Barbara Lykke Lind

Abstract Multimodal astrocyte–neuron communications govern brain circuitry assembly and function 1 . For example, through rapid glutamate release, astrocytes can control excitability, plasticity and synchronous activity 2,3 of synaptic networks, while also contributing to their dysregulation in neuropsychiatric conditions 4–7 . For astrocytes to communicate through fast focal glutamate release, they should possess an apparatus for Ca 2+ -dependent exocytosis similar to neurons 8–10 . However, the existence of this mechanism has been questioned 11–13 owing to inconsistent data 14–17 and a lack of direct supporting evidence. Here we revisited the astrocyte glutamate exocytosis hypothesis by considering the emerging molecular heterogeneity of astrocytes 18–21 and using molecular, bioinformatic and imaging approaches, together with cell-specific genetic tools that interfere with glutamate exocytosis in vivo. By analysing existing single-cell RNA-sequencing databases and our patch-seq data, we identified nine molecularly distinct clusters of hippocampal astrocytes, among which we found a notable subpopulation that selectively expressed synaptic-like glutamate-release machinery and localized to discrete hippocampal sites. Using GluSnFR-based glutamate imaging 22 in situ and in vivo, we identified a corresponding astrocyte subgroup that responds reliably to astrocyte-selective stimulations with subsecond glutamate release events at spatially precise hotspots, which were suppressed by astrocyte-targeted deletion of vesicular glutamate transporter 1 (VGLUT1). Furthermore, deletion of this transporter or its isoform VGLUT2 revealed specific contributions of glutamatergic astrocytes in cortico-hippocampal and nigrostriatal circuits during normal behaviour and pathological processes. By uncovering this atypical subpopulation of specialized astrocytes in the adult brain, we provide insights into the complex roles of astrocytes in central nervous system (CNS) physiology and diseases, and identify a potential therapeutic target.

Increased neuron and astrocyte activity triggers increased brain blood flow, but controversy exists over whether stimulation-induced changes in astrocyte activity are rapid and widespread enough to contribute to brain blood flow control. Here, we provide evidence for stimulus-evoked Ca(2+) elevations with rapid onset and short duration in a large proportion of cortical astrocytes in the adult mouse somatosensory cortex. Our improved detection of the fast Ca(2+) signals is due to a signal-enhancing analysis of the Ca(2+) activity. The rapid stimulation-evoked Ca(2+) increases identified in astrocyte somas, processes, and end-feet preceded local vasodilatation. Fast Ca(2+) responses in both neurons and astrocytes correlated with synaptic activity, but only the astrocytic responses correlated with the hemodynamic shifts. These data establish that a large proportion of cortical astrocytes have brief Ca(2+) responses with a rapid onset in vivo, fast enough to initiate hemodynamic responses or influence synaptic activity.

Cerebral blood flow (CBF) is controlled by arterial blood pressure, arterial CO 2 , arterial O 2 , and brain activity and is largely constant in the awake state. Although small changes in arterial CO 2 are particularly potent to change CBF (1 mmHg variation in arterial CO 2 changes CBF by 3%–4%), the coupling mechanism is incompletely understood. We tested the hypothesis that astrocytic prostaglandin E 2 (PgE 2 ) plays a key role for cerebrovascular CO 2 reactivity, and that preserved synthesis of glutathione is essential for the full development of this response. We combined two-photon imaging microscopy in brain slices with in vivo work in rats and C57BL/6J mice to examine the hemodynamic responses to CO 2 and somatosensory stimulation before and after inhibition of astrocytic glutathione and PgE 2 synthesis. We demonstrate that hypercapnia (increased CO 2 ) evokes an increase in astrocyte [Ca 2+ ] i and stimulates COX-1 activity. The enzyme downstream of COX-1 that synthesizes PgE 2 (microsomal prostaglandin E synthase-1) depends critically for its vasodilator activity on the level of glutathione in the brain. We show that, when glutathione levels are reduced, astrocyte calcium-evoked release of PgE 2 is decreased and vasodilation triggered by increased astrocyte [Ca 2+ ] i in vitro and by hypercapnia in vivo is inhibited. Astrocyte synthetic pathways, dependent on glutathione, are involved in cerebrovascular reactivity to CO 2 . Reductions in glutathione levels in aging, stroke, or schizophrenia could lead to dysfunctional regulation of CBF and subsequent neuronal damage. SIGNIFICANCE STATEMENT Neuronal activity leads to the generation of CO 2 , which has previously been shown to evoke cerebral blood flow (CBF) increases via the release of the vasodilator PgE 2 . We demonstrate that hypercapnia (increased CO 2 ) evokes increases in astrocyte calcium signaling, which in turn stimulates COX-1 activity and generates downstream PgE 2 production. We demonstrate that astrocyte calcium-evoked production of the vasodilator PgE 2 is critically dependent on brain levels of the antioxidant glutathione. These data suggest a novel role for astrocytes in the regulation of CO 2 -evoked CBF responses. Furthermore, these results suggest that depleted glutathione levels, which occur in aging and stroke, will give rise to dysfunctional CBF regulation and may result in subsequent neuronal damage.

Abstract Cerebral blood flow (CBF) is regulated by the activity of neurons and astrocytes. Understanding how these cells control activity‐dependent increases in CBF is crucial to interpreting functional neuroimaging signals. The relative importance of neurons and astrocytes is debated, as are the functional implications of fast Ca 2+ changes in astrocytes versus neurons. Here, we used two‐photon microscopy to assess Ca 2+ changes in neuropil, astrocyte processes, and astrocyte end‐feet in response to whisker pad stimulation in mice. We also developed a pixel‐based analysis to improve the detection of rapid Ca 2+ signals in the subcellular compartments of astrocytes. Fast Ca 2+ responses were observed using both chemical and genetically encoded Ca 2+ indicators in astrocyte end‐feet prior to dilation of arterioles and capillaries. A low dose of the NMDA receptor antagonist (5R,10s)‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine‐hydrogen‐maleate (MK801) attenuated fast Ca 2+ responses in the neuropil and astrocyte processes, but not in astrocyte end‐feet, and the evoked CBF response was preserved. In addition, a low dose of 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP), an agonist for the extrasynaptic GABA A receptor (GABA A R), increased CBF responses and the fast Ca 2+ response in astrocyte end‐feet but did not affect Ca 2+ responses in astrocyte processes and neuropil. These results suggest that fast Ca 2+ increases in the neuropil and astrocyte processes are not necessary for an evoked CBF response. In contrast, as local fast Ca 2+ responses in astrocyte end‐feet are unaffected by MK801 but increase via GABA A R‐dependent mechanisms that also increased CBF responses, we hypothesize that the fast Ca 2+ increases in end‐feet adjust CBF during synaptic activity.