Astrocytes functionally integrate multiple synapses via specialized leaflet domains.

Accumulating studies have shown that astrocytes are essential for regulating neurons at both synaptic and circuit levels. The main mechanism of brain astrocytic intracellular Ca2+ activity is through the release of Ca2+ via the inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) from the endoplasmic reticulum (ER). Studies using IP3R2 knockout mouse models (Itpr2-/-) have shown that eliminating IP3R2 leads to a significant reduction in astrocytic Ca2+ activity However, there is ongoing controversy regarding the effect of this IP3R2-dependent reduction in astrocytic Ca2+ transients on neuronal activity. In our study, we employed dual-color two-photon Ca2+ imaging to study astrocytes and neurons simultaneously in vibrissa somatosensory cortex (vS1) in awake-behaving wild-type and Itpr2-/- mice. We systematically characterized and compared both recorded astrocytic and neuronal Ca2+ activities in wild-type and Itpr2-/- mice during various animal behaviors, particularly during the transition period from stillness to locomotion. We report that vS1 astrocytic Ca2+ elevation in both wild-type and Itpr2-/- mice was significantly modulated by free whisking and locomotion. However, vS1 neurons were only significantly modulated by locomotion in wild-type mice, but not in Itpr2-/- mice. Our study suggests a non-synaptic modulatory mechanism on functions of astrocytic IP3R2-dependent Ca2+ transients to local neurons.

Astrocytic Ca2 + waves mediate activation of extrasynaptic NMDA receptors in hippocampal neurons to aggravate brain damage during ischemia

Defining the Role of Astrocytes in Neuromodulation

Astrocytes can control basal synaptic strength and arteriole tone via their resting Ca2+ activity. However, whether resting astrocyte Ca2+ can adjust to a new steady-state level, with an impact on surrounding brain cells, remains unknown. Using two-photon Ca2+ imaging in male rat acute brain slices of the somatosensory neocortex, we found that theta burst neural activity produced an unexpected long-lasting reduction in astrocyte free Ca2+ in the soma and endfeet. The drop in intracellular Ca2+ was attenuated by antagonists targeting multiple ionotropic and metabotropic glutamate receptors, and intracellular cascades involved Ca2+ stores and nitric oxide. The reduction in astrocyte endfoot Ca2+ was coincident with an increase in arteriole tone, and both the Ca2+ drop and the tone change were prevented by an NMDA receptor antagonist. Astrocyte patch-clamp experiments verified that the glutamate receptors in question were located on astrocytes and that Ca2+ changes within astrocytes were responsible for the long-lasting change in arteriole diameter caused by theta burst neural activity. In astrocytes from animals that lived in an enriched environment, we measured a relatively lower resting Ca2+ level that occluded any further drop in Ca2+ in response to theta burst activity. These data suggest that electrically evoked patterns of neural activity or natural experience can adjust steady-state resting astrocyte Ca2+ and that the effect has an impact on basal arteriole diameter.SIGNIFICANCE STATEMENT The field of astrocyte-neuron and astrocyte-arteriole interactions is currently in a state of refinement. Experimental evidence ex vivo suggests that direct manipulation of astrocyte-free Ca2+ regulates synaptic signaling and local blood flow control; however, in vivo experiments fail to link synaptically evoked astrocyte Ca2+ transients and immediate changes to various astrocyte-mediated processes. To clarify this discrepancy, we examined a different aspect of astrocyte Ca2+: the resting, steady-state free Ca2+ of astrocytes, its modulation, and its potential role in the tonic regulation of surrounding brain cells. We found that ex vivo or in vivo neural activity induced a long-lasting reduction in resting free astrocyte Ca2+ and that this phenomenon changed arteriole tone.

Despite extensive research on astrocytic Ca2+ in synaptic transmission, its contribution to the modulation of sensory transmission during different brain states remains largely unknown. Here, by using two-photon microscopy and whole-cell recordings, we show two distinct astrocytic Ca2+ signals in the murine barrel cortex: a small, long-lasting Ca2+ increase during sleep and a large, widespread but short-lasting Ca2+ spike when aroused. The large Ca2+ wave in aroused mice was inositol trisphosphate (IP3)-dependent, evoked by the locus coeruleus-norepinephrine system, and enhanced sensory input, contributing to reliable sensory transmission. However, the small Ca2+ transient was IP3-independent and contributed to decreased extracellular K+, hyperpolarization of the neurons, and suppression of sensory transmission. These events respond to different pharmacological inputs and contribute to distinct sleep and arousal functions by modulating the efficacy of sensory transmission. Together, our data demonstrate an important function for astrocytes in sleep and arousal states via astrocytic Ca2+ waves.

