabeling, we document that astrocytes constitute a smaller fraction of total cell number than previously estimated in the normal adult mouse brain.

Metabolically, the brain is a highly active organ that relies almost exclusively on glucose as its energy source. According to the astrocyte-to-neuron lactate shuttle hypothesis, glucose is taken up by astrocytes and converted to lactate, which is then oxidized by neurons. Here we show, using 2-photon imaging of a near-infrared 2-deoxyglucose analogue (2DG-IR), that glucose is taken up preferentially by neurons in awake behaving mice. Anesthesia suppressed neuronal 2DG-IR uptake and sensory stimulation was associated with a sharp increase in neuronal, but not astrocytic, 2DG-IR uptake. Moreover, hexokinase, which catalyze the first enzymatic steps in glycolysis, was highly enriched in neurons compared with astrocytes, in mouse as well as in human cortex. These observations suggest that brain activity and neuronal glucose metabolism are directly linked, and identifies the neuron as the principal locus of glucose uptake as visualized by functional brain imaging.