Astrocytes, the most abundant non-neuronal cell type in the mammalian brain, are crucial circuit components that respond to and modulate neuronal activity through calcium (Ca2+) signalling1–7. Astrocyte Ca2+ activity is highly heterogeneous and occurs across multiple spatiotemporal scales—from fast, subcellular activity3,4 to slow, synchronized activity across connected astrocyte networks8–10—to influence many processes5,7,11. However, the inputs that drive astrocyte network dynamics remain unclear. Here we used ex vivo and in vivo two-photon astrocyte imaging while mimicking neuronal neurotransmitter inputs at multiple spatiotemporal scales. We find that brief, subcellular inputs of GABA and glutamate lead to widespread, long-lasting astrocyte Ca2+ responses beyond an individual stimulated cell. Further, we find that a key subset of Ca2+ activity—propagative activity—differentiates astrocyte network responses to these two main neurotransmitters, and may influence responses to future inputs. Together, our results demonstrate that local, transient neurotransmitter inputs are encoded by broad cortical astrocyte networks over a minutes-long time course, contributing to accumulating evidence that substantial astrocyte–neuron communication occurs across slow, network-level spatiotemporal scales12–14. These findings will enable future studies to investigate the link between specific astrocyte Ca2+ activity and specific functional outputs, which could build a consistent framework for astrocytic modulation of neuronal activity.

Gap junctions and the intercellular communication syncytium they form between glial cells are thought to play a critical role in glial maintenance of appropriate metabolic environments in neural tissues. We have previously suggested (Yamamoto et al., Brain Res. 508:313-319, '90) that the vast majority of astrocytes in rat brain express connexin43, one of several recently identified gap junction proteins. Here, we confirm ultrastructurally that astrocytes in a number of brain regions of rat are immunolabelled with an antibody against connexin43 and that neurons and oligodendrocytes are devoid of labelling. The distribution of connexin43 immunoreactivity throughout the brain is presented at the light microscope (LM) level. By LM, immunoreactive structures consisted primarily of round or elongated puncta ranging from 0.3 microns to 4 microns in length and of annular profiles ranging from 1 to 10 microns in diameter. Immunolabelled fibrous processes were only occasionally seen and no labelling was observed in astrocytic cell bodies. Long, linear arrays of puncta were rare in gray matter but were common in white matter where they were arranged parallel to myelinated fibers. Puncta organized in a honeycomb pattern were seen near the cerebral cortical surface and frequently around blood vessels. Regional immunoreaction density, which was a reflection of either the concentration or staining intensity of immunoreactive elements, was remarkably heterogeneous; dramatic differences in labelling intensity frequently delineated anatomical boundaries between adjacent nuclei. Abrupt as well as graded fluctuations of immunoreaction intensity were also observed within nuclear structures. By electron microscopy (EM), gap junctions of fibrous and protoplasmic astrocytes were intensely stained and labelled organelles were often observed intracellularly in areas near gap junctions. These junctions and the spread of immunoreaction product to perijunctional organelles in their vicinity were considered to correspond to puncta seen by LM. Labelling within astrocytic cell bodies was seen in only a few instances. In some brain areas, astrocytic processes commonly gave rise to immunoreactive lamellae that partially ensheathed neuronal cell bodies, axon terminals, dendrites, and synaptic glomeruli. Such lamellae were considered to correspond to immunoreactive annular profiles seen by LM. Perivascular endfoot processes of astrocytes displayed intense staining of their gap junctions and portions of their apposing membranes.(ABSTRACT TRUNCATED AT 400 WORDS)

A series of biochemical determinations was performed on five cellular fractions obtained from the cerebellum of 8- to 14-day-old mice. Cerebellar tissue was dissociated by mild trypsinization and mechanical agitation. The dissociated cellular material was separated into five fractions using a series of continuous density gradients formed with Percoll. Three of the fractions were comprised primarily of cell bodies. One of these was dominated by cells having the size and morphological appearance of granule cells, and based on several criteria the other two were were enriched in astrocyte cell bodies. Morphological analysis indicated that the remaining two fractions were enriched, respectively, in nerve terminals and large nucleated cell bodies. The uptake of 12 amino acids and 4 other metabolites by these cellular fractions was examined, and Km and Vmax values were determined for 10 of the compounds studied. High affinity transport carriers (Km approximately 1 to 20 microM) for most of the compounds studied were evident in neuronal and astrocyte-enriched fractions; however, for glutamate and gamma-aminobutyric acid (GABA) additional carriers with higher substrate affinities (Km approximately 0.1 to 0.3 microM) were evident in the astrocyte-enriched fraction. The fraction enriched in granule cell bodies was distinguished by an exceptionally low uptake of GABA and citrate, and a comparatively low uptake of beta-alanine, taurine, alpha-ketoglutarate, and glutamate. An analysis of the content of nine amino acids in the five fractions revealed that only glutamate, aspartate, and GABA were concentrated in the fraction enriched in nerve terminals. GABA was concentrated also in the fraction enriched in large cell bodies and was present at a low concentration in the fraction enriched in granule cell bodies. The other amino acids measured were distributed nearly evenly among the five fractions. Several differences in metabolic activity among the five fractions were observed. Radiolabel from several precursors was incorporated into GABA preferentially in the fractions enriched in large cell bodies and nerve terminals. In contrast, the accumulation of label in glutamine occurred preferentially in the fractions enriched in astrocytes and granule cell bodies. Labeling of alanine from [14C]pyruvate and of proline from [14C]ornithine was most prominent in the fractions enriched in astrocytes and granule cell bodies.

Behavior arises from coordinated brain-wide neural and glial networks, enabling organisms to perceive, interpret, and respond to stimuli. Astrocytes play an important role in shaping behavioral output, yet the underlying molecular mechanisms are not fully understood. Astrocytes respond to intrinsic and extrinsic cues with calcium (Ca2+) fluctuations, which are highly heterogeneous across spatio-temporal scales, contexts, and brain regions. This heterogeneity allows astrocytes to exert dynamic regulatory effects on neuronal function but has made it challenging to understand the precise mechanisms and pathways linking astrocytic Ca2+ to specific behavioral outcomes, and the functional relevance of these signals remains unclear. Here, we review recent literature uncovering roles for astrocytic Ca2+ signaling in a wide array of behaviors, including cognitive, homeostatic, and affective focusing on its physiological roles, and potential pathological implications. We specifically highlight how different types of astrocytic Ca2+ signals are linked to distinct behavioral outcomes and discuss limitations and unanswered questions that remain to be addressed.

Astrocyte pathology in the ventral prefrontal white matter in depression

Whole brain irradiation (WBI) therapy is an important treatment for brain metastases and potential microscopic malignancies. WBI promotes progressive cognitive dysfunction in over half of surviving patients, yet, the underlying mechanisms remain obscure. Astrocytes play critical roles in the regulation of neuronal activity, brain metabolism, and cerebral blood flow, and while neurons are considered radioresistant, astrocytes are sensitive to γ-irradiation. Hallmarks of astrocyte function are the ability to generate stimulus-induced intercellular Ca2+ signals and to move metabolic substrates through the connected astrocyte network. We tested the hypothesis that WBI-induced cognitive impairment associates with persistent impairment of astrocytic Ca2+ signaling and/or gap junctional coupling. Mice were subjected to a clinically relevant protocol of fractionated WBI, and 12 to 15months after irradiation, we confirmed persistent cognitive impairment compared to controls. To test the integrity of astrocyte-to-astrocyte gap junctional coupling postWBI, astrocytes were loaded with Alexa-488-hydrazide by patch-based dye infusion, and the increase of fluorescence signal in neighboring astrocyte cell bodies was assessed with 2-photon microscopy in acute slices of the sensory-motor cortex. We found that WBI did not affect astrocyte-to-astrocyte gap junctional coupling. Astrocytic Ca2+ responses induced by bath administration of phenylephrine (detected with Rhod-2/AM) were also unaltered by WBI. However, an electrical stimulation protocol used in long-term potentiation (theta burst), revealed attenuated astrocyte Ca2+ responses in the astrocyte arbor and soma in WBI. Our data show that WBI causes a long-lasting decrement in synaptic-evoked astrocyte Ca2+ signals 12-15months postirradiation, which may be an important contributor to cognitive decline seen after WBI.

Astrocytes in the Epileptic Brain

Ischemia-induced cellular redistribution of the astrocytic gap junctional protein connexin43 in rat brain

Aspartate-like and glutamate-like immunoreactivities in the inferior olive and climbing fibre system: A light microscopic and semiquantitative electron microscopic study in rat and baboon ( Papio anubis)

The uptake and metabolism of (U‐14C) L‐omithine by several cellular preparations was examined and compared to corresponding data for glutamine and α‐ketoglutarate. Five fractions were obtained from the cerebellum of 10‐to 14‐day‐old mice; two fractions were enriched in astrocyte cell bodies, whereas one was comprised primarily of granule cell bodies, and another was enriched in nerve terminals. Metabolic studies were also conducted on two synaptosomal preparations prepared from rat cerebral tissue.The uptake of ornithine by the cerebellar fractions was mediated by one or two saturable transport systems with apparent Km values between 50 and 200 μM. Uptake inhibition experiments indicated that ornithine is transported primarily, if not exclusively, by the basic amino acid carrier(s), and that glutamine is transported in part by this carrier. Ornithine was metabolized to proline, glutamate, and to a lesser extent aspartate, glutamine and GABA. Under the conditions of our experiments, the cell bodies metabolized ornithine somewhat more readily than did the nerve terminal enriched fraction obtained from the mouse cerebellum. Our data indicate that the conversion of ornithine to GABA occurs predominantly, if not exclusively, via the pathway involving glutamate rather than putrescine. In comparison to data for glutamine and α‐ketoglutarate, the metabolism of omithine to glutamate was slow.Although the results of our study are consistent with the hypothesis that ornithine serves as a metabolic precursor of the neurotransmitter pools of glutamate and GABA, our data do not support a major role for omithine in this capacity. Based on a comparison of the availability of omithine, glutamine and α‐ketoglutarate, and the rates at which they are each transported into synaptosomes and metabolized therein to glutamate and GABA, our data suggest that glutamine and α‐ketoglutarate are used more extensively to replenish these transmitter pools.